RESUMO
In the tetrapod limb, the digits (fingers or toes) are the elements most subject to morphological diversification in response to functional adaptations. However, despite their functional importance, the mechanisms controlling digit morphology remain poorly understood. Here we have focused on understanding the special morphology of the thumb (digit 1), the acquisition of which was an important adaptation of the human hand. To this end, we have studied the limbs of the Hoxa13 mouse mutant that specifically fail to form digit 1. We show that, consistent with the role of Hoxa13 in Hoxd transcriptional regulation, the expression of Hoxd13 in Hoxa13 mutant limbs does not extend into the presumptive digit 1 territory, which is therefore devoid of distal Hox transcripts, a circumstance that can explain its agenesis. The loss of Hoxd13 expression, exclusively in digit 1 territory, correlates with increased Gli3 repressor activity, a Hoxd negative regulator, resulting from increased Gli3 transcription that, in turn, is due to the release from the negative modulation exerted by Hox13 paralogs on Gli3 regulatory sequences. Our results indicate that Hoxa13 acts hierarchically to initiate the formation of digit 1 by reducing Gli3 transcription and by enabling expansion of the 5'Hoxd second expression phase, thereby establishing anterior-posterior asymmetry in the handplate. Our work uncovers a mutual antagonism between Gli3 and Hox13 paralogs that has important implications for Hox and Gli3 gene regulation in the context of development and evolution.
Assuntos
Extremidades/crescimento & desenvolvimento , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Proteína Gli3 com Dedos de Zinco/metabolismo , Animais , Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Transcriptoma , Proteína Gli3 com Dedos de Zinco/genéticaRESUMO
The fin-to-limb transition represents one of the major vertebrate morphological innovations associated with the transition from aquatic to terrestrial life and is an attractive model for gaining insights into the mechanisms of morphological diversity between species. One of the characteristic features of limbs is the presence of digits at their extremities. Although most tetrapods have limbs with five digits (pentadactyl limbs), palaeontological data indicate that digits emerged in lobed fins of early tetrapods, which were polydactylous. How the transition to pentadactyl limbs occurred remains unclear. Here we show that the mutually exclusive expression of the mouse genes Hoxa11 and Hoxa13, which were previously proposed to be involved in the origin of the tetrapod limb, is required for the pentadactyl state. We further demonstrate that the exclusion of Hoxa11 from the Hoxa13 domain relies on an enhancer that drives antisense transcription at the Hoxa11 locus after activation by HOXA13 and HOXD13. Finally, we show that the enhancer that drives antisense transcription of the mouse Hoxa11 gene is absent in zebrafish, which, together with the largely overlapping expression of hoxa11 and hoxa13 genes reported in fish, suggests that this enhancer emerged in the course of the fin-to-limb transition. On the basis of the polydactyly that we observed after expression of Hoxa11 in distal limbs, we propose that the evolution of Hoxa11 regulation contributed to the transition from polydactyl limbs in stem-group tetrapods to pentadactyl limbs in extant tetrapods.
Assuntos
Evolução Biológica , Extremidades/anatomia & histologia , Proteínas de Homeodomínio/metabolismo , Vertebrados/anatomia & histologia , Vertebrados/genética , Nadadeiras de Animais/anatomia & histologia , Nadadeiras de Animais/metabolismo , Animais , Elementos Facilitadores Genéticos/genética , Extinção Biológica , Feminino , Íntrons/genética , Camundongos , RNA Antissenso/biossíntese , RNA Antissenso/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/genéticaRESUMO
Analysis of the human and murine transcriptomes has identified long noncoding RNAs (lncRNAs) as major functional components in both species. Transcriptional profiling of the murine limb led to our discovery of lncRNA-HIT, which our previous in vitro analyses suggested a potential role for this lncRNA in the development of limb, craniofacial, and genitourinary tissues (Carlson et al., 2015). To test this hypothesis, we developed a conditional lncRNA-HIT loss of function allele which uses Cre recombinase to activate an shRNA specific for lncRNA-HIT. Activation of the lncRNA-HIT shRNA allele resulted in a robust knock-down of lncRNA-HIT as well as co-activation of a mCherry reporter, confirming the efficacy of the shRNA allele to reduce endogenous lncRNA levels in a tissue- and cell-type specific manner. Developmental analyses of embryos expressing the activated shRNA and mCherry co-reporter revealed multiple malformations corresponding to the sites of shRNA activation, affecting craniofacial, limb, and genitourinary tissue development. These results confirm the efficacy of lncRNA-HIT shRNA allele to knock-down endogenous transcripts in tissue- and cell type specific manner and indicate a requirement for lncRNA-HIT in the development of these tissues.
Assuntos
Alelos , Perfilação da Expressão Gênica , Inativação Gênica , RNA Longo não Codificante/genética , Transcriptoma , Animais , Biologia Computacional/métodos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Humanos , Camundongos , Especificidade de Órgãos , Fenótipo , RNA Interferente Pequeno/genética , Ativação TranscricionalRESUMO
The long tendons of the limb extend from muscles that reside in the zeugopod (arm/leg) to their skeletal insertions in the autopod (paw). How these connections are established along the length of the limb remains unknown. Here, we show that mouse limb tendons are formed in modular units that combine to form a functional contiguous structure; in muscle-less limbs, tendons develop in the autopod but do not extend into the zeugopod, and in the absence of limb cartilage the zeugopod segments of tendons develop despite the absence of tendons in the autopod. Analyses of cell lineage and proliferation indicate that distinct mechanisms govern the growth of autopod and zeugopod tendon segments. To elucidate the integration of these autopod and zeugopod developmental programs, we re-examined early tendon development. At E12.5, muscles extend across the full length of a very short zeugopod and connect through short anlagen of tendon progenitors at the presumptive wrist to their respective autopod tendon segment, thereby initiating musculoskeletal integration. Zeugopod tendon segments are subsequently generated by proximal elongation of the wrist tendon anlagen, in parallel with skeletal growth, underscoring the dependence of zeugopod tendon development on muscles for tendon anchoring. Moreover, a subset of extensor tendons initially form as fused structures due to initial attachment of their respective wrist tendon anlage to multiple muscles. Subsequent individuation of these tendons depends on muscle activity. These results establish an integrated model for limb tendon development that provides a framework for future analyses of tendon and musculoskeletal phenotypes.
Assuntos
Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Músculo Esquelético/embriologia , Tendões/embriologia , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cartilagem/metabolismo , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Deleção de Genes , Proteínas de Fluorescência Verde/metabolismo , Articulação Metacarpofalângica/patologia , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Músculo Esquelético/metabolismo , Fenótipo , Fatores de Transcrição SOX9/genética , Tendões/metabolismoRESUMO
Gene expression profiling in E 11 mouse embryos identified high expression of the long noncoding RNA (lncRNA), LNCRNA-HIT in the undifferentiated limb mesenchyme, gut, and developing genital tubercle. In the limb mesenchyme, LncRNA-HIT was found to be retained in the nucleus, forming a complex with p100 and CBP. Analysis of the genome-wide distribution of LncRNA-HIT-p100/CBP complexes by ChIRP-seq revealed LncRNA-HIT associated peaks at multiple loci in the murine genome. Ontological analysis of the genes contacted by LncRNA-HIT-p100/CBP complexes indicate a primary role for these loci in chondrogenic differentiation. Functional analysis using siRNA-mediated reductions in LncRNA-HIT or p100 transcripts revealed a significant decrease in expression of many of the LncRNA-HIT-associated loci. LncRNA-HIT siRNA treatments also impacted the ability of the limb mesenchyme to form cartilage, reducing mesenchymal cell condensation and the formation of cartilage nodules. Mechanistically the LncRNA-HIT siRNA treatments impacted pro-chondrogenic gene expression by reducing H3K27ac or p100 activity, confirming that LncRNA-HIT is essential for chondrogenic differentiation in the limb mesenchyme. Taken together, these findings reveal a fundamental epigenetic mechanism functioning during early limb development, using LncRNA-HIT and its associated proteins to promote the expression of multiple genes whose products are necessary for the formation of cartilage.
Assuntos
Diferenciação Celular/genética , Condrogênese/genética , RNA Longo não Codificante/genética , Proteína p120 Ativadora de GTPase/genética , Animais , Epigênese Genética/genética , Extremidades/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Botões de Extremidades/crescimento & desenvolvimento , Mesoderma/crescimento & desenvolvimento , Camundongos , RNA Longo não Codificante/biossíntese , Proteína p120 Ativadora de GTPase/biossínteseRESUMO
BACKGROUND: Retinoic acid (RA), plays an essential role in the growth and patterning of vertebrate limb. While the developmental processes regulated by RA are well understood, little is known about the transcriptional mechanisms required to precisely control limb RA synthesis. Here, Aldh1a2 functions as the primary enzyme necessary for RA production which regulates forelimb outgrowth and hindlimb digit separation. Because mice lacking HOXA13 exhibit similar defects in digit separation as Aldh1a2 mutants, we hypothesized that HOXA13 regulates Aldh1a2 to facilitate RA-mediated interdigital programmed cell death (IPCD) and digit separation. RESULTS: In this report, we identify Aldh1a2 as a direct target of HOXA13. In absence of HOXA13 function, Aldh1a2 expression, RA signaling, and IPCD are reduced. In the limb, HOXA13 binds a conserved cis-regulatory element in the Aldh1a2 locus that can be regulated by HOXA13 to promote gene expression. Finally, decreased RA signaling and IPCD can be partially rescued in the Hoxa13 mutant hindlimb by maternal RA supplementation. CONCLUSIONS: Defects in IPCD and digit separation in Hoxa13 mutant mice may be caused in part by reduced levels of RA signaling stemming from a loss in the direct regulation of Aldh1a2. These findings provide new insights into the transcriptional regulation of RA signaling necessary for limb morphogenesis.
Assuntos
Aldeído Desidrogenase/metabolismo , Apoptose , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Família Aldeído Desidrogenase 1 , Animais , Sequência de Bases , Padronização Corporal , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Retinal Desidrogenase , Ácido Retinoico 4 Hidroxilase , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Transgenes , Tretinoína/metabolismoRESUMO
The realisation of a novel concept for automated on-line monitoring of enzymatic activities in water was successfully demonstrated by long-term field testing at two remote Austrian ground water resources. The ß-D-glucuronidase (GLUC) activity was selected as a representative enzymatic model parameter for the on-line determination. But the device can be adapted for any enzymatic reaction with diagnostic relevance for microbial water quality monitoring, as demonstrated for the ß-D-galactosidase activity. Automated filtration of volumes up to 5 litres supports sensitive quantification of enzymatic activities. Internet-based data transfer, using internal control parameters for verification and a dynamic determination of the limit of quantification, enabled robust enzymatic on-line monitoring during a 2-year period. A proportion of 5,313 out of 5,506 GLUC activity measurements (96.5%) could be positively verified. Hydrological (discharge, gauge, turbidity, temperature, pH, electric conductivity, spectral absorbance coefficient at 254 nm) as well as microbiological parameters (Escherichia coli, coliforms) were concurrently determined to characterise the investigated ground water resources. The enzymatic on-line measurements closely reflected the different hydrological conditions and contamination patterns of the test sites. Contrary to expectations, GLUC did not qualify as a proxy-parameter for the occurrence of cultivation-based E. coli contamination and warrants further detailed investigations on its indication capacity as a rapid means for microbial faecal pollution detection in such aquatic habitats. Microbial on-line monitoring is likely to become more important in the future, complementing existing surveillance strategies for water safety management. Further perspectives on the application of such analytical on-line technologies, such as their connection with event-triggered sampling and standardised diagnostics, are discussed.
Assuntos
Monitoramento Ambiental/instrumentação , Glucuronidase/análise , Água Subterrânea/análise , Microbiologia da Água , Abastecimento de Água/análise , Qualidade da ÁguaRESUMO
Polyalanine repeat expansion diseases are hypothesized to result from unequal chromosomal recombination, yet mechanistic studies are lacking. We identified two de novo cases of hand-foot-genital syndrome (HFGS) associated with polyalanine expansions in HOXA13 that afforded rare opportunities to investigate the mechanism. The first patient with HFGS was heterozygous for a de novo nine codon polyalanine expansion. Haplotype investigation showed that the expansion arose on the maternally inherited chromosome but not through unequal crossing over between homologs, leaving unequal sister chromatid exchange during mitosis or meiosis or slipped mispairing as possible explanations. The asymptomatic father of the second patient with HFGS was mosaic for a six codon polyalanine expansion. Multiple tissue PCR and clonal analysis of paternal fibroblasts showed only expansion/WT and WT/WT clones, and haplotype data showed that two unaffected offspring inherited the same paternal allele without the expansion, supporting a postzygotic origin. Absence of the contracted allele in the mosaic father does not support sister chromatid exchange in the origin of the expansion. Mosaicism for HOXA13 polyalanine expansions may be associated with a normal phenotype, making examination of parental DNA essential in apparently de novo HFGS cases to predict accurate recurrence risks. We could not find an example in the literature where unequal sister chromatid exchange has been proven for any polyalanine expansion, suggesting that the principal mechanism for polyalanine expansions (and contractions) is slipped mispairing without repair or that the true frequency of unequal sister chromatid exchange involving these repeats is low.
Assuntos
Anormalidades Múltiplas/genética , Expansão das Repetições de DNA/genética , Deformidades Congênitas do Pé/genética , Deformidades Congênitas da Mão/genética , Proteínas de Homeodomínio/genética , Anormalidades Urogenitais/genética , Adolescente , Adulto , Feminino , Humanos , Recém-Nascido , Masculino , Mutação , Peptídeos , FenótipoRESUMO
The mechanical properties of bitumen, such as elasticity/Young's modulus, stickiness/adhesion, hardness and energy loss, and sample deformation were acquired quantitatively and simultaneously with the topology at the microscale, discriminating clearly two separate phases within the bitumen. Temperature-dependent measurements revealed detailed and specific data about the changes of these properties with temperature, enabling the development of predictive models for the performance and durability of asphalt.
RESUMO
In vitro models allow for the study of developmental processes outside of the embryo. To gain access to the cells mediating digit and joint development, we identified a unique property of undifferentiated mesenchyme isolated from the distal early autopod to autonomously re-assemble forming multiple autopod structures including: digits, interdigital tissues, joints, muscles and tendons. Single-cell transcriptomic analysis of these developing structures revealed distinct cell clusters that express canonical markers of distal limb development including: Col2a1, Col10a1, and Sp7 (phalanx formation), Thbs2 and Col1a1 (perichondrium), Gdf5, Wnt5a, and Jun (joint interzone), Aldh1a2 and Msx1 (interdigital tissues), Myod1 (muscle progenitors), Prg4 (articular perichondrium/articular cartilage), and Scx and Tnmd (tenocytes/tendons). Analysis of the gene expression patterns for these signature genes indicates that developmental timing and tissue-specific localization were also recapitulated in a manner similar to the initiation and maturation of the developing murine autopod. Finally, the in vitro digit system also recapitulates congenital malformations associated with genetic mutations as in vitro cultures of Hoxa13 mutant mesenchyme produced defects present in Hoxa13 mutant autopods including digit fusions, reduced phalangeal segment numbers, and poor mesenchymal condensation. These findings demonstrate the robustness of the in vitro digit system to recapitulate digit and joint development. As an in vitro model of murine digit and joint development, this innovative system will provide access to the developing limb tissues facilitating studies to discern how digit and articular joint formation is initiated and how undifferentiated mesenchyme is patterned to establish individual digit morphologies. The in vitro digit system also provides a platform to rapidly evaluate treatments aimed at stimulating the repair or regeneration of mammalian digits impacted by congenital malformation, injury, or disease.
RESUMO
Water resource management must strive to link catchment information with water quality monitoring. The present study attempted this for the field of microbial fecal source tracking (MST). A fecal pollution source profile based on catchment data (e.g., prevalence of fecal sources) was used to formulate a hypothesis about the dominant sources of pollution in an Austrian mountainous karst spring catchment. This allowed a statistical definition of methodical requirements necessary for an informed choice of MST methods. The hypothesis was tested in a 17-month investigation of spring water quality. The study followed a nested sampling design in order to cover the hydrological and pollution dynamics of the spring and to assess effects such as differential persistence between parameters. Genetic markers for the potential fecal sources as well as microbiological, hydrological, and chemo-physical parameters were measured. The hypothesis that ruminant animals were the dominant sources of fecal pollution in the catchment was clearly confirmed. It was also shown that the concentration of ruminant markers in feces was equally distributed in different ruminant source groups. The developed approach provides a tool for careful decision-making in MST study design and might be applied on various types of catchments and pollution situations.
Assuntos
Monitoramento Ambiental/métodos , Fezes/microbiologia , Microbiologia da Água , Poluição da Água/análise , Abastecimento de Água/análise , Animais , Áustria , Bacteroidetes/isolamento & purificação , Ruminantes , Microbiologia do SoloRESUMO
In eutherian mammals, embryonic growth and survival is dependent on the formation of the placenta, an organ that facilitates the efficient exchange of oxygen, nutrients, and metabolic waste between the maternal and fetal blood supplies. Key to the placenta's function is the formation of its vascular labyrinth, a series of finely branched vessels whose molecular ontogeny remains largely undefined. In this report, we demonstrate that HOXA13 plays an essential role in labyrinth vessel formation. In the absence of HOXA13 function, placental endothelial cell morphology is altered, causing a loss in vessel wall integrity, edema of the embryonic blood vessels, and mid-gestational lethality. Microarray analysis of wild-type and mutant placentas revealed significant changes in endothelial gene expression profiles. Notably, pro-vascular genes, including Tie2 and Foxf1, exhibited reduced expression in the mutant endothelia, which also exhibited elevated expression of genes normally expressed in lymphatic or sinusoidal endothelia. ChIP analysis of HOXA13-DNA complexes in the placenta confirmed that HOXA13 binds the Tie2 and Foxf1 promoters in vivo. In vitro, HOXA13 binds sequences present in the Tie2 and Foxf1 promoters with high affinity (K(d) = 27-42 nM) and HOXA13 can use these bound promoter regions to direct gene expression. Taken together, these findings demonstrate that HOXA13 directly regulates Tie2 and Foxf1 in the placental labyrinth endothelia, providing a functional explanation for the mid-gestational lethality exhibited by Hoxa13 mutant embryos as well as a novel transcriptional program necessary for the specification of the labyrinth vascular endothelia.
Assuntos
Endotélio Vascular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Placenta/irrigação sanguínea , Animais , Sequência de Bases , Endotélio Vascular/embriologia , Endotélio Vascular/crescimento & desenvolvimento , Fatores de Transcrição Forkhead/genética , Genes Reporter , Proteínas de Homeodomínio/genética , Homozigoto , Técnicas In Vitro , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Neovascularização Fisiológica , Análise de Sequência com Séries de Oligonucleotídeos , Placenta/embriologia , Placenta/metabolismo , Regiões Promotoras Genéticas , Receptor TIE-2/genética , Trofoblastos/fisiologiaRESUMO
The loss of HOXA13 function severely disrupts embryonic limb development. However, because embryos lacking HOXA13 die by mid-gestation, the defects present in the mutant limb could arise as a secondary consequence of failing embryonic health. In our analysis of the mutant Hoxa13(GFP) allele, we identified a surviving cohort of homozygous mutants exhibiting severe limb defects including: missing phalanx elements, fusions of the carpal/tarsal elements, and significant reductions in metacarpal/metatarsal length. Characterization of prochondrogenic genes in the affected carpal/tarsal regions revealed significant reduction in Gdf5 expression, whereas Bmp2 expression was significantly elevated. Analysis of Gdf5 mRNA localization also revealed diffuse expression in the carpal/tarsal anlagen, suggesting a role for HOXA13 in the organization of the cells necessary to delineate individual carpal/tarsal elements. Together these results identify Gdf5 as a potential target gene of HOXA13 target gene and confirm a specific role for HOXA13 during appendicular skeletal development.
Assuntos
Ossos do Pé/embriologia , Fator 5 de Diferenciação de Crescimento/metabolismo , Proteínas de Homeodomínio/metabolismo , Esqueleto , Animais , Morte Celular , Proliferação de Células , Feminino , Proteínas de Homeodomínio/genética , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fatores de Transcrição SOX9/metabolismoRESUMO
In most tetrapod vertebrates, limb skeletal progenitors condense with postaxial dominance. Posterior elements (such as ulna and fibula) appear prior to their anterior counterparts (radius and tibia), followed by digit-appearance order with continuing postaxial polarity. The only exceptions are urodele amphibians (salamanders), whose limb elements develop with preaxial polarity and who are also notable for their unique ability to regenerate complete limbs as adults. The mechanistic basis for this preaxial dominance has remained an enigma and has even been proposed to relate to the acquisition of novel genes involved in regeneration. However, recent fossil evidence suggests that preaxial polarity represents an ancestral rather than derived state. Here, we report that 5'Hoxd (Hoxd11-d13) gene deletion in mouse is atavistic and uncovers an underlying preaxial polarity in mammalian limb formation. We demonstrate this shift from postaxial to preaxial dominance in mouse results from excess Gli3 repressor (Gli3R) activity due to the loss of 5'Hoxd-Gli3 antagonism and is associated with cell-cycle changes promoting precocious cell-cycle exit in the anterior limb bud. We further show that Gli3 knockdown in axolotl results in a shift to postaxial dominant limb skeleton formation, as well as expanded paddle-shaped limb-bud morphology and ensuing polydactyly. Evolutionary changes in Gli3R activity level, which also played a key role in the fin-to-limb transition, appear to be fundamental to the shift from preaxial to postaxial polarity in formation of the tetrapod limb skeleton.
Assuntos
Extremidades , Botões de Extremidades , Animais , Evolução Biológica , Extremidades/anatomia & histologia , Mamíferos , Camundongos , Fatores de Transcrição/genética , Urodelos/anatomia & histologiaRESUMO
Barrett's esophagus in gastrointestinal reflux patients constitutes a columnar epithelium with distal characteristics, prone to progress to esophageal adenocarcinoma. HOX genes are known mediators of position-dependent morphology. Here we show HOX collinearity in the adult gut while Barrett's esophagus shows high HOXA13 expression in stem cells and their progeny. HOXA13 overexpression appears sufficient to explain both the phenotype (through downregulation of the epidermal differentiation complex) and the oncogenic potential of Barrett's esophagus. Intriguingly, employing a mouse model that contains a reporter coupled to the HOXA13 promotor we identify single HOXA13-positive cells distally from the physiological esophagus, which is mirrored in human physiology, but increased in Barrett's esophagus. Additionally, we observe that HOXA13 expression confers a competitive advantage to cells. We thus propose that Barrett's esophagus and associated esophageal adenocarcinoma is the consequence of expansion of this gastro-esophageal HOXA13-expressing compartment following epithelial injury.
Assuntos
Esôfago de Barrett/genética , Carcinogênese/genética , Proteínas de Homeodomínio/genética , Oncogenes/genética , Adulto , Animais , Esôfago de Barrett/metabolismo , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/metabolismo , Trato Gastrointestinal/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Família Multigênica/genética , RNA-Seq/métodosRESUMO
Because spring water quality from alpine karst aquifers can change very rapidly during event situations, water abstraction management has to be performed in near real-time. Four summer events (2005-2008) at alpine karst springs were investigated in detail in order to evaluate the spectral absorption coefficient at 254 nm (SAC254) as a real-time early warning proxy for faecal pollution. For the investigation Low-Earth-Orbit (LEO) Satellite-based data communication between portable hydrometeorological measuring stations and an automated microbiological sampling device was used. The method for event triggered microbial sampling and analyzing was already established and described in a previous paper. Data analysis including on-line event characterisation (i.e. precipitation, discharge, turbidity, SAC254) and comprehensive E. coli determination (n>800) indicated that SAC254 is a useful early warning proxy. Irrespective of the studied event situations SAC254 always increased 3 to 6 hours earlier than the onset of faecal pollution, featuring different correlation phases. Furthermore, it seems also possible to use SAC254 as a real-time proxy parameter for estimating the extent of faecal pollution after establishing specific spring and event-type calibrations that take into consideration the variability of the occurrence and the transferability of faecal material It should be highlighted that diffuse faecal pollution from wildlife and live stock sources was responsible for spring water contamination at the investigated catchments. In this respect, the SAC254 can also provide useful information to support microbial source tracking efforts where different situations of infiltration have to be investigated.
Assuntos
Monitoramento Ambiental/métodos , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Água Doce/microbiologia , Microbiologia da Água , Poluição da Água/análise , Absorção , Altitude , Áustria , Contagem de Colônia Microbiana , Monitoramento Ambiental/estatística & dados numéricos , Inundações , Água Doce/análise , Hidrodinâmica , Estações do Ano , Fatores de Tempo , Microbiologia da Água/normas , Abastecimento de Água/normasRESUMO
A long non-coding RNA called GRASLND is essential to help stem cells create stable cartilage.
Assuntos
Cartilagem Articular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , RNA Longo não Codificante , Condrogênese , Interferon gama , Transdução de Sinais , Engenharia TecidualRESUMO
Congenital anomalies of the external genitalia (CAEG) are a prevalent and serious public health concern with lifelong impacts on the urinary function, sexual health, fertility, tumor development, and psychosocial wellbeing of affected individuals. Complications of treatment are frequent, and data reflecting long-term outcomes in adulthood are limited. To identify a path forward to improve treatments and realize the possibility of preventing CAEG, the National Institute of Diabetes and Digestive and Kidney Diseases and the American Urological Association convened researchers from a range of disciplines to coordinate research efforts to fully understand the different etiologies of these common conditions, subsequent variation in clinical phenotypes, and best practices for long term surgical success. Meeting participants concluded that a central data hub for clinical evaluations, including collection of DNA samples from patients and their parents, and short interviews to determine familial penetrance (small pedigrees), would accelerate research in this field. Such a centralized datahub will advance efforts to develop detailed multi-dimensional phenotyping and will enable access to genome sequence analyses and associated metadata to define the genetic bases for these conditions. Inclusion of tissue samples and integration of clinical studies with basic research using human cells and animal models will advance efforts to identify the developmental mechanisms that are disrupted during development and will add cellular and molecular granularity to phenotyping CAEG. While the discussion focuses heavily on hypospadias, this can be seen as a potential template for other conditions in the realm of CAEG, including cryptorchidism or the exstrophy-epispadias complex. Taken together with long-term clinical follow-up, these data could inform surgical choices and improve likelihood for long-term success.
Assuntos
Extrofia Vesical , Epispadia , Adulto , Animais , Genitália , Humanos , Masculino , National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) , Pesquisa Translacional Biomédica , Estados UnidosRESUMO
Collagen requires hydroxylation of its proline residues to achieve proper assembly, structure, and function. Prolyl 4-hydroxylase catalyzes formation of 4-hydroxyproline, which is essential for collagen triple helix formation and stability. Prolyl 3-hydroxylase catalyzes formation of 3-hydroxyproline, which is far less abundant in collagens and whose function remains unclear. Recently mutations in prolyl 3-hydroxylase 1 have been associated with osteogenesis imperfecta, yet the temporal and spatial expression patterns of the prolyl 3-hydroxylase family members during development and in adult tissues remain undefined. By northern blot analysis distinct differences in transcript sizes of the three prolyl 3-hydroxylase genes were detected. Quantitative RTPCR demonstrated tissue-specific differences in prolyl 3-hydroxylase expression, most notable of which were high levels of prolyl 3-hydroxylase 2 in kidney and prolyl 3-hydroxylase 1 expression in embryonic tissues. Finally, in situ hybridization was used to assess spatio-temporal distribution of three prolyl 3-hydroxylases at embryonic days 11-15. Importantly, prolyl 3-hydroxylase 1 was expressed within cartilage condensations of the vertebral bodies and in the aortic arch of the developing heart, whereas prolyl 3-hydroxylase 2 was expressed in developing lens capsule. The prolyl 3-hydroxylase 3 gene showed more generalized expression overlapping somewhat with the other two genes. This report characterizes expression of the three prolyl 3-hydroxylase genes in embryonic and adult mice. Overall these data demonstrate tissue specific prolyl 3-hydroxylase gene expression in both fetal and adult tissues indicating a developmental role for prolyl 3-hydroxylase activity.
Assuntos
Embrião de Mamíferos/enzimologia , Feto/enzimologia , Glicoproteínas de Membrana/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Proteoglicanas/genética , Animais , Colágeno/metabolismo , Desenvolvimento Embrionário/genética , Camundongos , RNA Mensageiro/metabolismoRESUMO
Pulmonary arterial hypertension (PAH) results in chronic right heart failure, which is associated with an increase in sympathetic tone. This may adversely affect cardiac autonomic control. We investigated the changes in cardiac autonomic nervous activity in relation to disease severity in patients with PAH. In 48 patients with PAH (median World Health Organization class III, pulmonary artery pressure 52+/-14 mmHg, pulmonary vascular resistance 1,202+/-718 dyn x s x cm(-5), cardiac index 2.0+/-0.8 L x min(-1) x m(-2)) and 41 controls, cardiac autonomic nervous activity was evaluated by measurement of heart rate variability (HRV) and baroreflex sensitivity. All patients underwent cardiopulmonary exercise testing (peak oxygen uptake 13.2+/-5.1 mL x kg(-1) x min(-1), minute ventilation/carbon dioxide production slope 47+/-16). In patients with PAH, spectral power of HRV was reduced in the high-frequency (239+/-64 versus 563+/-167 ms2), low-frequency (245+/-58 versus 599+/-219 ms2) and very low-frequency bands (510+/-149 versus 1106+/-598 ms2; all p<0.05). Baroreflex sensitivity was also blunted (5.8+/-0.6 versus 13.9+/-1.2 ms x mmHg(-1); p<0.01). The reduction in high-frequency (r = 0.3, p = 0.04) and low-frequency (r = 0.33, p = 0.02) spectral power and baroreflex sensitivity (r = 0.46, p<0.01) was related to the reduction in peak oxygen uptake. Patients with PAH have a marked alteration in cardiac autonomic control that is related to exercise capacity and may, therefore, serve as an additional marker of disease severity.