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1.
Am J Hum Genet ; 110(11): 1903-1918, 2023 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-37816352

RESUMO

Despite whole-genome sequencing (WGS), many cases of single-gene disorders remain unsolved, impeding diagnosis and preventative care for people whose disease-causing variants escape detection. Since early WGS data analytic steps prioritize protein-coding sequences, to simultaneously prioritize variants in non-coding regions rich in transcribed and critical regulatory sequences, we developed GROFFFY, an analytic tool that integrates coordinates for regions with experimental evidence of functionality. Applied to WGS data from solved and unsolved hereditary hemorrhagic telangiectasia (HHT) recruits to the 100,000 Genomes Project, GROFFFY-based filtration reduced the mean number of variants/DNA from 4,867,167 to 21,486, without deleting disease-causal variants. In three unsolved cases (two related), GROFFFY identified ultra-rare deletions within the 3' untranslated region (UTR) of the tumor suppressor SMAD4, where germline loss-of-function alleles cause combined HHT and colonic polyposis (MIM: 175050). Sited >5.4 kb distal to coding DNA, the deletions did not modify or generate microRNA binding sites, but instead disrupted the sequence context of the final cleavage and polyadenylation site necessary for protein production: By iFoldRNA, an AAUAAA-adjacent 16-nucleotide deletion brought the cleavage site into inaccessible neighboring secondary structures, while a 4-nucleotide deletion unfolded the downstream RNA polymerase II roadblock. SMAD4 RNA expression differed to control-derived RNA from resting and cycloheximide-stressed peripheral blood mononuclear cells. Patterns predicted the mutational site for an unrelated HHT/polyposis-affected individual, where a complex insertion was subsequently identified. In conclusion, we describe a functional rare variant type that impacts regulatory systems based on RNA polyadenylation. Extension of coding sequence-focused gene panels is required to capture these variants.


Assuntos
Proteína Smad4 , Telangiectasia Hemorrágica Hereditária , Humanos , Sequência de Bases , DNA , Leucócitos Mononucleares/patologia , Nucleotídeos , Poliadenilação/genética , RNA , Proteína Smad4/genética , Telangiectasia Hemorrágica Hereditária/genética , Sequenciamento Completo do Genoma
2.
Hum Mol Genet ; 29(22): 3717-3728, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33105483

RESUMO

Talipes equinovarus (clubfoot, TEV) is a congenital rotational foot deformity occurring in 1 per 1000 births with increased prevalence in males compared with females. The genetic etiology of isolated clubfoot (iTEV) remains unclear. Using a genome-wide association study, we identified a locus within FSTL5, encoding follistatin-like 5, significantly associated with iTEV. FSTL5 is an uncharacterized gene whose potential role in embryonic and postnatal development was previously unstudied. Utilizing multiple model systems, we found that Fstl5 was expressed during later stages of embryonic hindlimb development, and, in mice, expression was restricted to the condensing cartilage anlage destined to form the limb skeleton. In the postnatal growth plate, Fstl5 was specifically expressed in prehypertrophic chondrocytes. As Fstl5 knockout rats displayed no gross malformations, we engineered a conditional transgenic mouse line (Fstl5LSL) to overexpress Fstl5 in skeletal osteochondroprogenitors. We observed that hindlimbs were slightly shorter and that bone mineral density was reduced in adult male, but not female, Prrx1-cre;Fstl5LSL mice compared with control. No overt clubfoot-like deformity was observed in Prrx1-cre;Fstl5LSL mice, suggesting FSTL5 may function in other cell types to contribute to iTEV pathogenesis. Interrogating published mouse embryonic single-cell expression data showed that Fstl5 was expressed in cell lineage subclusters whose transcriptomes were associated with neural system development. Moreover, our results suggest that lineage-specific expression of the Fstl genes correlates with their divergent roles as modulators of transforming growth factor beta and bone morphogenetic protein signaling. Results from this study associate FSTL5 with iTEV and suggest a potential sexually dimorphic role for Fstl5 in vivo.


Assuntos
Pé Torto Equinovaro/genética , Proteínas Relacionadas à Folistatina/genética , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Animais , Pé Torto Equinovaro/patologia , Modelos Animais de Doenças , Extremidades/patologia , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Estudos de Associação Genética , Humanos , Camundongos , Ratos
3.
J Biomed Sci ; 30(1): 16, 2023 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-36872339

RESUMO

BACKGROUND: Quelling microglial-induced excessive neuroinflammation is a potential treatment strategy across neurological disorders, including traumatic brain injury (TBI), and can be achieved by thalidomide-like drugs albeit this approved drug class is compromised by potential teratogenicity. Tetrafluorobornylphthalimide (TFBP) and tetrafluoronorbornylphthalimide (TFNBP) were generated to retain the core phthalimide structure of thalidomide immunomodulatory imide drug (IMiD) class. However, the classical glutarimide ring was replaced by a bridged ring structure. TFBP/TFNBP were hence designed to retain beneficial anti-inflammatory properties of IMiDs but, importantly, hinder cereblon binding that underlies the adverse action of thalidomide-like drugs. METHODS: TFBP/TFNBP were synthesized and evaluated for cereblon binding and anti-inflammatory actions in human and rodent cell cultures. Teratogenic potential was assessed in chicken embryos, and in vivo anti-inflammatory actions in rodents challenged with either lipopolysaccharide (LPS) or controlled cortical impact (CCI) moderate traumatic brain injury (TBI). Molecular modeling was performed to provide insight into drug/cereblon binding interactions. RESULTS: TFBP/TFNBP reduced markers of inflammation in mouse macrophage-like RAW264.7 cell cultures and in rodents challenged with LPS, lowering proinflammatory cytokines. Binding studies demonstrated minimal interaction with cereblon, with no resulting degradation of teratogenicity-associated transcription factor SALL4 or of teratogenicity in chicken embryo assays. To evaluate the biological relevance of its anti-inflammatory actions, two doses of TFBP were administered to mice at 1 and 24 h post-injury following CCI TBI. Compared to vehicle treatment, TFBP reduced TBI lesion size together with TBI-induction of an activated microglial phenotype, as evaluated by immunohistochemistry 2-weeks post-injury. Behavioral evaluations at 1- and 2-weeks post-injury demonstrated TFBP provided more rapid recovery of TBI-induced motor coordination and balance impairments, versus vehicle treated mice. CONCLUSION: TFBP and TFNBP represent a new class of thalidomide-like IMiDs that lower proinflammatory cytokine generation but lack binding to cereblon, the main teratogenicity-associated mechanism. This aspect makes TFBP and TFNBP potentially safer than classic IMiDs for clinical use. TFBP provides a strategy to mitigate excessive neuroinflammation associated with moderate severity TBI to, thereby, improve behavioral outcome measures and warrants further investigation in neurological disorders involving a neuroinflammatory component.


Assuntos
Lesões Encefálicas Traumáticas , Lesões Encefálicas , Embrião de Galinha , Humanos , Animais , Camundongos , Talidomida , Doenças Neuroinflamatórias , Agentes de Imunomodulação , Lipopolissacarídeos , Inflamação
4.
Dev Dyn ; 250(9): 1340-1357, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33347679

RESUMO

BACKGROUND: Slits (1-3) and their Robo (1-3) receptors play multiple non-neuronal roles in development, including in development of muscle, heart and mammary gland. Previous work has demonstrated expression of Slit and Robo family members during limb development, where their functions are unclear. RESULTS: In situ hybridisation confirmed strong expression of Slit2, Slit3, Robo1, and Robo2 throughout mouse limb and joint development. No expression of Slit1 or Robo3 was detected. Analysis of Slit1/2 or Slit3 knockout mice revealed normal limb development. In contrast, locally blocking Slit signaling though grafting of cells expressing a dominant-negative Robo2 construct in the proximo-central region of developing chicken limb buds caused significant shortening of the humerus. CONCLUSIONS: These findings demonstrate an essential role for Slit/Robo signaling in regulating bone length during chicken limb development.


Assuntos
Proteínas do Tecido Nervoso , Receptores Imunológicos , Animais , Galinhas , Úmero/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais/genética
5.
J Cell Sci ; 132(13)2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31138678

RESUMO

VGLL proteins are transcriptional co-factors that bind TEAD family transcription factors to regulate events ranging from wing development in fly, to muscle fibre composition and immune function in mice. Here, we characterise Vgll3 in skeletal muscle. We found that mouse Vgll3 was expressed at low levels in healthy muscle but that its levels increased during hypertrophy or regeneration; in humans, VGLL3 was highly expressed in tissues from patients with various muscle diseases, such as in dystrophic muscle and alveolar rhabdomyosarcoma. Interaction proteomics revealed that VGLL3 bound TEAD1, TEAD3 and TEAD4 in myoblasts and/or myotubes. However, there was no interaction with proteins from major regulatory systems such as the Hippo kinase cascade, unlike what is found for the TEAD co-factors YAP (encoded by YAP1) and TAZ (encoded by WWTR1). Vgll3 overexpression reduced the activity of the Hippo negative-feedback loop, affecting expression of muscle-regulating genes including Myf5, Pitx2 and Pitx3, and genes encoding certain Wnts and IGFBPs. VGLL3 mainly repressed gene expression, regulating similar genes to those regulated by YAP and TAZ. siRNA-mediated Vgll3 knockdown suppressed myoblast proliferation, whereas Vgll3 overexpression strongly promoted myogenic differentiation. However, skeletal muscle was overtly normal in Vgll3-null mice, presumably due to feedback signalling and/or redundancy. This work identifies VGLL3 as a transcriptional co-factor operating with the Hippo signal transduction network to control myogenesis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Muscular , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Neoplasias/metabolismo , Ligação Proteica , Fatores de Transcrição de Domínio TEA , Transcriptoma/genética
6.
Development ; 145(19)2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30305274

RESUMO

Absence of the developing lens results in severe eye defects, including substantial reductions in eye size. How the lens controls eye expansion and the underlying signalling pathways are very poorly defined. We identified RDH10, a gene crucial for retinoic acid synthesis during embryogenesis, as a key factor downregulated in the peripheral retina (presumptive ciliary body region) of lens-removed embryonic chicken eyes prior to overt reductions in eye size. This is associated with a significant decrease in retinoic acid synthesis by lens-removed eyes. Restoring retinoic acid signalling in lens-removed eyes by implanting beads soaked in retinoic acid or retinal, but not vitamin A, rescued eye size. Conversely, blocking retinoic acid synthesis decreased eye size in lens-containing eyes. Production of collagen II and collagen IX, which are major vitreal proteins, is also regulated by the lens and retinoic acid signalling. These data mechanistically link the known roles of both the lens and retinoic acid in normal eye development, and support a model whereby retinoic acid production by the peripheral retina acts downstream of the lens to support vitreous production and eye expansion.


Assuntos
Cristalino/embriologia , Cristalino/metabolismo , Transdução de Sinais , Tretinoína/metabolismo , Aldeído Desidrogenase/metabolismo , Animais , Padronização Corporal/efeitos dos fármacos , Embrião de Galinha , Corpo Ciliar/efeitos dos fármacos , Corpo Ciliar/metabolismo , Colágeno/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Cristalino/anatomia & histologia , Cristalino/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Tenascina/metabolismo , Tretinoína/farmacologia , Vitamina A/farmacologia
7.
Development ; 145(3)2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29439133

RESUMO

Genetic factors underlying the human limb abnormality congenital talipes equinovarus ('clubfoot') remain incompletely understood. The spontaneous autosomal recessive mouse 'peroneal muscular atrophy' mutant (PMA) is a faithful morphological model of human clubfoot. In PMA mice, the dorsal (peroneal) branches of the sciatic nerves are absent. In this study, the primary developmental defect was identified as a reduced growth of sciatic nerve lateral motor column (LMC) neurons leading to failure to project to dorsal (peroneal) lower limb muscle blocks. The pma mutation was mapped and a candidate gene encoding LIM-domain kinase 1 (Limk1) identified, which is upregulated in mutant lateral LMC motor neurons. Genetic and molecular analyses showed that the mutation acts in the EphA4-Limk1-Cfl1/cofilin-actin pathway to modulate growth cone extension/collapse. In the chicken, both experimental upregulation of Limk1 by electroporation and pharmacological inhibition of actin turnover led to defects in hindlimb spinal motor neuron growth and pathfinding, and mimicked the clubfoot phenotype. The data support a neuromuscular aetiology for clubfoot and provide a mechanistic framework to understand clubfoot in humans.


Assuntos
Doença de Charcot-Marie-Tooth/embriologia , Pé Torto Equinovaro/embriologia , Pé Torto Equinovaro/genética , Quinases Lim/genética , Mutação , Animais , Axônios , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Embrião de Galinha , Mapeamento Cromossômico , Pé Torto Equinovaro/patologia , Modelos Animais de Doenças , Feminino , Membro Posterior/anormalidades , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Neurônios Motores/patologia , Músculo Esquelético/anormalidades , Músculo Esquelético/inervação , Nervo Fibular/anormalidades , Fenótipo , Gravidez , Receptor EphA4/deficiência , Receptor EphA4/genética , Nervo Isquiático/anormalidades , Regulação para Cima
8.
FASEB J ; 34(9): 11395-11404, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32677118

RESUMO

Cereblon (CRBN) is a substrate recruiter element of the E3 cullin 4-RING ubiquitin ligase complex, and a binding target of immunomodulatory agents (IMiDs). CRBN is responsible for the pleiotropic effects of IMiDs, yet its function in angiogenesis and in mediating the antiangiogenic effects of IMiDs remains unclear. We investigated the role of CRBN in the angiogenic process and in propagating the antiangiogenic effects of IMiDs in vitro. siRNA-mediated CRBN knock down in human endothelial cells (HUVEC and HMVEC-L), did not affect endothelial cell proliferation, migration, or tube formation. Using CRBN-deficient mice, we further demonstrated that microvessal formation can occur independently of cereblon in the ex vivo mouse aortic ring model. The cereblon E3 ubiquitin ligase complex can recruit endothelial cell-specific factors, AGO2 (associated with angiogenesis), and SALL4 (associated with embryogenesis/angiogenesis), for ubiquitin-mediated degradation. Knockdown of CRBN caused a corresponding increase in AGO2 and SALL4 protein expression and IMiD treatment was able to rescue the siCRBN effect to increase the CRBN expression. These findings suggest one potential mechanism of action that likely involves a tightly coordinated regulation of CRBN with endothelial cell targets and highlight the need to further elucidate the mechanism(s), which could include cereblon-independent pathways, through which IMiDs exert their antiangiogenic effects.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Inibidores da Angiogênese/farmacologia , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Lenalidomida/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Interferência de RNA , Ubiquitina-Proteína Ligases/genética
9.
Dev Dyn ; 249(3): 298-312, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31566855

RESUMO

It is now 50 years since Lewis Wolpert published the paper in which he set out the concept of Positional Information to explain how spatial patterns of cellular differentiation are generated. This concept has provided a universal model for pattern formation in embryonic development and regeneration and become part of the fabric of the field of developmental biology. Here I outline how Wolpert devised the concept of Positional Information and describe landmark studies from his lab investigating how Positional Information is specified in the developing chick limb.


Assuntos
Biologia do Desenvolvimento/métodos , Animais , Padronização Corporal/fisiologia , Embrião de Galinha , Extremidades/embriologia , Humanos , Morfogênese/fisiologia
10.
Development ; 144(13): 2504-2516, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28676569

RESUMO

Visual information is relayed from the eye to the brain via retinal ganglion cell (RGC) axons. Mice lacking NRP1 or NRP1-binding VEGF-A isoforms have defective RGC axon organisation alongside brain vascular defects. It is not known whether axonal defects are caused exclusively by defective VEGF-A signalling in RGCs or are exacerbated by abnormal vascular morphology. Targeted NRP1 ablation in RGCs with a Brn3bCre knock-in allele reduced axonal midline crossing at the optic chiasm and optic tract fasciculation. In contrast, Tie2-Cre-mediated endothelial NRP1 ablation induced axon exclusion zones in the optic tracts without impairing axon crossing. Similar defects were observed in Vegfa120/120 and Vegfa188/188 mice, which have vascular defects as a result of their expression of single VEGF-A isoforms. Ectopic midline vascularisation in endothelial Nrp1 and Vegfa188/188 mutants caused additional axonal exclusion zones within the chiasm. As in vitro and in vivo assays demonstrated that vessels do not repel axons, abnormally large or ectopically positioned vessels are likely to present physical obstacles to axon growth. We conclude that proper axonal wiring during brain development depends on the precise molecular control of neurovascular co-patterning.


Assuntos
Axônios/metabolismo , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Neuropilina-1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Padronização Corporal , Diencéfalo/embriologia , Diencéfalo/metabolismo , Células Endoteliais/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Neovascularização Fisiológica , Quiasma Óptico/embriologia , Quiasma Óptico/metabolismo , Células Ganglionares da Retina/metabolismo , Fator de Transcrição Brn-3B/metabolismo , Vias Visuais/metabolismo
11.
Molecules ; 25(23)2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-33276504

RESUMO

Due to its antiangiogenic and anti-immunomodulatory activity, thalidomide continues to be of clinical interest despite its teratogenic actions, and efforts to synthesize safer, clinically active thalidomide analogs are continually underway. In this study, a cohort of 27 chemically diverse thalidomide analogs was evaluated for antiangiogenic activity in an ex vivo rat aorta ring assay. The protein cereblon has been identified as the target for thalidomide, and in silico pharmacophore analysis and molecular docking with a crystal structure of human cereblon were used to investigate the cereblon binding abilities of the thalidomide analogs. The results suggest that not all antiangiogenic thalidomide analogs can bind cereblon, and multiple targets and mechanisms of action may be involved.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Inibidores da Angiogênese/farmacologia , Aorta/efeitos dos fármacos , Simulação de Acoplamento Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Talidomida/análogos & derivados , Talidomida/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Inibidores da Angiogênese/química , Animais , Simulação por Computador , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
12.
Dev Dyn ; 247(11): 1217-1226, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30225906

RESUMO

BACKGROUND: While data regarding expression of limb element and tissue markers during normal mouse limb development exist, few studies show expression patterns in upper and lower limbs throughout key limb development stages. A comparison to normal developmental events is essential when analyzing development of the limb in mutant mice models. RESULTS: Expression patterns of the joint marker Gdf5, tendon and ligament marker Scleraxis, early muscle marker MyoD1, and blood vessel marker Cadherin5 (Cdh5) are presented during the most active phases of embryonic mouse limb patterning. Anti-neurofilament staining of developing nerves in the fore- and hindlimbs and cartilage formation and progression also are described. CONCLUSIONS: This study demonstrates and describes a range of key morphological markers and methods that together can be used to assess normal and abnormal limb development. Developmental Dynamics 247:1217-1226, 2018. © 2018 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.


Assuntos
Desenvolvimento Embrionário/fisiologia , Extremidades/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Animais , Antígenos CD/metabolismo , Biomarcadores , Padronização Corporal , Caderinas/metabolismo , Condrogênese , Embrião de Mamíferos , Extremidades/inervação , Fator 5 de Diferenciação de Crescimento/metabolismo , Filamentos Intermediários/metabolismo , Camundongos , Proteína MyoD/metabolismo
13.
J Anat ; 232(4): 568-574, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29023763

RESUMO

Thalidomide notoriously caused severe birth defects, particularly to the limbs, in those exposed in utero following maternal use of the drug to treat morning sickness. How the drug caused these birth defects remains unclear. Many theories have been proposed including actions on the forming blood vessels. However, thalidomide survivors also have altered nerve patterns and the drug is known for its neurotoxic actions in adults following prolonged use. We have previously shown that CPS49, an anti-angiogenic analog of thalidomide, causes a range of limb malformations in a time-sensitive manner in chicken embryos. Here we investigated whether CPS49 also is neurotoxic and whether effects on nerve development impact upon limb development. We found that CPS49 is neurotoxic, just like thalidomide, and can cause some neuronal loss late developing chicken limbs, but only when the limb is already innervated. However, CPS49 exposure does not cause defects in limb size when added to late developing chicken limbs. In contrast, in early limb buds which are not innervated, CPS49 exposure affects limb area significantly. To investigate in more detail the role of neurotoxicity and its impact on chicken limb development we inhibited nerve innervation at a range of developmental timepoints through using ß-bungarotoxin. We found that neuronal inhibition or ablation before, during or after limb outgrowth and innervation does not result in obvious limb cartilage patterning or number changes. We conclude that while CPS49 is neurotoxic, given the late innervation of the developing limb, and that neuronal inhibition/ablation throughout limb development does not cause similar limb patterning anomalies to those seen in thalidomide survivors, nerve defects are not the primary underlying cause of the severe limb patterning defects induced by CPS49/thalidomide.


Assuntos
Inibidores da Angiogênese/toxicidade , Padronização Corporal/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Deformidades Congênitas dos Membros/induzido quimicamente , Crescimento Neuronal/efeitos dos fármacos , Teratogênicos/toxicidade , Talidomida/análogos & derivados , Animais , Bungarotoxinas/farmacologia , Embrião de Galinha , Extremidades/embriologia , Extremidades/inervação , Feminino , Botões de Extremidades/inervação , Camundongos , Camundongos Endogâmicos C57BL , Síndromes Neurotóxicas , Talidomida/toxicidade
14.
Stem Cells ; 35(8): 1958-1972, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28589555

RESUMO

Hippo pathway downstream effectors Yap and Taz play key roles in cell proliferation and regeneration, regulating gene expression especially via Tead transcription factors. To investigate their role in skeletal muscle stem cells, we analyzed Taz in vivo and ex vivo in comparison with Yap. Small interfering RNA knockdown or retroviral-mediated expression of wild-type human or constitutively active TAZ mutants in satellite cells showed that TAZ promoted proliferation, a function shared with YAP. However, at later stages of myogenesis, TAZ also enhanced myogenic differentiation of myoblasts, whereas YAP inhibits such differentiation. Functionally, while muscle growth was mildly affected in Taz (gene Wwtr1-/- ) knockout mice, there were no overt effects on regeneration. Conversely, conditional knockout of Yap in satellite cells of Pax7Cre-ERT2/+ : Yapfl °x/fl °x :Rosa26Lacz mice produced a regeneration deficit. To identify potential mechanisms, microarray analysis showed many common TAZ/YAP target genes, but TAZ also regulates some genes independently of YAP, including myogenic genes such as Pax7, Myf5, and Myod1 (ArrayExpress-E-MTAB-5395). Proteomic analysis revealed many novel binding partners of TAZ/YAP in myogenic cells, but TAZ also interacts with proteins distinct from YAP that are often involved in myogenesis and aspects of cytoskeleton organization (ProteomeXchange-PXD005751). Neither TAZ nor YAP bind members of the Wnt destruction complex but both regulated expression of Wnt and Wnt-cross talking genes with known roles in myogenesis. Finally, TAZ operates through Tead4 to enhance myogenic differentiation. In summary, Taz and Yap have overlapping functions in promoting myoblast proliferation but Taz then switches to enhance myogenic differentiation. Stem Cells 2017;35:1958-1972.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Músculo Esquelético/citologia , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células-Tronco/metabolismo , Animais , Proteínas de Ciclo Celular , Diferenciação Celular/genética , Fusão Celular , Proliferação de Células , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Via de Sinalização Hippo , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Regeneração/genética , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Células-Tronco/citologia , Transativadores , Via de Sinalização Wnt/genética , Proteínas de Sinalização YAP
15.
Bioorg Med Chem ; 26(8): 1547-1559, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29472124

RESUMO

A library of 15 novel and heretofore uncharacterized adamantyl and noradamantyl phthalimidines was synthesized and evaluated for neuroprotective and anti-angiogenic properties. Phthalimidine treatment in LPS-challenged cells effected reductions in levels of secreted TNF-α and nitrite relative to basal amounts. The primary SAR suggests nitration of adamantyl phthalimidines has marginal effect on TNF-α activity but promotes anti-nitrite activity; thioamide congeners retain anti-nitrite activity but are less effective reducing TNF-α. Site-specific nitration and thioamidation provided phthalimidine 24, effecting an 88.5% drop in nitrite concurrent with only a 4% drop in TNF-α. Notable anti-angiogenesis activity was observed for 20, 21 and 22.


Assuntos
Inibidores da Angiogênese/farmacologia , Desenho de Fármacos , Fármacos Neuroprotetores/farmacologia , Nitritos/antagonistas & inibidores , Ftalimidas/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Nitritos/metabolismo , Ftalimidas/síntese química , Ftalimidas/química , Células RAW 264.7 , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismo
16.
Proc Natl Acad Sci U S A ; 110(31): 12703-8, 2013 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-23858438

RESUMO

Thalidomide and its analog, Lenalidomide, are in current use clinically for treatment of multiple myeloma, complications of leprosy and cancers. An additional analog, Pomalidomide, has recently been licensed for treatment of multiple myeloma, and is purported to be clinically more potent than either Thalidomide or Lenalidomide. Using a combination of zebrafish and chicken embryos together with in vitro assays we have determined the relative anti-inflammatory activity of each compound. We demonstrate that in vivo embryonic assays Pomalidomide is a significantly more potent anti-inflammatory agent than either Thalidomide or Lenalidomide. We tested the effect of Pomalidomide and Lenalidomide on angiogenesis, teratogenesis, and neurite outgrowth, known detrimental effects of Thalidomide. We found that Pomalidomide, displays a high degree of cell specificity, and has no detectable teratogenic, antiangiogenic or neurotoxic effects at potent anti-inflammatory concentrations. This is in marked contrast to Thalidomide and Lenalidomide, which had detrimental effects on blood vessels, nerves, and embryonic development at anti-inflammatory concentrations. This work has implications for Pomalidomide as a treatment for conditions Thalidomide and Lenalidomide treat currently.


Assuntos
Inibidores da Angiogênese/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Neuritos/metabolismo , Neurotoxinas , Teratogênicos , Talidomida/análogos & derivados , Peixe-Zebra/embriologia , Animais , Embrião de Galinha , Galinhas , Lenalidomida , Especificidade da Espécie , Talidomida/farmacologia
17.
Birth Defects Res C Embryo Today ; 105(2): 140-56, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26043938

RESUMO

Nearly 60 years ago thalidomide was prescribed to treat morning sickness in pregnant women. What followed was the biggest man-made medical disaster ever, where over 10,000 children were born with a range of severe and debilitating malformations. Despite this, the drug is now used successfully to treat a range of adult conditions, including multiple myeloma and complications of leprosy. Tragically, a new generation of thalidomide damaged children has been identified in Brazil. Yet, how thalidomide caused its devastating effects in the forming embryo remains unclear. However, studies in the past few years have greatly enhanced our understanding of the molecular mechanisms the drug. This review will look at the history of the drug, and the range and type of damage the drug caused, and outline the mechanisms of action the drug uses including recent molecular advances and new findings. Some of the remaining challenges facing thalidomide biologists are also discussed.


Assuntos
Anormalidades Induzidas por Medicamentos/história , Teratogênese/efeitos dos fármacos , Teratogênicos/farmacologia , Talidomida/efeitos adversos , Anormalidades Induzidas por Medicamentos/etiologia , Adulto , Animais , Feminino , História do Século XX , História do Século XXI , Humanos , Masculino , Gravidez
18.
Nat Genet ; 37(4): 373-81, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15735646

RESUMO

Autosomal dominant mutations in the gene encoding the basic helix-loop-helix transcription factor Twist1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome. The molecular mechanism underlying these phenotypes is poorly understood. We show that ectopic expression of the related basic helix-loop-helix factor Hand2 phenocopies Twist1 loss of function in the limb and that the two factors have a gene dosage-dependent antagonistic interaction. Dimerization partner choice by Twist1 and Hand2 can be modulated by protein kinase A- and protein phosphatase 2A-regulated phosphorylation of conserved helix I residues. Notably, multiple Twist1 mutations associated with Saethre-Chotzen syndrome alter protein kinase A-mediated phosphorylation of Twist1, suggesting that misregulation of Twist1 dimerization through either stoichiometric or post-translational mechanisms underlies phenotypes of individuals with Saethre-Chotzen syndrome.


Assuntos
Acrocefalossindactilia/metabolismo , Sequências Hélice-Alça-Hélice , Membro Posterior/anormalidades , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Acrocefalossindactilia/genética , Acrocefalossindactilia/patologia , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Embrião de Galinha/virologia , Galinhas , Sequência Conservada , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Dimerização , Humanos , Rim/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação/genética , Proteínas Nucleares/genética , Fenótipo , Fosfoproteínas Fosfatases/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2 , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Proteína 1 Relacionada a Twist , Proteínas de Peixe-Zebra
19.
Methods Mol Biol ; 2753: 251-260, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38285343

RESUMO

Pre-clinical trials are an essential step that underpins the drug discovery, development, and safety process. During this process, animal testing is performed to determine the safety of new compounds and any potential adverse effects. Developmental toxicity tests are carried out to verify whether the drug has potential to cause congenital anomalies to the developing embryo/fetus. Chicken embryos are very useful for these purposes and present several advantages, such as low cost of production and housing, easy handling and manipulation, and rapid development in addition to sharing similarities to the human embryo at molecular, cellular, and anatomical levels. In this chapter, we bring methods for using the chicken embryo model for testing the teratogenic effects of drugs and assessing the main outcomes of them.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Teratogênese , Embrião de Galinha , Animais , Humanos , Galinhas , Descoberta de Drogas , Embrião de Mamíferos
20.
J Anat ; 223(1): 1-13, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23678942

RESUMO

The developing chick limb has the remarkable ability to regulate for the loss of large amounts of mesenchyme and maintain a normal limb pattern in early (Hamburger and Hamilton Stage 19; E3) limbs. How the limb can regulate for tissue loss and why this ability is lost as development proceeds (after Hamburger and Hamilton Stage 21; E3.5) is unclear. We have investigated the origins of cells involved in regulative processes and, for the first time, the molecular changes occurring, and find striking differences between developmental time points just 0.5 days apart. We demonstrate that subtle changes in cell dispersal and cell proliferation occur in HH St21 limbs but not in HH St19 limbs and also demonstrate that there is no net replacement of removed tissue at either HH St21 or St19. We further show that changes in the Fgf8/Shh/Bmp4/Gremlin signaling pathway together with the appearance of distal Hox gene activation coincide with the limbs' ability to regulate for large tissue loss. We also demonstrate that following small tissue loss, limbs can regulate for missing tissue to produce normal pattern with no net replacement of missing tissue, as seen in limbs following large tissue loss. Our results indicate the regulative ability of the limb is not due to changes in cell proliferation, cell lineage nor replacement of the missing tissue - regulative ability is reliant upon the signaling environment remaining.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Botões de Extremidades/embriologia , Mesoderma/embriologia , Animais , Proteína Morfogenética Óssea 4/fisiologia , Proliferação de Células , Embrião de Galinha , Fator 8 de Crescimento de Fibroblasto/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox/fisiologia , Proteínas Hedgehog/fisiologia , Mesoderma/fisiologia , Organogênese/fisiologia , Transdução de Sinais/fisiologia
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