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The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdxC57BL. Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdxC57BL presents three times more differentially expressed genes (DEGs) than the mdx129 (371 and 137 DEGs respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4,8 times higher on western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process.
Citation: Calyjur PC, Almeida CdF, Ayub-Guerrieri D, Ribeiro AF Junior, Fernandes SdA, Ishiba R, et al. (2016) The mdx Mutation in the 129/Sv Background Results in a Milder Phenotype: Transcriptome Comparative Analysis Searching for the Protective Factors. PLoS ONE 11(3): e0150748. https://doi.org/10.1371/journal.pone.0150748
Editor: Maurilio Sampaolesi, Stem Cell Research Institute, BELGIUM
Received: July 23, 2015; Accepted: February 18, 2016; Published: March 8, 2016
Copyright: © 2016 Calyjur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are all contained within the paper and/or Supporting Information files.
Funding: This work was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq projetos 404664/2012-1 e 456522/2014-0), Fundação de Amparo a Pesquisa do Estado de São Paulo - Centro de Pesquisa, Inovação e Difusão (FAPESP-CEPID- projeto 2013/08028-1), CNPq-INCT (Projeto no: 610031/2009-0), Financiadora de Estudos e Projetos (FINEP – projeto 01.08.0579.00) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Comitê Francês de Avaliação da Cooperação Universitária com o Brasil (CAPES-COFECUB).
Competing interests: The authors have declared that no competing interests exist.
Neuromuscular disorders are a heterogeneous group of genetic diseases, causing progressive loss of motor ability. More than 30 genetically defined forms are recognized and in the last decade mutations in several genes have been reported that result in deficiency or loss of function of different important muscle proteins.
Duchenne muscular dystrophy (DMD) is the most common and severe human muscular dystrophy, affecting 1 in 3500 male births. It is caused by mutations in the dystrophin gene which result in the absence of this important sarcolemmal protein and consequent muscle degeneration. The clinical course of DMD is severe and progressive, starting with muscle weakness at the age of five and loss of ambulation around 12 years; without special care, death occurs due to respiratory failure or cardiomyopathy in the late teens [1]. There is no effective cure for patients suffering from this type of dystrophy.
Several animal models manifesting phenotypes observed in neuromuscular diseases have been identified in nature or generated in laboratory. These models generally present physiological alterations observed in human patients and can be used as important tools for pathophysiological studies and therapy testing [2]. The C57BL/10ScSn-Dmdmdx/J, named here as mdx, is the most widely used animal model for DMD, bearing a non-sense point mutation in exon 23 of the dystrophin gene which causes lack of this protein in the skeletal muscle [3]. However, differently from human DMD patients, the mdx presents a mild phenotype, with normal lifespan and reproductive capacity [4]. Therefore, this model is not effective in clinical trials to track possible functional benefits of tested therapies.
Studies in animal models by insertion of a human pathogenic mutation in distinct mouse backgrounds have shown differences in phenotypical manifestation according to the mouse strain. Previously, a group introduced the mgΔ mutation from a mouse model for Marfan syndrome in two different genetic backgrounds: C57BL and 129/Sv [5]. The animals with 129/Sv background presented a more severe and earlier phenotype than those with C57BL background.
Considering that the increased severity of muscle damage is extremely useful in assessing how effective a novel therapy might be at halting human disease and in order to obtain a more reliable animal model for DMD, we decided to transfer the mdx mutation to the 129/Sv background expecting that the resulting animals would present a more severe DMD phenotype.
Unexpectedly, the newly created mdx129 model showed a progressive amelioration of the phenotype. Thus, a functional analysis, followed by transcriptome comparative gene expression analysis, histological and protein studies were performed to identify and characterize what is protecting this new model from the dystrophic characteristics. We identified the overexpression of the Spp1 gene, which codes for the osteopontin (OPN) protein, as the most significant candidate to cause this benefit to the dystrophic phenotype.
Materials and Methods
The 129/Sv male mice were obtained from the ICB USP experimentation housing facility, while C57BL and the mdx females were obtained from the Center for Human Genome and Stem Cell Researches (IB USP) experimentation housing facility. The animals were kept under controlled temperature and light conditions and were fed with pellets and water ad libitum. All experimental procedures were analyzed and approved by the Institute of Biosciences Ethics Commission in the Use of Animals (Permit Number: CEUA/IBUSP 201/2014).
Transferring the mdx mutation to the 129/Sv phenotype
The first breeding pairs consisted of 129/Sv males and mdx females. Their offspring (generation mdx129 F1) consisted of affected males and carrier females. The carrier females were backcrossed with the 129/Sv males, and their offspring (generation mdx129 F2) consisted of wild-type males and females, affected males and carrier females, according to Mendelian proportions (Fig 1A). From this generation on, the litters were genotyped for the mdx mutation to select only the affected males and carrier females (Fig 1B). These carrier females were then backcrossed with the 129/Sv male, generating mdx129 F3.
(A) Schematic representation of the cross-breeding. (B) Genotyping for the mdx mutation: acrylamide gel electrophoresis of the PCR competitive reaction showing the presence of the 134 pb band in two wild type normal DNA (N), a 117 pb band in the mdx (M) and both bands in two carrier females (H). (C) Dystrophin immunofluorescence analysis with DYS2 antibody showing the presence of dystrophin in the muscle membrane of normal control, and the absence of dystrophin in the mdx129.
DNA was extracted from a 0.5cm piece of tail using Proteinase K (Promega, Madison, WI, USA) as described [6]. The genotyping was done by PCR competitive reaction, using specific primers for the exon 23 of the murine dystrophin gene. The product was applied in 10% acrylamide gel where different band patterns can be identified for wild type (134 pb), heterozygous (134 and 117 pb) and affected animals (117 pb) (Fig 1B), according to a previously described protocol [7]. Dystrophin analysis confirmed the deficiency of the protein in F3 males, carrying the pathogenic mutation in the dystrophin gene (Fig 1C).
Functional evaluations
All mdx129 male mice obtained in the three generations were monthly evaluated during the period of six months, using mdxC57BL mice as controls. The number of studied male animals was: F1 = 9, F2 = 13, F3 = 14.
The used tests were previously described [8] and validated in our colonies of neuromuscular disease mice models. In addition, the same researcher performed the analysis in all mice, avoiding intra-personal variability. Briefly:
- The animals capacity of hanging from a bar by their fore limbs and by all four limbs—the animal is positioned hanging from a 3mm metal bar by its fore limbs or by all four limbs and the time it keeps hanging is counted. We consider 60 seconds to be the maximum time and the test is successively repeated for three times, after which we calculate the average.
- Fore limbs and hind limbs grip strength—the animal is positioned so that it will grip the grid attached to a dynamometer with its fore limbs or with its hind limbs and then is pulled by the tail until it releases it. This procedure is successively repeated for five times and the mean is calculated.
Histological and immunohistochemical analyses
For these analyses, three male animals from each group were used, all in the age of six months. After animals were euthanized, muscles of the posterior portion of the leg (mainly encompassing the gastrocnemius muscle) and the diaphragm were dissected, fixed in cork blocks with Tissue-Tek® O.C.T Compound (Sakura Finetek USA, Torrance, CA, USA), cryoprotected with talc and frozen in liquid nitrogen.
For histopathology analysis, the following parameters were used: percentage of centronucleated fibers (evaluated using hematoxilin/eosin (HE) staining), fibrosis (evaluated using the quantification of picrossirius staining) and regenerating fibers (quantified using immunofluorescence staining for mouse monoclonal myosin heavy chain (developmental) antibody (VP-M664, Vector Laboratories, Burlingame, CA, USA), in a double reaction with antibody to muscle laminin (rat monoclonal anti-laminin gamma 1 antibody, ab80580, Abcam, Cambridge, MA, USA). Additionally, a mouse monoclonal anti-dystrophin DYS2 antibody was used to identify this protein (VP-D505, Vector Laboratories, Burlingame, CA, USA). The slides were examined and photographed using a Zeiss AxioImager.Z1 microscope.
For positive developmental myosin fibers quantification, fiber numbers from at least five different fields in the cross-sections from each animal were measured, and the total number of positive fibers was compared to the total number of counted fibers. The number of counted fibers was in a total of 600 to 2000 fibers per animal. The area of fibrotic tissue using picrossirius-stained sections was relatively measured using ImageJ software (http://rsb.info.nih.gov/ij/) and using area of control mouse as 1.
Western blotting analysis
Total proteins were extracted from the gastrocnemius muscle and separated by 13% SDS-PAGE polyacrylamide gel electrophoresis and were transferred onto a nitrocellulose membrane (GE Healthcare Biosciences, Pittsburgh, PA, USA) for 60 min at 0.35A at 4°C. Membranes were then pre-stained in 0.2% Ponceau S, to ensure protein transfer and equal protein loading of the lanes. Membranes were blocked with 5% non fat milk in PBS, 0.1% Tween 20 (TBS-T) for 1 h and probed using the primary antibodies against OPN sc-21742 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). After an overnight period of incubation, membranes were washed three times with TBS-T for 10 min. The blots were then immunostained with Pierce® Anti-mouse IgG (HRP) Polyclonal antibody (Thermo Fisher Scientific, Waltham, MA, USA) and posterior detection of protein was done using Novex® ECL Chemiluminescent Substrate Reagent Kit (Invitrogen, Waltham, MA, USA).
Quantitative analysis of mouse OPN present in total protein extracted was performed using the ImageJ software (http://rsb.info.nih.gov/ij/), with myosin at the Ponceau staining as a protein loading control. The value of each animal was normalized to the normal control within the same blot.
RNA extraction and purification
For RNA extraction, frozen gastrocnemius muscle of three animals from each strain was used, at the age of six months. Frozen muscles were finely powdered using a mortar and total RNA was extracted using RNeasy Microarray Tissue Mini Kit (Qiagen, Valencia, CA, USA) according to manufacturer instructions. RNA contamination by DNA was verified in 1% agarose gel.
Hybridization and Microarray data analysis
Samples for hybridization were prepared using Ambion® WT Expression and GeneChip® WT Terminal Labeling (Life Technologies, Waltham, MA, USA) and hybridized in GeneChip® Mouse Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA) chips, all according to instructions provided by the manufacturer. Pre-analysis and data normalization were performed in the Expression Console™ (Affymetrix, Santa Clara, CA, USA) software using the RMA (Robust Multi-array Average) algorithm. Normalized data were uploaded to the MeV software where differentially expressed genes (DEGs) were determined by the SAM algorithm. To study functional networks among the identified DEGs, we used the IPA software (Ingenuity® Systems, www.ingenuity.com). Gene ontology function enrichment analysis was performed with the DAVID tool (Database for Annotation, Visualization and Integrate Discovery http://david.abcc.ncifcrf.gov/). Raw data has been deposited at Gene Expression Omnibus (GEO) database (accession number GSE77126).
qPCR analysis
RNA was quantified and normalized for cDNA synthesis using oligo dT and random primers, and MMLV enzyme (Invitrogen, Waltham, MA, USA), in a quick 3-step protocol of incubation in 65°C or 37°C. For qPCR, we used the protocol previously described [9]. Samples of cDNA of each animal were applied in triplicate in 96 wells plates. At each sample was added the pair of primers of the gene of interest and Sybr Green Master Mix (Applied Biosystems, Carlsbad, CA, USA) in a total volume of 25μL. Each plate was run in the 7500 Fast Applied Biosystems thermocycler for real-time PCR.
The relative expression of the following genes was measured:
- Pathway of muscle regeneration: MyoD, Myf5 and Myogenin (Myog);
- Validation of microarray data: Spp1 and Col5a2.
The used primers are described in Table 1: we used previously described primers for Spp1 [10] and the others were selected from a real-time primer database with validation results (Primerbank). The chosen gene for use as an endogenous control was Gapdh.
As the analysis uses one unique calibrator control, it was necessary to select one strain, for this purpose. We decided to use the wild type mouse 129/Sv sample with the nearest expression of the most genes in all of them.
Statistical Analysis
Functional evaluations were statistically analyzed using Mann-Whitney test. For the other parameters, due to the small size of the samples, Mann-Whitney and Kruskal Wallis corrected with Dunn-Bonferroni non parametric tests were performed, but with no statistically significant differences detected. All calculations were performed using the Minitab 17 software.
Evidences from functional evaluations
As shown in Fig 2, in the first generation of mdx129 (F1) forelimbs bar test, the animals already demonstrated more resistance than mdxC57BL animals, yet, the results were significant merely in the ages two, four and five months. In the grip strength test, F1 animals were stronger than mdxC57BL mice, but the results were only significant at 30 days, three and four months. The second mdx129 generation (F2) showed improved performance in all tests compared to mdxC57BL. In both bar tests, F2 mice hang on the bar for the whole time of the test (60 seconds), and the values were statistically significant at all ages. The strength was also increased in this group. Noticing the results from the third mdx129 generation (F3), we could confirm the progressive increase both in resistance and strength of mdx129 animals. The F3 mice hang on the bar for 60 seconds with no sign of fatigue, and grip strength results were better than in generations F1 and F2. The same results were observed in the test applied for the four limbs resistance and hind limb strength.
Data of the fore limbs in the bar test and grip strength test are shown with the median expression and standard error range for each age/strain. The number of tested animals in each generation is also presented. * p<0,05.
Therefore, the observed results were the opposite of what was previously described [5] and which we were expecting. Considering the amelioration of the mdx phenotype in the 129/Sv background, we hypothesized that factors in this strain could act protecting the mdx129 from the dystrophic effect. Consequently, the study was directed to identify these factors as possible positive modifiers of the dystrophic phenotype.
Muscle Analysis
Evidence from histological analysis.
Histological analysis using HE stain showed that the degenerative/regenerative processes were similar in all mdxC57BL mice as compared to the three generations of mdx129 mice, both in gastrocnemius and diaphragm muscles (Fig 3). In all strains, degenerated fibers, regenerating fibers and connective tissue infiltration were identified, with almost 100% of centrally nucleated fibers.
HE staining of gastrocnemius and diaphragm muscles, in mdxC57BL and mdx129 mice in the age of 6 months.
Evidence of reduced degeneration.
The quantification of connective tissue replacement, measured by picrossirius staining, demonstrated a reduction of the amount of endomysial and perimysial connective tissues in mdx129 in relation to mdxC57BL, both in gastrocnemius (27%) as well as in the diaphragm (44%) muscles (Fig 4).
(A) Representation of picrossirius staining of gastrocnemius and diaphragm muscles from mdxC57BL and mdx129 models. (B) Graphic representing the quantitative comparison between the two mdx strains.
Evidence of regeneration.
The regeneration findings obtained for gastrocnemius samples of normal (129/Sv) and the two mdx models are shown in Fig 5. The average relative expression for regenerative genes (Myf5, MyoD and Myog) was obtained in qPCR analysis (Fig 5A). As to myogenic factors related to early regeneration, Myf5 expression was higher in mdx129 mice versus control, while MyoD expression was higher in mdxC57BL. However, for the late stage of myogenic differentiation, Myog expression presented higher expression in the two mdx models when compared to control. Meanwhile, immunohistochemical analysis for developmental myosin in gastrocnemius muscle of the two affected mice models showed that mdx129 presented an increase of about 50% in the number of these fibers in relation to the mdxC57BL, suggesting a more active regeneration in mdx129 (Fig 5B and 5C).
(A) Results of the qPCR expression of genes involved with the regeneration process (Myf5, MyoD and Myog). (B) Representation of the comparative immunohistochemical analysis of gastrocnemius for developmental myosin (red) in double reaction with laminin (green), showing the proportion of positive fibers in mdxC57BL and mdx129 mice in the age of 6 months. (C) Graphic representing the quantitative comparison between the two groups of mdx with normal control (con).
Evidence from transcriptome analysis.
We used expression microarray to investigate mdx129 transcriptome comparing it to mdxC57BL and each of these models to their same background wild type.
C57BL and 129/Sv background comparison.
First, we compared both wild types to verify the difference between them. In the comparison of C57BL/ x 129/Sv we found 44 differentially expressed genes (DEGs), and 13 DEGs among the muscle expressed genes. The majority of these DEGs were downregulated in 129/Sv in comparison to C57BL (Table 2).
Comparing both mdx models.
Comparing C57BL and mdxC57Bl, we found 371 DEGs: 320 upregulated and 51 downregulated in mdxC57BL. When comparing 129/Sv with mdx129 F3, we found 137 DEGs: 130 upregulated and 7 downregulated in mdx129 F3 mice. In both backgrounds, the number of upregulated genes was higher than the downregulated ones (Table 2).
The DEGs were classified according to their functional categories and we observed that the immune system category was the most significant, gathering 60% of the genes in C57BL x mdxC57BL and 80% of the genes in 129/Sv x mdx129.
Since this category is so predominant, we decided to remove it in order to verify the significance of the participation of other categories in the two mdx models (Fig 6).
(A) Total identified DEGs in the comparisons C57BL x mdxC57BL and 129/Sv x mdx129 F3. (B) Categories excluding the immune system.
Both models showed corresponding categories, except homeostasis, which only appear in C57BL x mdxC57BL. Otherwise, the proportion in which these categories appear is different. Genes related to endo/exocytic pathways demonstrated a decreased expression in 129/Sv x mdx129, while an increased expression was found in genes of other categories, such as extracellular matrix, binding to molecules/cells and enzymatic activity.
Comparing both mdx models for genes expressed in muscle.
To better visualize the changes in mdx129 expression profile, we filtered the lists C57BL x mdxC57BL, 129/Sv x mdx129 and mdxC57BL x mdx129 for genes that, according to the literature, act in skeletal muscle. We found 85 DEGs that were exclusive to C57BL x mdxC57BL, 13 DEGs unique to 129/Sv x mdx129 and 12 DEGs exclusive to mdxC57BL x mdx129 (Fig 7).
Of the 13 genes exclusively expressed in 129/Sv x mdx129 (Table 3), all of them presented an upregulated expression, and two important DEGs were identified: Spp1 and Ilrn. Comparing mdxC57BL x mdx129 we found 12 exclusive genes (Table 3), where Klk3 showed the most upregulated expression and Mup1, the most downregulated expression. qPCR validated the results observed for Col5a2 and Spp1.
The most highly induced transcript in the mdx129 data set was Spp1, which is a strong indicator of muscle injury [11] and an important gene for the dystrophic process. Based on this, Spp1 was also studied through qPCR for mRNA relative quantification and protein quantification through western blotting (Fig 8). An elevation of ~200X in mRNA expression was observed in mdx129 as compared to 129/Sv (Fig 8A). At the protein level, the used OPN antibody recognized both the full-length protein (66 kDa band) and one fragment of 32 kDa corresponding to a cleaved product. Those findings revealed the presence of the same concentration of cleaved product in all mdx models, but a stronger concentration of the full protein (4,8 times higher) in mdx129 (Fig 8B and 8C), showing a direct correlation with transcript findings.
(A) Fold changes in qPCR relative expression levels of osteopontin mRNA, as compared to the control group (129/Sv). (B) Western blotting analysis for OPN protein showing both the full length protein (66 kDa band) and one fragment of 32 kDa (cleaved product) in mdxC57BL and mdx129 as compared to normal control 129/Sv (con); M—myosin band. (C) Western blotting quantification showing the mean of each group for the band of 32 kDa and 66 kDa. An increase of 4,8 times of the 66 kDA band is observed in the mdx129 group using myosin band as a protein loading control.
Studies in animal models are essential for testing therapies, mainly in diseases still with no effective cure, such as Duchenne Muscular Dystrophy (DMD). Considering that the genetic background can significantly affect phenotype in mouse models of human diseases, and that the most common animal model for DMD, the mdx mouse in the C57BL background, shows a very mild phenotype, we aimed to create a mdx model with a different genetic background, expecting that the resulting animals would present a more severe DMD phenotype. Surprisingly, the opposite results were obtained, with mdx129 mice presenting a significantly amelioration of phenotype in all functional tests, in successive generations, when compared to mdxC57BL. We could attribute this crescent improvement of the phenotype to the increasing proportion of the 129/Sv background.
We speculated if in the same background of the mouse, primary mutations in different genes could affect phenotype in a different way. Mainly because the mgΔ mutation for Marfan syndrome resulted in a more severe phenotype in the 129/Sv than in the C57BL background [5], while with the mdx mutation the result was inverse: a better phenotype in the 129/Sv than in the C57BL background.
Histological analysis was not informative to differentiate the two mdx strains, since both mdx models presented a similar histopathological pattern in hind limbs as well as in diaphragm muscles. However, a lower proportion of connective tissue and an intense regeneration were observed in mdx129 strain, as revealed by higher expression of muscle myogenesis factors and the presence of a higher number of developmental myosin positive fibers in this model.
To identify the protective factors that could be involved, we used an expression microarray analysis to investigate mdx129 transcriptome comparing it to mdxC57BL, in six-month old animals, when the disease is fully established.
A first remarkable observation was the similarity of C57BL versus 129/Sv wild backgrounds, presenting a small number of DEGs among the mapped genes. Besides, we did not find among the DEGs expressed in the muscle anything which could be, somehow, associated to a directly related muscular function that could eventually provide protection against dystrophy.
As to the comparison between the complete transcriptome of the two mdx strains, we verified that DEGs number is about three times higher in mdxC57BL than in mdx129 when compared to wild type from the same background animals. Nevertheless, in both backgrounds the dystrophin gene mutation causes more upregulation than downregulation of genes expression. These results suggest that, in the new background, the dystrophin mutation clearly induced fewer mechanisms of action that could be responsible for the installation and maintenance of the dystrophic process.
The comparison of the three generations of mdx129 exhibited a small number of DEGs, and we could therefore conclude that in the expression level, the background effect is present since the first generation, and it does not increase with 129/Sv background (Table 2).
In both models, the participation of genes involved in immune system was clearly predominant, which was expected, once chronic inflammation is a dystrophic muscle characteristic [12]. Nevertheless, when excluding this category, some differences were noted between the two deficient dystrophin models, including a reduction in endo/exocytic pathway but an increase in the participation of the extracellular matrix, binding to molecules/cells and enzymatic activity pathways in mdx129. Vesicle trafficking is a necessary process for membrane repair [13] and several studies have shown its importance in the dystrophic phenotype [14, 15]. Our findings suggest that the decreased participation of vesicle-related genes in mdx129 animals is due to a more stable membrane, which can present, consequently, better fiber conservation and functional performance. The membrane stability can also explain why the homeostasis category disappeared, since repair mechanism is calcium dependent [16]. Among the functional categories that increased in mdx129, the most remarkable was the extracellular matrix. As seen on histological slides, regeneration is present in these animals up to six months; consequently, matrix remodeling is probably occurring, which can explain the increased participation of genes related to this process [17].
When the analysis was focused on genes expressed in muscle, we identified DEGs that were exclusive for some of the compared strains. Among the exclusive DEGs in the comparison of mdxC57BL x mdx129 the more upregulated gene was Klk3 and the more downregulated was Mup1. Klk3 codes for PSA (prostate-specific antigen) which is a prostate cancer biomarker [18]. That being, we could not find a correlation between this gene and mdx129 phenotype. Mup1 is part of a gene family involved in chemical communication among animals (mice and rats) [19] and few studies were conducted about its metabolic functions. A group [20] observed that the skeletal muscle was a major target for Mup1. In their study with obese mice, which present lower levels of the protein, partial level corrections of the protein alleviated insulin resistance and glucose intolerance, ameliorating skeletal muscle mitochondrial function. In our study, mdx129 presented a significant decrease of Mup1 expression than mdxC57BL, suggesting a beneficial effect of this lower expression in their phenotype. More studies will be needed to explain Klk3 and Mup1 roles in muscle function.
Of the 13 genes exclusively expressed in 129/Sv x mdx129, two deserve to be highlighted: the upregulated Spp1 and Il1rn. Il1rn gene codes for IL-1Ra, an interleukin-1 antagonist. Considering interleukins pro-fibrotic and pro-inflammatory effects, the increased expression of its antagonist suggests that mdx129 animals could be more protected from the inflammatory process generated by these molecules.
Spp1 gene codes for osteopontin (OPN), which can be found as an extracellular matrix component and a soluble molecule with cytokine properties [21]. OPN is a multifunctional molecule that is involved in both physiological and pathological processes, including tissue repair, inflammation and fibrosis [22, 23, 24, 25, 26, 27, 28, 29, 30], and its effects in muscular dystrophy are not yet clear. OPN alterations have been described as a dystrophic and injured muscle component [11, 12, 31].
Our study showed a higher expression of Spp1 gene in mdx129 model in the transcriptomic analysis, and this data was confirmed through mRNA relative quantification and protein quantification through western blotting. OPN overexpression could, therefore, be considered a good candidate to explain the better phenotype of this model. A 100-fold increase in the expression of osteopontin mRNA in regenerating muscle of mice with muscle induced damage, lead Hoffman et al. [32] to suggest that OPN is involved in the inflammatory, degenerative and regenerative events that occur in early skeletal muscle regeneration. Our results strongly support this hypothesis, once we found differences of expression of early regenerative genes (Myod and Myf5) between the mdx129 mice and the other mice strains, suggesting that OPN contributes with muscle regeneration. Additionally, according to the model proposed by Pagel et al. in 2014 [33], at later stages in muscle regeneration, osteopontin may be increasingly incorporated into the remodeled extracellular matrix and support their terminal differentiation into new muscle fibers [34]. As OPN is immobilized by incorporation into the extracellular matrix in terminal differentiation, the fusion of myoblasts is favored, thereby promoting the formation of myotubes. In our study, among the functional categories that increased in mdx129, the most remarkable was the extracellular matrix. Also, differences in Myog expression and the higher number of positive developmental myosin fibers in the mdx129 model support a stimulus for a better terminal differentiation into new muscle fibers in this model. Hence, it is quite likely that the enhancement in OPN expression might play a part in improving the dystrophic pathology in mdx129 mice stimulating late regeneration. These findings points to a potential value of OPN as a skeletal muscle disease progression biomarker.
Phenotypic variability due to genetic modifiers that regulate disease process acting in the regeneration process has been demonstrated recently in the DBA/2J background. The mdx mutation in this background confers a more severe muscular dystrophy phenotype than the original strain, demonstrating the presence of genetic modifier loci in the DBA/2J background [35]. Interestingly, self-renewal efficiency of satellite cells in this background is lower than that of C57BL strain [36], demonstrating a direct effect of modifier genes in the capacity of muscle regeneration, an important factor in muscular dystrophy prognosis.
At the molecular level, Spp1 gene is expressed by a single copy gene as a 34 kDa nascent OPN protein composed of 300 amino acids residues. In mammalian cells, the final size of OPN can ranges from 44 to 75 kDA after the post-translational modifications (glycosylation, sulphatation and phosphorylation), which under certain circumstances influence its function [37, 38]. Western blotting results in our mdx models showed that the full length form of OPN (66 kDa) was present in a higher quantity in mdx129 mice suggesting also a more effective post-translational process in this model. On the other hand, OPN is also a substrate for some matrix metalloproteinases (MMPs) [39] and the cleavage by these MMPs occurs at a limited number of sites. It has been reported that OPN proteolytic fragmentation is a biological process having physiological importance [40] and the fragments possess greater activity than the full-length forms [41]. The small ~32 kDa fragment observed in all animals in the same intensity could reflect this proteolytic fragment, and in this case, it would be similar in both models. The larger band would be a repository for this protein in the muscle.
To date, studies on the role of osteopontin in skeletal muscle pose almost as many questions as they answer. In dystrophic mouse muscles chronic overexpression of osteopontin appears to be related to decreased muscle strength and fibrosis in mice [33, 42], whereas in muscles from patients with Duchenne muscular dystrophy a SNP associated with osteopontin overexpression in vitro has been found to be a significant positive modifier of the disease [43]. Therefore, one possible explanation for the apparently contradictory observations is that osteopontin plays many roles, some of them antagonistic to others in injured muscle, and that post-translational modification, processing, timing, as well as absolute level of osteopontin expression influence the role that it plays.
The results observed in our more benign mdx129 model, expressing higher levels of Spp1 than the mdxC57BL, are suggestive of a positive role of this gene as a prognostic biomarker in humans, since it is more expressed in the mildly affected model.
In conclusion, modeling the expression of these differentially expressed genes involved in the benign course of the mdx mutation, in particular Spp1 gene, should be tested as possible therapeutic targets for the dystrophic process.
The authors would like to thank the following researchers for scientific and technical support: Dr. Lydia Yamamoto, Dr. Mayana Zatz, Marta Canovas, Heloísa Maria Siqueira Bueno.
Author Contributions
Conceived and designed the experiments: PCC MV DAG. Performed the experiments: PCC CFA ALFS AFRJ DAG SAF RI. Analyzed the data: PCC CFA ALFS AFRJ DAG SAF. Contributed reagents/materials/analysis tools: POO CFA. Wrote the paper: MV PCC CFA DAG SAF.
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- 5. Lima BL, Santos EJ, Fernandes GR, Merkel C, Mello MR, Gomes JP, et al. A new mouse model for marfan syndrome presents phenotypic variability associated with the genetic background and overall levels of Fbn1 expression. PLoS One. 2010; 5(11): e14136. pmid:21152435
- 6. Zangala T. Isolation of genomic DNA from mouse tails. J Vis Exp. 2007; 6: 246. pmid:18997894
- 7. Shin JH, Hakim CH, Zhang K, Duan D. Genotyping mdx, mdx3cv, and mdx4cv mice by primer competition polymerase chain reaction. Muscle Nerve. 2011; 43: 283–286. pmid:21254096
- 8. Chiavegatto S, Sun J, Nelson RJ, Schnaar RL. A functional role for complex gangliosides: motor deficits in GM2/GD2 synthase knockout mice. Exp Neurol. 2000; 166: 227–234. pmid:11085888
- 9. Gosselin LE, Williams JE, Brazeau D, Koury S, Martinez DA. Localization and early time course of TGF-beta 1 mRNA expression in dystrophic muscle. Muscle and Nerve. 2004; 30(5): 645–53.6 pmid:15389721
- 10. Szulzewsky F, Pelz A, Feng X, Synowitz M, Markovic D, Langmann T, et al. Glioma associated microglia/macrophages display an expression profile different from M1 and M2 polarization and highly express Gpnmb and Spp1. PLoS One. 2015; 10(2): e0116644. pmid:25658639
- 11. Hirata A, Masuda S, Tamura T, Kai K, Ojima K, Fukase A, et al. Expression profiling of cytokines and related genes in regenerating skeletal muscle after cardiotoxin injection: a role for osteopontin. Am J Pathol, 2003; 163: 203–215. pmid:12819025
- 12. Porter JD, Khanna S, Kaminski HJ, Rao JS, Merriam AP, Richmonds CR, et al. A chronic inflammatory response dominates the skeletal muscle molecular signature in dystrophin-deficient mdx mice. Hum Mol Genet. 2002; 11: 263–272. pmid:11823445
- 13. Han R. Muscle membrane repair and inflammatory attack in dysferlinopathy. Skelet Muscle. 2011; 1: 10. pmid:21798087
- 14. He B, Tang RH, Weisleder N, Xiao B, Yuan Z, Cai C, et al. Enhancing muscle membrane repair by gene delivery of MG53 ameliorates muscular dystrophy and heart failure in d-Sarcoglycan-deficient hamsters. Mol Ther. 2012; 20: 727–735. pmid:22314291
- 15. Swaggart KA, Demonbreun AR, Vo AH, Swanson KE, Kim EY, Cai C, et al. Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair. Proc Natl Acad Sci USA. 2014; 111: 6004–6009. pmid:24717843
- 16. Reddy A, Caler EV, Andrews NW. Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell. 2001; 106: 157–169. pmid:11511344
- 17. Gillies AR, Lieber RL. Structure and function of the skeletal muscle extracellular matrix. Muscle Nerve. 2011; 44: 318–331. pmid:21949456
- 18. Amaro A, Esposito AI, Gallina A, Nees M, Angelini G, Albini A, et al. Validation of proposed prostate cancer biomarkers with gene expression data: a long road to travel. Cancer Metastasis Rev. 2014; 33: 657–671. pmid:24477410
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- 20. Hui X, Zhu W, Wang Y, Lam KS, Zhang J, Wu D, et al. Major urinary protein-1 increases energy expenditure and improves glucose intolerance through enhancing mitochondrial function in skeletal muscle of diabetic mice. J Biol Chem. 2009; 284: 14050–14057. pmid:19336396
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- 24. Duvall CL, Taylor WR, Weiss D, Wojtowicz AM, Guldberg RE. Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of osteopontin-deficient mice. J Bone Miner Res. 2007; 22: 286–297. pmid:17087627
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- 26. Hashimoto M, Sun D, Rittling SR, Denhardt DT, Young W. Osteopontin-deficient mice exhibit less inflammation, greater tissue damage, and impaired locomotor recovery from spinal cord injury compared with wild-type controls. J Neurosci. 2007; 27: 3603–3611. pmid:17392476
- 27. Liaw L, Almeida M, Hart CE, Schwartz SM, Giachelli CM. Osteopontin promotes vascular cell adhesion and spreading and is chemotactic for smooth muscle cells in vitro. Circ Res. 1994; 74: 214–224. pmid:8293561
- 28. Mori R, Shaw TJ, Martin P. Molecular mechanisms linking wound inflammation and fibrosis: knockdown of osteopontin leads to rapid repair and reduced scarring. J Exp Med. 2008; 205: 43–51. pmid:18180311
- 29. O'Regan A, Berman JS. Osteopontin: a key cytokine in cell-mediated and granulomatous inflammation. Int J Exp Pathol. 2000; 81: 373–390. pmid:11298186
- 30. Sam F, Xie Z, Ooi H, Kerstetter DL, Colucci WS, Singh M, et al. Mice lacking osteopontin exhibit increased left ventricular dilation and reduced fibrosis after aldosterone infusion. Am J Hypertens. 2004; 17: 188–193. pmid:14751663
- 31. Haslett JN, Sanoudou D, Kho AT, Bennett RR, Greenberg SA, Kohane IS, et al. Gene expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal skeletal muscle. Proc Natl Acad Sci USA. 2002; 99: 15000–15005. pmid:12415109
- 32. Hoffman EP, Gordish-Dressman H, McLane VD, Devaney JM, Thompson PD, Visich P, et al. Alterations in osteopontin modify muscle size in females in both humans and mice. Med Sci Sports Exerc. 2013; 45: 1060–1068. pmid:23274598
- 33. Pagel CN, Wijesinghe DKW, Esfandouni NT, Mackie EJ. Osteopontin, inflammation and myogenesis: influencing regeneration, fibrosis and size of skeletal muscle. J Cell Commun Signal. 2014; 8(2): 95–103. pmid:24318932
- 34. Uaesoontrachoon K, Yoo HJ, Tudor EM, Pike RN, Mackie EJ, Pagel CN. Osteopontin and skeletal muscle myoblasts: association with muscle regeneration and regulation of myoblast function in vitro. Int J Biochem Cell Biol. 2008; 40: 2303–2314. pmid:18490187
- 35. Coley WD, Bogdanik L, Vila MC, Yu Q, Van Der Meulen JH, Rayavarapu S, et al. Effect of genetic background on the dystrophic phenotype in mdx mice. Hum Mol Genet. 2016; 25(1): 130–145. pmid:26566673
- 36. Fukada S, Morikawa D, Yamamoto Y, Yoshida T, Sumie N, Yamaguchi M, et al. Genetic background affects properties of satellite cells and mdx phenotypes. Am J Pathol. 2010; 176(5): 2414–2424. pmid:20304955
- 37. Fatherazi S, Matsa-Dunn D, Foster BL, Rutherford RB, Somerman MJ, Preland RB. Phosphate regulates osteopontin Gene transcription. J Dent Res. 2009; 88(1): 39–44. pmid:19131315
- 38. Subraman V, Thiyagarajan M, Malathi N, Rajan ST. OPN–Revisited. J Clin Diagn Res. 2015; 9(6): ZE10–ZE13. pmid:26266236
- 39. Agnihotri R, Crawford HC, Haro H, Matrisian LM, Havrda MC, Liaw L. Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin). J Biol Chem. 2001; 276: 28261–28267. pmid:11375993
- 40. Takafuji V, Forgues M, Unsworth E, Goldsmith P, Wang XW. An osteopontin fragment is essential for tumor cell invasion in hepatocellular carcinoma. Oncogene. 2007; 26(44): 6361–6371. pmid:17452979
- 41. Lund AK, Lucero J, Lucas S, Madden MC, McDonald JD, Seagrave JC, et al. Vehicular emissions induce vascular MMP-9 expression and activity associated with endothelin-1-mediated pathways. Arterioscler Thromb Vasc Biol. 2009; 29(4): 511–517. pmid:19150882
- 42. Vetrone SA, Montecino-Rodriguez E, Kudryashova E, Kramerova I, Hoffman EP, Liu SD, et al. Osteopontin promotes fibrosis in dystrophic mouse muscle by modulating immune cell subsets and intramuscular TGF-beta. J Clin Invest. 2009; 119: 1583–1594. pmid:19451692
- 43. Pegoraro E, Hoffman EP, Piva L, Gavassini BF, Cagnin S, Ermani M, et al. Cooperative International Neuromuscular Research. SPP1 genotype is a determinant of disease severity in Duchenne muscular dystrophy. Neurology. 2011; 76: 219–226.


text: Superhuman Durability Category page 1,596pages on this wiki Add New Page Edit History Talk0 Sorting type: Most visited Most visited Alphabetical | Pages in category "Superhuman Durability" Wolverine Caliban X-23 Mr. Sinister Magik Sabretooth Jubilee Beast Selene Wolfsbane Callisto Feral Marrow Magma Albert Warpath Back 1 2 Next Subcategories Demons Werewolves Vampires Retrieved from "http://x-men.wikia.com/wiki/Category:Superhuman_Durability?oldid=19567" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Powers Characters by Power

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Pakistan has said India has no evidence to back its assertions about Islamabad's involvement in the July Mumbai blasts and dismissed as "propaganda" claims of any proof against it.
A day after National Security Advisor MK Narayanan said India has "pretty good" evidence of Pakistan's involvement in the Mumbai blasts, Pakistan Foreign Office Spokesperson Tasnim Aslam said "It is a propagandist statement as they have no evidence whatsoever".
"If they have any evidence they should furnish it, then we will take action," she was quoted as saying by The Nation newspaper.


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Staff & Directors
Meet our Staff
Jaime Herrero, Executive Director
Jaime is a native Houstonian who is passionate about providing young people with opportunities for growth and creative self-expression. Prior to joining Workshop Houston as Executive Director, Jaime founded his own community-based arts organization in Brooklyn called Xmental. He also spent two years at Carnegie Hall as the chief architect of NeON Arts: the first grant-making program in the Hall's 125 year history. A graduate of the University of Texas School of Law, Jaime started his career as a staff attorney at the Bronx Defenders, an innovative, holistic, and client-centered legal office in the Bronx.
LaTasha Brown, Volunteer Coordinator
A Missouri City native, LaTasha attended Sam Houston State University where she received a B.S. in Psychology with a minor in Mass Communication and also received a M.A. in Psychology from Texas Southern University. Her interest in psychology comes from a desire to understand the actions of others and to promote the unity of family. LaTasha has previously worked for a non-profit organization that focused on improving literacy for elementary students, as well as other important life-skills. Actively involved in supporting youth since she was in high school, LaTasha has continued this journey in her role at Workshop Houston.
Bass Heavy, Beat Shop Coordinator and Director of Student Life
Bass Heavy hails from the musical marshes of Louisiana. Bass attended performing arts schools from the age of ten. He went on to The University of New Orleans where he double majored in jazz studies and engineering finishing with a B.S. in electrical engineering in 2000. Most of Bass Heavy's production gigs have come at the hands of some of New Orleans most successful Rap Artists including Former No Limit and Cash Money artists such as Mannie Fresh, Juvenile, B.G., Mia X, C-Murder, and Soulja Slim. However, Bass is no one trick pony. His productions offer a wide range of musical landscapes thanks to his broad musical background.
Bryant Livingston, Scholar Shop Coordinator
Bryant Livingston was raised in San Antonio, Texas. In high school, he often taught youth groups and volunteered at local charities. He received a BS in Social Studies Education from Baylor University in 2008. He continued his philanthropic efforts as he volunteered and tutored in many Waco-area middle and high schools. Upon graduation, Bryant discovered that the educational system was not doing enough to help America's future leaders. Here at Workshop Houston, Bryant is able to give essential one-on-one time with each student and provide them with tools to be successful both academically and socially.
Crystal Green, Style Shop Coordinator
An Illinois native, the self-proclaimed fashionista relocated to Houston in 2009 after graduating Columbia College-Chicago with a degree in Fashion Design (BFA.) Crystal's experience includes an impressive mix of fashion and education via retail sales, professional wardrobe styling, photography and serving as a modeling and acting instructor. Crystal uses her passions to expose students to different possibilities through art and style. At Workshop Houston, Crystal helps our students see all that the world of fashion potentially has to offer.
Meet our Board
John Blackmon, President
A North Carolina native, John holds a BA in Architecture from the School of Design at North Carolina State University and a Masters Degree from Columbia University. After spending 10 years in New York, John relocated to Houston in 1997, opening his own practice in 2001. In addition to sitting on Workshop Houston's board, John also sits on the Modern & Contemporary Arts Subcomittee at Houston's Museum of Fine Arts.
Andrew Davis
Andrew Davis is Dean of the College of the Arts at the University of Houston. His work focuses on integrating interdisciplinary training opportunities into the curriculum and on establishing the arts as a leading force for social engagement and community impact. He serves on the Boards of Directors of the Houston Symphony Orchestra, the Immanuel and Helen B. Olshan Foundation, Music Doing Good, the PhiBeta Kappa Alumni Association of Greater Houston, and the Texas Society for Music Theory. A music theorist by training, he is the author of two books and numerous articles on the opera and instrumental music of the 19th century.
Jennifer Ford
A Houston native, Jennifer has been actively involved in volunteering with inner city children since her undergraduate years Emory University in Atlanta. After moving back to Houston in 2004 she opened Premium Goods, the first sneaker boutique in Texas known for in the shoe industry as a southern tastemaker. An extreme lover of the arts, fashion and music, Jennifer loves using her passions to assist Workshp Houston.
Helen Fosdick
Helen has been involved in various art organizations, beginning with John and Dominique de Menil at the University of St. Thomas, Rice and the Rothko Chapel. A founding director at the Dia Art Foundation, Helen has worked on various large-scale permanent projects with a number of artists. Through lending support to numerous non-profit projects, Helen learned about Workshop Houston early on and continues to admire and support their mission.
Elizabeth Herndon
A native Houstonian, Elizabeth received a B.A. from the University of Texas and an MBA from Duke University. She became interested in Workshop Houston after being introduced to the organization through The Children's Fund, where she and her husband Rogers have served on the board for over five years; including last year as co-presidents. A former Zoo Friends member, she has also served on the founding board for Heartgift Houston and enjoys volunteering at the Houston Foodbank and her children's school. She loves raising her three daughters, Lola, Charlotte, and Frances.
Patti Melcher
A graduate of the University of Virginia and Harvard Business School, Patti is the CEO of EIV Capital Management Co., LLC which manages a private equity fund focused on investments in the energy industry. She has been actively engaged in the non-profit community in Houston with a particular interest in organizations serving children and in education. In 1997, Patti was instrumental in co-founding the Joy School, which serves the needs of children with learning differences.
Frank Montgomery, Treasurer
Frank grew up in Houston and is a graduate of Wabash College in Indiana. He's currently a managing partner of a closely held real estate investment firm, which owns and operates income producing real estate throughout Houston's Museum District. Frank has an interest in early education and has been involved with several local non-profit organizations.
Holden Rushing
A native Houstonian, Holden is a graduate of Pepperdine University and Rhodes University. He's been actively involved with multiple non-profit projects and organizations for many years including finishing the 2,170 mile Appalachian Trail for St. Jude's Hospital in Memphis, TN; volunteering as an "On-Call Musician" for Musicians On Call in NYC and serving as a board member for MD Anderson Cancer Center. During the day Holden works as a commercial real estate for NAI Houston, and in the evenings plays in several bands around town. He enjoys travel, cycling, surfing and music, and believes that the education and encouragement provided by Workshop Houston is the key to building a better community.
Andrew "Andy" Stepanian, Secretary
Andy is the Senior Vice President, Chief Administrative Officer and General Counsel of Silver Eagle Distributors, L.P., one of the largest beer wholesalers in the nation. He is also the co-founder of lifestyle apparel company, the Howler Brothers, and a member of the band Wrinkle Neck Mules. In addition to Workshop Houston, Andy serves on the Board of Directors of Houston wilderness and is also a member of the Jackson Hole Land Trust Open Space Council. He lives in Houston with his wife, Elizabeth, and their three children, Katharine, Reese and Driggs.
Jill Whitten
A paintings conservator at Whitten & Proctor Fine Art Conservation, Jill studied Studio Art at UT and received a Masters in Conversation from Buffalo State College in New York. After moving to Houston in 1998, she worked with Legacy Land Trust and the Houston Parks Board to found Freed Art and Nature Park in the Woodland Heights. For the past two years Jill has served on the board of the foundation that oversees Peckerwood Garden. She is enthusiastic about the creative youth-centered mission of Workshop Houston.


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Raje, who returned home after completing two-day Madhya Pradesh tour, reached Kota on Tuesday, Aug 5. Raje while coming out of her private helicopter noticed the cop and complained to his senior officials.
Complaining to senior police officials Govind Gupta, Raje said, "Had there been a Central leader or a VIP in my place, what type of impression of the state police he or she would have carried back?"
Policemen were served showcause notice and charge-sheeted as Raje was upset
Citing the wrath of the CM, Gupta issued a show-cause notice to his subordinates and charge-sheeted the officer who had picked up the team of the cops to present the guard of honour.
According to sources, the Rajasthan CM was returning to Jaipur. However, her helicopter was running behind of the schedule due to heavy rains. Later, Raje came to Kota for a brief stopover to refuel her helicopter on way to Jaipur, added sources.
Despite Raje's complaint, senior police officials claimed that 10-member team, which was selected for CM's guard-of-honour, was fit, except one person.
Media reports quoted the officials as saying that it was mandatory for policemen to attend PT exercises and parades held on Mondays and Fridays. For office staff, however, to attend at least 25 per cent of these exercises were mandatory. Moreover, all the officers volunteered themselves for jogging on Saturdays.
OneIndia News


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If you follow me, you've probably noticed the lack of posts on all my social platforms. As written in my previous blog post, I was afraid my blogging would be slowed down by an immense amount of work due to college. However, I paid a visit to my doctor's for some pain and was told that I now have a virus, preventing me from attending school. This has also prevented me from going out, or for staying awake for long hours. It's like I'm tired all the time, and I can't even do anything about it. I can't shoot, I can't draw, I can't eat, I can't sleep, I can't stay awake. It's ridiculous, and I feel so lackadaisical that It's driving me crazy. Bear with me.
Assymetric dress by Style Moi
Plaid collar top by Deandri
Grail Platforms by Unif
Earrings by Mikukumi


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Get a shipping estimateZip Code:
- Description
- View Full Specifications
- Year:
- 2003
- Length:
- 15'
- Engine/Fuel Type:
- Single / Gas/Petrol
- Located In:
- Forked River, NJ
- Hull Material:
- Fiberglass
- 78826-3011314
- Last Listed Price:
- Sold
Come see this well cared for low hour 150 Boston Whaler sport powered by a mercury 60 HP.
Call or Text Jason @ 732-684-0710
Please contact Jason Cavaleri at (732) 684-0710
Additional Specs, Equipment and Information:
Warranty: 10 years
Hull Shape: Deep Vee
Beam: 6 ft 6 in
Maximum Draft: 0 ft 7 in
Bridge Clearance: 3 ft 2 in
Dry Weight: 900 lbs
Max Load Capacity: 2900 lbs
Total Power: 60 HP
Engine 1:
Engine Brand: Mercury
Engine Model: 60
Engine Type: Outboard 2S
Engine/Fuel Type: Gas/Petrol
Propeller: 3 blade propeller
Engine Power: 60 HP
Fuel Tanks: (6 Gallons)
Seating Capacity: 6
Manufacturer Provided Description
Introducing a brand-new Classic Whaler - the 150 Sport. This model is as easy to use and as fuss-free as the Boston Whaler 130 Sport, but bigger and with extra features for every kind of fun on the water. Exclusive Unibond construction makes the 150 Sport unsinkable. And its wider beam, combined with a twin sponson hull, gives it extra stability and quick planing. And it has plenty of power to spare with a standard 60-horsepower Mercury outboard - more than enough to get an average adult up on waterskis fast. Passengers will appreciate its strategically placed drink holders and built-in rod holders. For added convenience, aft port and starboard boarding steps simplify getting in and out of the boat. The roomy, open cockpit layout features a side console, helm bench seat with backrest and storage, and a removable forward bench seat. Even a 15-foot Whaler features yacht-quality finish and components like stainless steel side rails, a console grab rail, a finished bow locker with a fiberglass cover and overboard drain, bow and stern lifting eyes, navigation lights and plenty more. All standard, including a galvanized EZ Loader trailer.
The Company offers the details of this vessel in good faith but cannot guarantee or warrant the accuracy of this information nor warrant the condition of the vessel. A buyer should instruct his agents, or his surveyors, to investigate such details as the buyer desires validated. This vessel is offered subject to prior sale, price change, or withdrawal without notice.
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by beredim in Strange Animals
Bees latch on to similarly-sized nectarless flowers to unpick pollen - like keys fitting into locks, according to a new study by researchers at the University of Stirling.
The research shows the right size of bee is needed to properly pollinate a flower. The bee fits tightly with the flower's anthers, to vibrate and release the pollen sealed within.
"We found that a pollinator's size, ... Read more »
Solís-Montero, L., & Vallejo-Marín, M. (2017) Does the morphological fit between flowers and pollinators affect pollen deposition? An experimental test in a buzz-pollinated species with anther dimorphism. Ecology and Evolution. DOI: 10.1002/ece3.2897
by Jente Ottenburghs in Evolutionary Stories
What to do when your prey refuses to be swallowed? Eurasian Spoonbills (Platalea leucordia) in Hungary have a solution…... Read more »
Pigniczki, C. (2017) Unique Prey Handling of Eurasian Spoonbill ( ) . Waterbirds, 40(1), 74-76. DOI: 10.1675/063.040.0111
by beredim in Strange Animals
Meet the Moroccan flic-flac spider (Cebrennus rechenbergi), a truly unique spider that when provoked or threatened escapes by doubling its normal walking speed using forward or backward flips similar to acrobatic flic-flac movements used by gymnasts.
C. rechenbergi is a species of huntsman spider indigenous to Morocco and can be found in the sand dunes of the Erg Chebbi desert . The spider ... Read more »
Ralf Simon King. (2013) BiLBIQ: A Biologically Inspired Robot with Walking and Rolling Locomotion. Biosystems . info:/10.1007/978-3-642-34682-8
by Piter Boll in Earthling Nature
by Piter Kehoma Boll Charles Darwin had already noticed that small animals, such as those found in zooplankton, are widely distributed around the world, even those that are found in small ponds of freshwater. This seemed to go against the … Continue reading →... Read more »
Gómez, A., Serra, M., Carvalho, G., & Lunt, D. (2002) Speciation in ancient cryptic species complexes: evidence from the molecular phylogeny of Brachionus plicatilis(Rotifera). Evolution, 56(7), 1431. DOI: 10.1554/0014-3820(2002)056[1431:SIACSC]2.0.CO;2
Øie, G., Makridis, P., Reitan, K., & Olsen, Y. (1997) Protein and carbon utilization of rotifers (Brachionus plicatilis) in first feeding of turbot larvae (Scophthalmus maximus L.). Aquaculture, 153(1-2), 103-122. DOI: 10.1016/S0044-8486(96)01514-1
Suatoni, E., Vicario, S., Rice, S., Snell, T., & Caccone, A. (2006) An analysis of species boundaries and biogeographic patterns in a cryptic species complex: The rotifer—Brachionus plicatilis. Molecular Phylogenetics and Evolution, 41(1), 86-98. DOI: 10.1016/j.ympev.2006.04.025
Walker, K. (1981) 13. A synopsis of ecological information on the saline lake rotifer Brachionus plicatilis Müller 1786. Hydrobiologia, 81-82(1), 159-167. DOI: 10.1007/BF00048713
by beredim in Strange Animals
In a recent study, researchers provide a new theory for the reason we walk the Earth
A new provocative study suggests it was the power of the eyes and not the limbs that first led our ancient aquatic ancestors to make the momentous leap from water to land. According to it, crocodile-like animals first saw easy meals on land and consequently evolved limbs that enabled them to get there, ... Read more »
MacIver MA, Schmitz L, Mugan U, Murphey TD, & Mobley CD. (2017) Massive increase in visual range preceded the origin of terrestrial vertebrates. Proceedings of the National Academy of Sciences of the United States of America. PMID: 28270619
by CAPB in Companion Animal Psychology Blog
Do online pet obituaries reveal how we truly feel about our pets?Guest post by Jane Gething-Lewis (Hartpury College)."You were such a selfless and giving boy. Dad loves you with all his heart."A heartfelt online tribute to a dearly departed loved one – but this loved one had four legs, a tail and was called Cosmo. Over the top? Not necessarily. Research suggests that many people feel the loss of a beloved pet as keenly as the loss of a child.The bond people have with each other has long been debated and discussed. Generations of psychologists have attempted to explain and quantify the mechanics of attachment (or lack of) between fellow humans. But is it possible that we form similar bonds with our animal companions? Recently, researchers have been interested in exploring whether human theories of bonding apply to our relationships with our pets. No easy task, when only half of the bonding equation can talk.Now researchers at the University of Edinburgh believe they have identified a useful source of information about the nature of the human-animal bond – online pet obituaries."The digital environment is a really interesting place for people to talk about animals," said Dr Jill MacKay, who led a study of tributes posted on pet memorial websites. "Society has an expectation that animals are not as important as humans, but the online space has no rules. I suspect that people write more freely and honestly online about what they're really feeling." Dr MacKay and her team looked at obituaries for dogs on two dedicated websites, investigating the kind of information available in these tributes. They also assessed whether that data might prove useful in future studies into the nature of the human-animal bond when it comes to attachment, grief and bereavement.Pulling a sickie? Or sick with grief...Taking time off work following the death of a pet is generally frowned upon, and yet losing a pet can seriously disrupt our lives. Some people lose sleep, lose their appetite and are unable to carry on with normal life for some time; the higher the level of bonding with their pet, the deeper the mourning. "Our study shows that people feel real grief when they lose their animals," said Dr MacKay. Why do we feel so close to our pets?Here's the science bit. The Attachment Theory of human bonding is a widely used psychological model, coined by psychologist John Bowlby in the 1950's. It works on the principal that a strong emotional attachment to a primary caregiver (usually a parent) is essential for making us feel safe, secure and grounded. Put simply, we will form a deep bond with another person if one or more of four key elements are present in the relationship. Do you want to be near to someone you feel close to? That's Proximity Maintenance. Do you seek out that person when you need comfort or are a bit scared? They are a Safe Haven. Do like to know that person is nearby when you're chatting to others at a party? They are your Secure Base. Do you panic a little when you think that person has left the party without you? That's called Separation Anxiety. Congratulations – you are attached. The degree and the style to which we bond with other people will differ from person to person, and will affect the way we cope with life's ups and downs – including the way we feel grief.In her 2011 paper, Not Just a Dog, Dr Marilyn Kwong, a psychologist at the Simon Fraser University in Canada, looked at whether human attachment theory may explain why some people are so deeply affected by pet bereavement. The study built on existing research, which has established that intense bonds with our pets do exist; pets are often considered to be family members, and can fulfill emotional needs like love and companionship.Dr Kwong looked at grief experienced by 25 people with disabilities who had lost their Assistance Animal. Assistance animals included Guide Dogs and dogs specially trained to help with physical tasks. The team analysed the transcripts of open-ended phone interviews. They looked for – and found - evidence of three of the four attachment theory components: safe haven, secure base and separation anxiety. All participants reported feeling intense grief, even when the bereavement may have occurred many years previously. The team concluded that grief following the loss of a well-loved pet can be as strong as losing a human loved-one. "She was always there for me," said one participant. Another even took her dog into the operating theatre because she couldn't bear to be separated from him. Of course, all the dogs in Dr Kwong's study were not purely pets; they had a working function as caregiver. Therefore separation from them would likely generate additional anxiety above and beyond 'normal' levels.Deciphering the deadPhone interviews and questionnaires can provide a robust set of results for formal studies, but they depend on the researchers' personal interaction with the mourner, who may not feel able to be frank about their true feelings. Could online memorials offer a more unadulterated source of information?Dr Jill Mackay identified two websites, HeavenlyPaws.com and ImmortalPets.com. Focusing on dogs, she selected 130 obituaries of up to 400 words in length. The researchers identified and agreed a number of key concepts present in the texts relating to attachment theory and relationship with the pet. They grouped these concepts under three categories: owner-pet relationship, pet's actions, and owner's feelings.Where it was possible to identify the gender of the writer, Dr MacKay found that the majority of female memorial authors (34.6%) considered themselves to be 'mum' to their dearly departed dog. Tributes were also found from male parental figures, 'dad' (7.7%), and from children (5.4%).Nearly half (49%) of the tribute writers referred to their dog as part of the family – "my baby", "mummy's darling". The researchers identified this category as a potential predictor for the depth of grief experienced by the human owner. A little over half (51%) discussed the afterlife in relation to their pets, who were "in heaven", or "over the rainbow bridge". There was a strong association between the two concepts of afterlife and being part of the family, supporting attachment theory understanding that the loss of a pet can be akin for some to the loss of a child. If you're feeling a little depressed by now – there was some light at the end of the (white, brightly lit) tunnel. 11% of the dog obituaries celebrated happy times, writing of their pet's sense of humour and gratitude, 'laughing' at its own farts and 'grateful' for a play in the park.In contrast to Dr Kwong's study of bereaved Assistant Dog owners, Dr McKay's investigation does seem to have disinterred a rich source of natural information about the human-animal bond: genuine expressions of love and loss, expressed on a public forum, garnered from real-world texts and not under experimental conditions. However, scientists might see this as a problem. Little is known about the demographics of the tribute writer, making it difficult to breakdown the information as a potential predictor for people more likely to feel the deepest and most debilitating grief. Neither does this plundering-of-the-online-grave study relate directly to specific elements of the formal attachment theory model.RIP onlineThe internet has become the go-to place for information, support and emotional expression around grief and bereavement. Mainstream animal charities like Blue Cross, veterinary practices, rescue centres, and even universities like Glasgow offer bereavement advice, steering the mourner to create an online memorial to their pet.While Dr MacKay's study focuses solely on dedicated pet obituary websites, she plans to broaden her investigations into the world of social media: Facebook, Twitter and Instagram teem with emotion."We looked at dedicated pet obituary websites rather than social media because of ethical issues," explained Dr MacKay. "Pet obit sites are public, but facebook is personal. There are layers of privacy settings. However," she ad... Read more »
Kwong, M., & Bartholomew, K. (2011) "Not just a dog": an attachment perspective on relationships with assistance dogs. Attachment , 13(5), 421-436. DOI: 10.1080/14616734.2011.584410
MacKay, J., Moore, J., & Huntingford, F. (2016) Characterizing the Data in Online Companion-dog Obituaries to Assess Their Usefulness as a Source of Information about Human–Animal Bonds. Anthrozoös, 29(3), 431-440. DOI: 10.1080/08927936.2016.1181374
by beredim in Strange Animals
The group feautred in this videp was formed by three adult or sub-adult whales. Social behavior of the True's Beaked Whale is still unknown but the group seemed to dive in a coordinated manner, as has been observed in other species of beaked whales. Credit: Roland Edler
True's beaked whales (Mesoplodon mirus) are such an elusive species that it's only now that we finally have the ... Read more »
Aguilar de Soto, N., Martín, V., Silva, M., Edler, R., Reyes, C., Carrillo, M., Schiavi, A., Morales, T., García-Ovide, B., Sanchez-Mora, A.... (2017) True's beaked whale (Mesoplodon mirus) in Macaronesia. PeerJ. DOI: 10.7717/peerj.3059
by beredim in Strange Animals
New study by researchers at Queen Mary University of London (QMUL) shows how bumblebees (Bombus terrestris) can be trained to score goals with a mini-ball, revealing unprecedented learning abilities:
Researchers train bumblebees to move a ball in order to access a sugar solution as a reward.
The study, published in the journal Science, suggests that species whose lifestyle demands advanced ... Read more »
Loukola, O., Perry, C., Coscos, L., & Chittka, L. (2017) Bumblebees show cognitive flexibility by improving on an observed complex behavior. Science, 355(6327), 833-836. DOI: 10.1126/science.aag2360
by beredim in Strange Animals
Kristin Walovich holds the newly described species of ghost shark
Photo Credit: Kristin Walovich
Researchers recently announced the discovery of a new species of ghost shark, Hydrolagus erithacus. Ghost sharks - which aren't actually sharks but instead their closest living relatives - are an extraordinarily rare sighting. Actually, it was just a few months ago, when a ghost shark was filmed... Read more »
Walovich KA, Ebert DA, & Kemper JM. (2017) Hydrolagus erithacus sp. nov. (Chimaeriformes: Chimaeridae), a new species of chimaerid from the southeastern Atlantic and southwestern Indian oceans. Zootaxa, 4226(4). PMID: 28187604
by Jente Ottenburghs in Evolutionary Stories
Scientists have sequenced the DNA of two extinct birds: the moa and the elephantbird. Comparison with their living relatives led to some surprising findings.... Read more »
Maderspacher F. (2017) Evolution: Flight of the Ratites. Current biology : CB, 27(3). PMID: 28171755
Yonezawa T, Segawa T, Mori H, Campos PF, Hongoh Y, Endo H, Akiyoshi A, Kohno N, Nishida S, Wu J.... (2017) Phylogenomics and Morphology of Extinct Paleognaths Reveal the Origin and Evolution of the Ratites. Current biology : CB, 27(1), 68-77. PMID: 27989673
by beredim in Strange Animals
Deep sea creatures come with all kinds of strange features that help them to survive their cold, dark habitat.. Some have eyes the size of a basketball, others come with appendages that blink and glow, deep-sea dwellers have developed some strange features and the "cockeyed" squid Histioteuthis heteropsis has one normal eye and one giant, bulging, yellow eye.
Histioteuthis heteropsis
One ... Read more »
Thomas KN, Robison BH, & Johnsen S. (2017) Two eyes for two purposes: in situ evidence for asymmetric vision in the cockeyed squids Histioteuthis heteropsis and Stigmatoteuthis dofleini. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 372(1717). PMID: 28193814
by Piter Boll in Earthling Nature
by Piter Kehoma Boll If you have your face buried in the mud at the bottom of a European lake, you may end up finding some of those tiny little roundworms known as Monhystera stagnalis. As usual, there is no common … Continue reading →... Read more »
Pehofer, H. (1989) Spatial Distribution of the Nematode Fauna and Production of Three Nematodes (Tobrilus gracilis, Monhystera stagnalis, Ethmolaimus pratensis) in the Profundal of Piburger See (Austria, 913 m a.s.l). Internationale Revue der gesamten Hydrobiologie und Hydrographie, 74(2), 135-168. DOI: 10.1002/iroh.19890740203
Traunspurger, W. (1996) Autecology of Monhystera paludicola De Man, 1880 – Seasonal, Bathymetric and Vertical Distribution of a Free-living Nematode in an Oligotrophic Lake. Internationale Revue der gesamten Hydrobiologie und Hydrographie, 81(2), 199-211. DOI: 10.1002/iroh.19960810205
by beredim in Strange Animals
Flabegraviera fujiae (left), the new species described in the
in the new study, and Flabegraviera mundata (right).
(Scale bar: 1cm)
A few days ago, a team of Japanese scientists from the Hokkaido University announced the discovery a new species of polychaete, a type of marine annelid worm.
The discovery took place 9-meters deep underwater near Japan's Syowa Station in Antarctica and provides... Read more »
Jimi N, Tsujimoto M, Watanabe K, Kakui K, & Kajihara H. (2017) A new species and the shallowest record of Flabegraviera Salazar-Vallejo, 2012 (Annelida: Flabelligeridae) from Antarctica. Zootaxa, 4221(4). PMID: 28187651
by beredim in Strange Animals
Kingdom: Animalia
Phylum: Mollusca
Class: Cephalopoda
Order: Octopoda
Family: Octopodidae
Genus: Amphioctopus
Species: Amphioctopus marginatus
Conservation Status: Not yet assessed
Common Name: Coconut octopus, Veined octopus
Meet Amphioctopus marginatus a medium-sized octopus found in the tropical waters of the western Pacific Ocean The species is best known as the "coconut octopus" ... Read more »
Finn JK, Tregenza T, Norman MD. (2009) Defensive tool use in a coconut-carrying octopus. . Curr. Biol, 19(23). info:/10.1016/j.cub.2009.10.052
by Jason Organ in Eatlemania!
The Eatles are munching on several juvenile American alligator skulls... Read more »
George, I., & Holliday, C. (2013) Trigeminal Nerve Morphology in and Its Significance for Crocodyliform Facial Sensation and Evolution . The Anatomical Record, 296(4), 670-680. DOI: 10.1002/ar.22666
Holliday, C., & Witmer, L. (2007) Archosaur adductor chamber evolution: Integration of musculoskeletal and topological criteria in jaw muscle homology. Journal of Morphology, 268(6), 457-484. DOI: 10.1002/jmor.10524
by Piter Boll in Earthling Nature
by Piter Kehoma Boll If you think spiders are scary creatures, today you will learn that they are scared too. But what could scary a spider? Well, a web bug! We usually think of spider webs as an astonishing evolutionary … Continue reading →... Read more »
PAPE, R. (2013) Description and Ecology of A New Cavernicolous, Arachnophilous Thread-legged Bug (Hemiptera: Reduviidae: Emesini) from Kartchner Caverns, Cochise County, Arizona. Zootaxa, 3670(2), 137. DOI: 10.11646/zootaxa.3670.2.2
Resende, L., Zepon, T., Bichuette, M., Pape, R., & Gil-Santana, H. (2016) Associations between Emesinae heteropterans and spiders in limestone caves of Minas Gerais, southeastern Brazil. Neotropical Biology and Conservation, 11(3). DOI: 10.4013/nbc.2016.113.01
Wignall, A., & Taylor, P. (2010) Predatory behaviour of an araneophagic assassin bug. Journal of Ethology, 28(3), 437-445. DOI: 10.1007/s10164-009-0202-8
by Piter Boll in Earthling Nature
by Piter Kehoma Boll A fascinating group of animals that has not yet joined the Friday Fellows are the sponges. Different from all other animals, sponges have a unique body structure that behaves more like a plant or fungus. They … Continue reading →... Read more »
Hendler, G. (1984) The Association of Ophiothrix lineata and Callyspongia vaginalis: A Brittlestar-Sponge Cleaning Symbiosis?. Marine Ecology, 5(1), 9-27. DOI: 10.1111/j.1439-0485.1984.tb00304.x
Hoppe, W. (1988) Growth, regeneration and predation in three species of large coral reef sponges. Marine Ecology Progress Series, 117-125. DOI: 10.3354/meps050117
by Christian de Guttry in genome ecology evolution etc
The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons About 450 mya bony vertebrates radiated into Lobe-finned fish, from which tetrapods appeared later, and Ray-finned fish, which include Teleost (Fig.1). Nowadays they make up to 96 percent of … Continue reading →... Read more »
Braasch I, Gehrke AR, Smith JJ, Kawasaki K, Manousaki T, Pasquier J, Amores A, Desvignes T, Batzel P, Catchen J.... (2016) The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons. Nature genetics, 48(4), 427-37. PMID: 26950095
by beredim in Strange Animals
Researchers recently announced the discovery of a frog whose groin flashes orange to scare away predators! The species was discovered in Australia.
When biologist Simon Clulow spotted a frog with an unusual marble pattern on its belly, he knew it could be a new species. If that turned to be true, it would be very surprising as the sighting took place on land close to an airport and not some ... Read more »
CLULOW, S., ANSTIS, M., KEOGH, J., & CATULLO, R. (2016) A new species of Australian frog (Myobatrachidae: Uperoleia) from the New South Wales mid-north coast sandplains. Zootaxa, 4184(2), 285. DOI: 10.11646/zootaxa.4184.2.3
by Piter Boll in Earthling Nature
by Piter Kehoma Boll A flatworm again, at last! Not a land planarian, but a flatworm nonetheless. If there is a group of flatworms that may put land planarians in second plan regarding beauty, those are the polyclads. Living in … Continue reading →... Read more »
Whitfield, J. (2004) Everything You Always Wanted to Know about Sexes. PLoS Biology, 2(6). DOI: 10.1371/journal.pbio.0020183
Do you write about peer-reviewed research in your blog? Use ResearchBlogging.org to make it easy for your readers — and others from around the world — to find your serious posts about academic research.
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3 Days in Aix-en-Provence
Shopping Guide to Aix-en-Provence
Museum Guide to Aix-en-Provence
Excellent smothies ! Great choice for healthy breakfast. Friendly staff,nice place. Totally recommended
A nice lunch with the family on a rainy Saturday
A nice little restaurant with two 'salles' the service was nice and friendly, to be honest like many places in Aix we didn't have any starters (as 90mins earlier I'd had a light brunch) so I chose the LTV burger and naughty me with chips, meat was great, cooked as...
This is a popular restaurant for locals within a short walking distance of La Rotonde. The decor is simple, but stylish. The minute you walk in you are greeted by all the staff. Very professional and friendly. The evening menu isn't extensive but the variety is good. Given we were there on a cold night, the daily soup (minestrone) was...
Pleasant restaurant, with good efficient service.
We opted for the set menus (as it works out far more cost efficient than going a la carte). There were 2 options available - a 25 euro and a 35 euro three course selection.
The signature dish of Green Tomato Tatin is very tasty. Salmon main course with root veg was a little...
This great restaurant was recommended to us by our hotel. It is in an area with many restaurants to choose from, and at first glance it seems unassuming. However, you only need to look at the menu to see you're in for a treat. We ended up taking the tasting menu, but the à la carte choices were varied and...
We stumbled upon this resturant while wandering in Aix and it turned out to be amazing. Outstanding food, ambience and staff. Food is all local and menu changes to reflect season. Had the beef and the cod and both were delicious. Recommend one of the chef's menus. The entree with a tomatoe and basil sorbet was beautiful anlong with a...
Intimate, lovely vocal jazz this nite, service very good, pleasant plate arrangements and tasted just as good. I had a green tomato tartin, didn't really care for the spice in the sorbet (plate cleanser). My beef was perfect, mushroom and spinach, sautéed mold of potato. All appealed to me this evening as I wanted a steak. Dessert, a fig Tarte...
We ate here on two occasions. Our French is poor but the staff were well versed with helping the tourists 👍🏽. The food was top notch . Fresh and well presented. Would recommend in a heart beat to anyone
Such a cracking restaurant!!!! Wonderful food, great staff and fantastic owners!! I've had the pleasure of being down in Aix a few times over the summer with work and have eaten (and drunk!) here a few times. Marilyn and Sebastian really went the extra mile to look after myself and colleagues. Really looking forward to visiting again soon!! Cheers guys,...
Stumbled on this gem while wondering around. We stopped in for lunch and was very impressed at the fresh ingredients, friendly staff and great french regional cuisine. Would return to this place next time in Aix.
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this sub pounds. deep long holding bass. i had 2400watt amp hooked up iv entered soundoffs and placed 3r place. iv put all the power i could to this sub and it seems to just get louder with minimal distortion. what i didnt like about this woofer is i haveto invert mine beacuse the glue around the lip disconected on all 3 as12sqatlases iv owned.
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