Genetic Analysis of the Role of Proteolysis in the Activation of Latent Myostatin

Myostatin is a secreted protein that normally acts to limit skeletal muscle growth. As a result, there is considerable interest in developing agents capable of blocking myostatin activity, as such agents could have widespread applications for the treatment of muscle degenerative and wasting conditions. Myostatin normally exists in an inactive state in which the mature C-terminal portion of the molecule is bound non-covalently to its N-terminal propeptide. We previously showed that this latent complex can be activated in vitro by cleavage of the propeptide with members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteases. Here, I show that mice engineered to carry a germline point mutation rendering the propeptide protease-resistant exhibit increases in muscle mass approaching those seen in mice completely lacking myostatin. Mice homozygous for the point mutation have increased muscling even though their circulating levels of myostatin protein are dramatically increased, consistent with an inability of myostatin to be activated from its latent state. Furthermore, I show that a loss-of-function mutation in Tll2, which encodes one member of this protease family, has a small, but significant, effect on muscle mass, implying that its function is likely redundant with those of other family members. These findings provide genetic support for the hypothesis that proteolytic cleavage of the propeptide by BMP-1/TLD proteases plays a critical role in the activation of latent myostatin in vivo and suggest that targeting the activities of these proteases may be an effective therapeutic strategy for enhancing muscle growth in clinical settings of muscle loss and degeneration

Identification of genes differentially expressed in dorsal and ventral chick midbrain during early Development

Background: During the development of the central nervous system (CNS), patterning processes along the dorsoventral ( DV) axis of the neural tube generate different neuronal subtypes. As development progresses these neurons are arranged into functional units with varying cytoarchitecture, such as laminae or nuclei for efficient relaying of information. Early in development ventral and dorsal regions are similar in size and structure. Different proliferation rates and cell migration patterns are likely to result in the formation of laminae or nuclei, eventually. However, the underlying molecular mechanisms that establish these different structural arrangements are not well understood. We undertook a differential display polymerase chain reaction (DD-PCR) screen to identify genes with distinct expression patterns between dorsal and ventral regions of the chick midbrain in order to identify genes which regulate the sculpturing of such divergent neuronal organisation. We focused on the DV axis of the early chick midbrain since mesencephalic alar plate and basal plate develop into laminae and nuclei, respectively. Results: We identified 53 differentially expressed bands in our initial screen. Twenty-six of these could be assigned to specific genes and we could unambiguously show the differential expression of five of the isolated cDNAs in vivo by in situ mRNA expression analysis. Additionally, we verified differential levels of expression of a selected number of genes by using reverse transcriptase (RT) PCR method with gene-specific primers. One of these genes, QR1, has been previously cloned and we present here a detailed study of its early developmental time course and pattern of expression providing some insights into its possible function. Our phylogenetic analysis of QR1 shows that it is the chick orthologue of Sparc-like 1/Hevin/Mast9 gene in mice, rats, dogs and humans, a protein involved in cell adhesion. Conclusion: This study reveals some possible networks, which might be involved in directing the difference in neuronal specification and cytoarchitecture observed in the brain

Evolution of pigment synthesis pathways by gene and genome duplication in fish

<p>Abstract</p> <p>Background</p> <p>Coloration and color patterning belong to the most diverse phenotypic traits in animals. Particularly, teleost fishes possess more pigment cell types than any other group of vertebrates. As the result of an ancient fish-specific genome duplication (FSGD), teleost genomes might contain more copies of genes involved in pigment cell development than tetrapods. No systematic genomic inventory allowing to test this hypothesis has been drawn up so far for pigmentation genes in fish, and almost nothing is known about the evolution of these genes in different fish lineages.</p> <p>Results</p> <p>Using a comparative genomic approach including phylogenetic reconstructions and synteny analyses, we have studied two major pigment synthesis pathways in teleost fish, the melanin and the pteridine pathways, with respect to different types of gene duplication. Genes encoding three of the four enzymes involved in the synthesis of melanin from tyrosine have been retained as duplicates after the FSGD. In the pteridine pathway, two cases of duplicated genes originating from the FSGD as well as several lineage-specific gene duplications were observed. In both pathways, genes encoding the rate-limiting enzymes, tyrosinase and GTP-cyclohydrolase I (GchI), have additional paralogs in teleosts compared to tetrapods, which have been generated by different modes of duplication. We have also observed a previously unrecognized diversity of <it>gchI </it>genes in vertebrates. In addition, we have found evidence for divergent resolution of duplicated pigmentation genes, <it>i.e</it>., differential gene loss in divergent teleost lineages, particularly in the tyrosinase gene family.</p> <p>Conclusion</p> <p>Mainly due to the FSGD, teleost fishes apparently have a greater repertoire of pigment synthesis genes than any other vertebrate group. Our results support an important role of the FSGD and other types of duplication in the evolution of pigmentation in fish.</p

What Causes Partial F1 Hybrid Viability? Incomplete Penetrance versus Genetic Variation

Hernán López-Fernández is with Texas A&M University, Daniel I. Bolnick is with UT Austin.Background -- Interspecific hybrid crosses often produce offspring with reduced but non-zero survivorship. In this paper we ask why such partial inviability occurs. This partial inviability could arise from incomplete penetrance of lethal Dobzhansky-Muller incompatibilities (DMIs) shared by all members of a hybrid cross. Alternatively, siblings may differ with respect to the presence or number of DMIs, leading to genotype-dependent variation in viability and hence non-Mendelian segregation of parental alleles in surviving F1 hybrids. Methodology/Principal Findings -- We used amplified fragment length polymorphisms (AFLPs) to test for segregation distortion in one hybrid cross between green and longear sunfish (Lepomis cyanellus and L. megalotis). Hybrids showed partial viability, and twice as much segregation distortion (36.8%) of AFLPs as an intraspecific control cross (18.8%). Incomplete penetrance of DMIs, which should cause genotype-independent mortality, is insufficient to explain the observed segregation distortion. Conclusions/Significance -- We conclude that F1 hybrid sunfish are polymorphic for DMIs, either due to sex-linked DMI loci (causing Haldane's Rule), or polymorphic autosomal DMI loci. Because few AFLP markers were sex-linked (2%), the most parsimonious conclusion is that parents may have been heterozygous for loci causing hybrid inviability.The University of Texas at Austin funded DIB as assistant professor, HLF as a postdoctoral researcher at DIB's lab, and all experimental work. The National Science Foundation grant DEB 0516831 supported HLF as a postdoctoral researcher at Texas A&M University during the writing phase of this project.Biological Sciences, School o

Participants performance in the First Financial Education Olympiad in Rio Grande do Sul

A Educação Financeira permite escolhas conscientes e seguras sobre finanças, oportunidades e futuro. Este estudo objetivou analisar o desempenho dos participantes da I Olimpíada de Educação Financeira no Rio Grande do Sul (I OBEF RS) relacionado aos conteúdos de Educação Financeira. Realizou-se a análise das provas aplicadas em 2019 nas três fases, para os cinco níveis. Quanto aos procedimentos metodológicos, é uma pesquisa quantitativa, descritiva e documental. Participaram desta pesquisa 2.948 alunos, de escolas públicas e privadas, do 2º ano do ensino fundamental ao 3º do ensino médio, dos quais se constatou melhora no desempenho ao longo da olimpíada. Na primeira fase, as escolas públicas apresentaram o maior percentual de participantes classificados, 52,1% em relação a 47,9% das privadas, e o nível um pontuou o maior percentual de aprovados. Na segunda fase, embora as escolas privadas tenham apresentado o maior percentual de classificados (53,3%), não houve diferenças significativas. Os níveis três a cinco apresentaram o maior percentual de aprovação. Na terceira fase, que premiou os medalhistas nacionais, predominou as escolas públicas, com 81,8% dos alunos. Dentre os nove medalhistas das escolas públicas, sete são de escola militar e foram os que apresentaram o melhor desempenho, sendo estes dos níveis três a cinco. Em relação ao gênero, não foram identificadas diferenças estatisticamente significativas. Este estudo promoveu a inserção do tema Educação Financeira no contexto escolar, destacando a importância da participação dos alunos na I Olimpíada Brasileira de Educação Financeira (I OBEF) e fortalecendo o mapeamento das habilidades sobre assuntos relacionados à Educação Financeira.Financial education allows conscious and secure choices about finances, opportunities and future. This study aimed to analyze participants performance in Olympiad of Financial Education in Rio Grande do Sul (I OBEF RS), in relation to Financial Education contents. An analysis of the three phases tests applied in 2019 to five levels of students was carried out. As for the methodological procedures, it is a quantitative, descriptive and documentary research. There were 2,948 pupils participating, from public and private schools, from the 2nd year of elementary school up to the 3rd year of high school. It was noticed their performance’s improvement throughout the Olympiad. In the first stage, public schools had the highest percentage of participants classified (52.1%), and private schools had 47.9%, and level one had the highest percentage of those approved. In the second stage, although the private schools showed the highest percentage of students classified (53.3%), the differences found were not significant. Levels 3 to 5 had the highest approval percentage. In the third stage, which gave rise to the national medalists, public schools predominated, with 81.8% of the students. Among the nine public schools’ medalists, seven were from military school, and they performed better in comparison with the total medal-winners, from levels 3 to 5. Regarding gender, there were no statistically significant differences. This study promoted the inclusion of this theme in the classrooms, highlighting the importance of pupils’ participation in the I Brazilian Olympiad of Financial Education (I OBEF) and strengthening the skills mapping of financial education matters

Single-Molecule LATE-PCR Analysis of Human Mitochondrial Genomic Sequence Variations

It is thought that changes in mitochondrial DNA are associated with many degenerative diseases, including Alzheimer's and diabetes. Much of the evidence, however, depends on correlating disease states with changing levels of heteroplasmy within populations of mitochondrial genomes, rather than individual mitochondrial genomes. Thus these measurements are likely to either overestimate the extent of heteroplasmy due to technical artifacts, or underestimate the actual level of heteroplasmy because only the most abundant changes are observable. In contrast, Single Molecule (SM) LATE-PCR analysis achieves efficient amplification of single-stranded amplicons from single target molecules. The product molecules, in turn, can be accurately sequenced using a convenient Dilute-‘N’-Go protocol, as shown here. Using these novel technologies we have rigorously analyzed levels of mitochondrial genome heteroplasmy found in single hair shafts of healthy adult individuals. Two of the single molecule sequences (7% of the samples) were found to contain mutations. Most of the mtDNA sequence changes, however, were due to the presence of laboratory contaminants. Amplification and sequencing errors did not result in mis-identification of mutations. We conclude that SM-LATE-PCR in combination with Dilute-‘N’-Go Sequencing are convenient technologies for detecting infrequent mutations in mitochondrial genomes, provided great care is taken to control and document contamination. We plan to use these technologies in the future to look for age, drug, and disease related mitochondrial genome changes in model systems and clinical samples

The vertebrate makorin ubiquitin ligase gene family has been shaped by large-scale duplication and retroposition from an ancestral gonad-specific, maternal-effect gene

Background Members of the makorin (mkrn) gene family encode RING/C3H zinc finger proteins with U3 ubiquitin ligase activity. Although these proteins have been described in a variety of eukaryotes such as plants, fungi, invertebrates and vertebrates including human, almost nothing is known about their structural and functional evolution. Results Via partial sequencing of a testis cDNA library from the poeciliid fish Xiphophorus maculatus, we have identified a new member of the makorin gene family, that we called mkrn4. In addition to the already described mkrn1 and mkrn2, mkrn4 is the third example of a makorin gene present in both tetrapods and ray-finned fish. However, this gene was not detected in mouse and rat, suggesting its loss in the lineage leading to rodent murids. Mkrn2 and mkrn4 are located in large ancient duplicated regions in tetrapod and fish genomes, suggesting the possible involvement of ancestral vertebrate-specific genome duplication in the formation of these genes. Intriguingly, many mkrn1 and mkrn2 intronless retrocopies have been detected in mammals but not in other vertebrates, most of them corresponding to pseudogenes. The nature and number of zinc fingers were found to be conserved in Mkrn1 and Mkrn2 but much more variable in Mkrn4, with lineage-specific differences. RT-qPCR analysis demonstrated a highly gonad-biased expression pattern for makorin genes in medaka and zebrafish (ray-finned fishes) and amphibians, but a strong relaxation of this specificity in birds and mammals. All three mkrn genes were maternally expressed before zygotic genome activation in both medaka and zebrafish early embryos. Conclusion Our analysis demonstrates that the makorin gene family has evolved through large-scale duplication and subsequent lineage-specific retroposition-mediated duplications in vertebrates. From the three major vertebrate mkrn genes, mkrn4 shows the highest evolutionary dynamics, with lineage-specific loss of zinc fingers and even complete gene elimination from certain groups of vertebrates. Comparative expression analysis strongly suggests that the ancestral E3 ubiquitin ligase function of the single copy mkrn gene before duplication in vertebrates was gonad-specific, with maternal expression in early embryos. (Résumé d'auteur

Accumulation and Rapid Decay of Non-LTR Retrotransposons in the Genome of the Three-Spine Stickleback

The diversity and abundance of non–long terminal repeat (LTR) retrotransposons (nLTR-RT) differ drastically among vertebrate genomes. At one extreme, the genome of placental mammals is littered with hundreds of thousands of copies resulting from the activity of a single clade of nLTR-RT, the L1 clade. In contrast, fish genomes contain a much more diverse repertoire of nLTR-RT, represented by numerous active clades and families. Yet, the number of nLTR-RT copies in teleostean fish is two orders of magnitude smaller than in mammals. The vast majority of insertions appear to be very recent, suggesting that nLTR-RT do not accumulate in fish genomes. This pattern had previously been explained by a high rate of turnover, in which the insertion of new elements is offset by the selective loss of deleterious inserts. The turnover model was proposed because of the similarity between fish and Drosophila genomes with regard to their nLTR-RT profile. However, it is unclear if this model applies to fish. In fact, a previous study performed on the puffer fish suggested that transposable element insertions behave as neutral alleles. Here we examined the dynamics of amplification of nLTR-RT in the three-spine stickleback (Gasterosteus aculeatus). In this species, the vast majority of nLTR-RT insertions are relatively young, as suggested by their low level of divergence. Contrary to expectations, a majority of these insertions are fixed in lake and oceanic populations; thus, nLTR-RT do indeed accumulate in the genome of their fish host. This is not to say that nLTR-RTs are fully neutral, as the lack of fixed long elements in this genome suggests a deleterious effect related to their length. This analysis does not support the turnover model and strongly suggests that a much higher rate of DNA loss in fish than in mammals is responsible for the relatively small number of nLTR-RT copies and for the scarcity of ancient elements in fish genomes. We further demonstrate that nLTR-RT decay in fish occurs mostly through large deletions and not by the accumulation of small deletions

Investigation of the residual stress distribution in repairs in H13 steel by friction hydro pillar processing

The distribution of residual stresses (RS) in repairs generated by Friction Hydro-Pillar Processing (FHPP) in AISI H13 was investigated. Three axial force levels, with consequent different deposition rates, were applied to replicate possible repaired conditions. The contour (CM) and X-ray diffraction (XRD) methods were employed for RS analysis in samples that were also evaluated through metallography, microhardness analysis, micro-tensile and Charpy testing. CM produced 2D maps of the RS in the joints, showing symmetrical distributions around the welded rod for all welded conditions. Other common features for all conditions were the maximum level of compressive RS, which was found in the TMAZ of the rod, and the maximum tensile residual stress, which was found near the HAZ of the base block. There was good agreement between the RS measurement techniques. Mechanical tests show similar tensile resistance for all conditions and an apparent increase in toughness at higher force levels

DNA Barcodes Provide a Quick Preview of Mitochondrial Genome Composition

DNA barcodes have achieved prominence as a tool for species-level identifications. Consequently, there is a rapidly growing database of these short sequences from a wide variety of taxa. In this study, we have analyzed the correlation between the nucleotide content of the short DNA barcode sequences and the genomes from which they are derived. Our results show that such short sequences can yield important, and surprisingly accurate, information about the composition of the entire genome. In other words, for unsequenced genomes, the DNA barcodes can provide a quick preview of the whole genome composition