RESUMO
Nephronophthisis-related ciliopathies (NPHP-RC) are recessive diseases characterized by renal dysplasia or degeneration. We here identify mutations of DCDC2 as causing a renal-hepatic ciliopathy. DCDC2 localizes to the ciliary axoneme and to mitotic spindle fibers in a cell-cycle-dependent manner. Knockdown of Dcdc2 in IMCD3 cells disrupts ciliogenesis, which is rescued by wild-type (WT) human DCDC2, but not by constructs that reflect human mutations. We show that DCDC2 interacts with DVL and DCDC2 overexpression inhibits ß-catenin-dependent Wnt signaling in an effect additive to Wnt inhibitors. Mutations detected in human NPHP-RC lack these effects. A Wnt inhibitor likewise restores ciliogenesis in 3D IMCD3 cultures, emphasizing the importance of Wnt signaling for renal tubulogenesis. Knockdown of dcdc2 in zebrafish recapitulates NPHP-RC phenotypes, including renal cysts and hydrocephalus, which is rescued by a Wnt inhibitor and by WT, but not by mutant, DCDC2. We thus demonstrate a central role of Wnt signaling in the pathogenesis of NPHP-RC, suggesting an avenue for potential treatment of NPHP-RC.
Assuntos
Doenças Renais Císticas/genética , Proteínas Associadas aos Microtúbulos/genética , Via de Sinalização Wnt/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cílios/genética , Cílios/patologia , Biologia Computacional , Proteínas Desgrenhadas , Éxons , Células HEK293 , Humanos , Rim/patologia , Camundongos , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Células NIH 3T3 , Fenótipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Peixe-Zebra/genética , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismoRESUMO
Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.
Assuntos
Autofagia/genética , Cisteína Endopeptidases/genética , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Vacúolos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Encéfalo/patologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Cães , Dados de Sequência Molecular , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/veterinária , Vacúolos/genética , Peixe-ZebraRESUMO
BACKGROUND: DYX1C1 (DNAAF4) and DCDC2 are two of the most replicated dyslexia candidate genes in genetic studies. They both have demonstrated roles in neuronal migration, in cilia growth and function and they both are cytoskeletal interactors. In addition, they both have been characterized as ciliopathy genes. However, their exact molecular functions are still incompletely described. Based on these known roles, we asked whether DYX1C1 and DCDC2 interact on the genetic and the protein level. RESULTS: Here, we report the physical protein-protein interaction of DYX1C1 and DCDC2 as well as their respective interactions with the centrosomal protein CPAP (CENPJ) on exogenous and endogenous levels in different cell models including brain organoids. In addition, we show a synergistic genetic interaction between dyx1c1 and dcdc2b in zebrafish exacerbating the ciliary phenotype. Finally, we show a mutual effect on transcriptional regulation among DYX1C1 and DCDC2 in a cellular model. CONCLUSIONS: In summary, we describe the physical and functional interaction between the two genes DYX1C1 and DCDC2. These results contribute to the growing understanding of the molecular roles of DYX1C1 and DCDC2 and set the stage for future functional studies.
Assuntos
Cílios , Chaperonas Moleculares , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Movimento Celular/genética , Regulação da Expressão Gênica , Fenótipo , Peixe-Zebra/genética , Chaperonas Moleculares/genética , Proteínas de Peixe-Zebra/genética , Cílios/patologiaRESUMO
Glioblastoma multiforme (GBM) affects individuals above 65 years of age and has low median survival rate. Due to limited treatment options, lack of effective diagnosis, and palliative care, there is an urgent need to develop new therapeutic strategies to combat GBM. This review provides an overview of the current clinical trial scenario with a special focus on new targets, repurposed drugs, and technologies in the field of GBM. The use of technological advances and artificial intelligence in diagnosis and imaging is also discussed. In addition, this review also highlights the need to design a dynamic palliative care strategy for end-of-life management of patients with GBM.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Inteligência Artificial , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/terapia , Glioblastoma/tratamento farmacológico , Glioblastoma/terapia , Humanos , Cuidados Paliativos , Taxa de SobrevidaRESUMO
Corticotropin-releasing hormone (CRH) plays a central role in the physiological regulation of the hypothalamus-pituitary-adrenal/interrenal axis mediating endocrine, behavioral, autonomic, and immune responses to stress. Despite the wealth of knowledge about the physiological roles of CRH, the genetic mechanisms by which CRH neurons arise during development are poorly understood. As a first step toward analyzing the molecular and genetic pathways involved in CRH lineage specification, we describe the developmental distribution of CRH neurons in the embryonic zebrafish, a model organism for functional genomics and developmental biology. We searched available zebrafish expressed sequence tag (EST) databases for CRH-like sequences and identified one EST that contained the complete zebrafish CRH open reading frame (ORF). The CRH precursor sequence contained a signal peptide, the CRH peptide, and a cryptic peptide with a conserved sequence motif. RT-PCR analysis showed crh expression in a wide range of adult tissues as well as during embryonic and larval stages. By whole-mount in situ hybridization histochemistry, discrete crh-expressing cell clusters were found in different parts of the embryonic zebrafish brain, including telencephalon, preoptic region, hypothalamus, posterior tuberculum, thalamus, epiphysis, midbrain tegmentum, and rostral hindbrain and in the neural retina. The localization of crh mRNA within the preoptic region is consistent with the central role of CRH in the teleost stress response through activation of the hypothalamic-pituitary-interrenal axis. The widespread distribution of CRH-synthesizing cells outside the preoptic region suggests additional functions of CRH in the embryonic zebrafish brain.
Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra/fisiologia , Animais , Sequência de Bases , Química Encefálica/fisiologia , Hormônio Liberador da Corticotropina/genética , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The phenotype-based drug discovery (PDD) approach is re-emerging as an alternative platform for drug discovery. This review provides an overview of the various model systems and technical advances in imaging and image analyses that strengthen the PDD platform. In PDD screens, compounds of therapeutic value are identified based on the phenotypic perturbations produced irrespective of target(s) or mechanism of action. In this article, examples of phenotypic changes that can be detected and quantified with relative ease in a cell-based setup are discussed. In addition, a higher order of PDD screening setup using small animal models is also explored. As PDD screens integrate physiology and multiple signaling mechanisms during the screening process, the identified hits have higher biomedical applicability. Taken together, this review highlights the advantages gained by adopting a PDD approach in drug discovery. Such a PDD platform can complement target-based systems that are currently in practice to accelerate drug discovery.
Assuntos
Células Cultivadas , Modelos Animais de Doenças , Descoberta de Drogas/métodos , Fenótipo , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Modelos AnimaisRESUMO
The potential of multifunctional wound heal biomaterial relies on the optimal content of therapeutic constituents as well as the desirable physical, chemical, and biological properties to accelerate the healing process. Formulating biomaterials such as amnion or collagen based scaffolds with natural products offer an affordable strategy to develop dressing material with high efficiency in healing wounds. Using image based phenotyping and quantification, we screened natural product derived bioactive compounds for modulators of types I and III collagen production from human foreskin derived fibroblast cells. The identified hit was then formulated with amnion to develop a biomaterial, and its biophysical properties, in vitro and in vivo effects were characterized. In addition, we performed functional profiling analyses by PCR array to understand the effect of individual components of these materials on various genes such as inflammatory mediators including chemokines and cytokines, growth factors, fibroblast stimulating markers for collagen secretion, matrix metalloproteinases, etc., associated with wound healing. FACS based cell cycle analyses were carried out to evaluate the potential of biomaterials for induction of proliferation of fibroblasts. Western blot analyses was done to examine the effect of biomaterial on collagen synthesis by cells and compared to cells grown in the presence of growth factors. This work demonstrated an uncomplicated way of identifying components that synergistically promote healing. Besides, we demonstrated that modulating local wound environment using biomaterials with bioactive compounds could enhance healing. This study finds that the developed biomaterials offer immense scope for healing wounds by means of their skin regenerative features such as anti-inflammatory, fibroblast stimulation for collagen secretion as well as inhibition of enzymes and markers impeding the healing, hydrodynamic properties complemented with other features including non-toxicity, biocompatibility, and safety.
RESUMO
Stem cells display a fundamentally different mechanism of proliferation control when compared to somatic cells. Uncovering these mechanisms would maximize the impact in drug discovery with a higher translational applicability. The unbiased approach used in phenotype-based drug discovery (PDD) programs can offer a unique opportunity to identify such novel biological phenomenon. Here, we describe an integrated phenotypic screening approach, employing a combination of in vitro and in vivo PDD models to identify a small molecule increasing stem cell proliferation. We demonstrate that a combination of both in vitro and in vivo screening models improves hit identification and reproducibility of effects across various PDD models. Using cell viability and colony size phenotype measurement we characterize the structure activity relationship of the lead molecule, and identify that the small molecule inhibits phosphorylation of ERK2 and promotes stem cell proliferation. This study demonstrates a PDD approach that employs combinatorial models to identify compounds promoting stem cell proliferation.
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The cellular cytoskeleton forms the primary basis through which a cell governs the changes in size, shape, migration, proliferation, and forms the primary means through which the cells respond to their environment. Indeed, cell and tissue morphologies are used routinely not only to grade tumors but also in various high-content screening methods with an aim to identify new small molecules with therapeutic potential. This study examines the expression of various cytoskeleton regulators in glioblastoma multiforme (GBM). GBM is a very aggressive disease with a low life expectancy even after chemo- and radiotherapy. Cancer cells of GBM are notorious for their invasiveness, ability to develop resistance to chemo- and radiotherapy, and to form secondary site tumors. This study aims to gain insight into cytoskeleton regulators in GBM cells and to understand the effect of various oncology drugs, including temozolomide, on cytoskeleton regulators. We compare the expression of various cytoskeleton regulators in GBM-derived tumor and normal tissue, CD133-postive and -negative cells from GBM and neural cells, and GBM stem-like and differentiated cells. In addition, the correlation between the expression of cytoskeleton regulators with the clinical outcome was examined to identify genes associated with longer patient survival. This was followed by a small molecule screening with US Food and Drug Administration (FDA)-approved oncology drugs, and its effect on cellular cytoskeleton was compared to treatment with temozolomide. This study identifies various groups of cytoskeletal regulators that have an important effect on patient survival and tumor development. Importantly, this work highlights the advantage of using cytoskeleton regulators as biomarkers for assessing prognosis and treatment design for GBM.
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In this paper, we describe a method to obtain a relatively pure mitochondrial and microsomal fractions by subcellular fractionation of human hepatoma cell line C3A using sucrose as the hypoosmotic medium. The cells were subjected to osmotic stress with sucrose and homogenized. Osmolarity was then restored to the cells and the organelles were separated by density gradient centrifugation. The protein profiles were examined by SDS-PAGE and the purity was analysed by marker enzymes and Western blotting. Our results indicate a good separation of mitochondrial and microsomal fractions from human hepatoma C3A cells.
Assuntos
Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Sacarose/farmacologia , Western Blotting , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Redutases do Citocromo/metabolismo , Eletroforese em Gel de Poliacrilamida , Glutamato Desidrogenase/metabolismo , Humanos , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Microssomos/metabolismo , Microssomos Hepáticos/metabolismo , Mitocôndrias/metabolismo , Frações Subcelulares/metabolismoRESUMO
A novel approach to incorporate the macrocyclic rings onto the privileged substructure, i.e., tetrahydroquinoline scaffold, is developed. The presence of an amino acid-derived moiety in the macrocyclic skeleton provides an opportunity to modulate the nature of the chiral side chain. Further, evaluation in a zebrafish screen identified three active small molecules (2.5b, 3.2d, and 4.2) as antiangiogenesis agents at 2.5 µM.
RESUMO
DYX1C1, a susceptibility gene for dyslexia, encodes a tetratricopeptide repeat domain containing protein that has been implicated in neuronal migration in rodent models. The developmental role of this gene remains unexplored. To understand the biological function(s) of zebrafish dyx1c1 during embryonic development, we cloned the zebrafish dyx1c1 and used morpholino-based knockdown strategy. Quantitative real-time PCR analysis revealed the presence of dyx1c1 transcripts in embryos, early larval stages and in a wide range of adult tissues. Using mRNA in situ hybridization, we show here that dyx1c1 is expressed in many ciliated tissues in zebrafish. Inhibition of dyx1c1 produced pleiotropic phenotypes characteristically associated with cilia defects such as body curvature, hydrocephalus, situs inversus and kidney cysts. We also demonstrate that in dyx1c1 morphants, cilia length is reduced in several organs including Kupffer's vesicle, pronephros, spinal canal and olfactory placode. Furthermore, electron microscopic analysis of cilia in dyx1c1 morphants revealed loss of both outer (ODA) and inner dynein arms (IDA) that have been shown to be required for cilia motility. Considering all these results, we propose an essential role for dyx1c1 in cilia growth and function.
Assuntos
Cílios/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cílios/patologia , Clonagem Molecular , Dineínas/metabolismo , Embrião não Mamífero/anormalidades , Embrião não Mamífero/patologia , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células de Kupffer/patologia , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Morfolinos/genética , Especificidade de Órgãos , Pronefro/metabolismo , Pronefro/patologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Canal Medular/patologia , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
A practical and modular approach to obtain a diverse set of 14-membered macrocyclic compounds from carbohydrates is developed that utilizes functional groups at C-1 and C-5. The evaluation of this toolbox in various zebrafish assays led to the identification of 2.7f as an antiangiogenesis agent.
Assuntos
Inibidores da Angiogênese/síntese química , Carboidratos/química , Compostos Macrocíclicos/síntese química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Desenho de Fármacos , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Estrutura Molecular , Peixe-ZebraRESUMO
A highly practical and modular synthesis to obtain a diverse 14-membered ring-based macrocyclic toolbox is achieved. These compounds were further tested in zebrafish assays related to early embryonic development, angiogenesis, and neurogenesis, respectively. 1.4c was identified as an antiangiogenesis agent.
Assuntos
Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/farmacologia , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/farmacologia , Inibidores da Angiogênese/química , Animais , Desenho de Fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Compostos Macrocíclicos/química , Estrutura Molecular , Morfogênese/efeitos dos fármacos , Neovascularização Patológica , Gravidez , Relação Estrutura-Atividade , Peixe-Zebra/embriologiaRESUMO
The identification of normal and cancerous stem cells and the recent advances made in isolation and culture of stem cells have rapidly gained attention in the field of drug discovery and regenerative medicine. The prospect of performing screens aimed at proliferation, directed differentiation, and toxicity and efficacy studies using stem cells offers a reliable platform for the drug discovery process. Advances made in the generation of induced pluripotent stem cells from normal or diseased tissue serves as a platform to perform drug screens aimed at developing cell-based therapies against conditions like Parkinson's disease and diabetes. This review discusses the application of stem cells and cancer stem cells in drug screening and their role in complementing, reducing, and replacing animal testing. In addition to this, target identification and major advances in the field of personalized medicine using induced pluripotent cells are also discussed.
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4-Nonylphenol (4-NP), an estrogen mimicking compound is produced by biodegradation of alkylethoxylates. It is well established that 4-NP can affect the development of aquatic animals by disrupting the endocrine signals. Here we show for the first time in zebrafish that 4-NP does not only target the neuroendocrine system but also the notochord and the muscle. The notochord malformation was first evident as distortions at 24hourspostfertilization (hpf) which within 24h appeared as kinks and herniations. The notochord phenotype was accompanied by reduced motility and impaired swimming behavior. Whole-mount in situ hybridization using chordamesoderm markers and electron microscopic analysis showed failure in the notochord differentiation and disruption of the perinotochordal basement membrane. Late larval stages of 4-NP treated embryos displayed abnormal mineralization, vertebral curvature, fusion of vertebral bodies and abnormal extension of haemal arches. The muscle structure and the maximal active force in isolated muscle preparations were similar between 4-NP exposed and of control embryos, suggesting that 4-NP did not induce major changes in striated muscle function. However, repeated electrical stimulation (>40Hz) of the 4-NP exposed larvae revealed an impaired relaxation between stimuli, possibly reflecting an alteration in the relaxant mechanisms (e.g. in cellular Ca(2+) removal) which could explain the abnormal swimming pattern exhibited by 4-NP exposed larvae. Additionally, we demonstrate that the expression levels of the stress hormone, corticotropin releasing hormonewere elevated in the brain following 4-NP treatment. We also observed a significant decrease in the transcript levels of luteinizing hormone b at early larval stages. Collectively, our results show that 4-NP is able to disrupt the notochord morphogenesis, muscle function and the neuroendocrine system. These data suggest that 4-NP enduringly affects the embryonic development in zebrafish and that this compound might exert these deleterious effects through diverse signaling pathways.
Assuntos
Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Organogênese/efeitos dos fármacos , Fenóis/toxicidade , Peixe-Zebra/embriologia , Animais , Apoptose/efeitos dos fármacos , Embrião não Mamífero/anormalidades , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica de Transmissão , Músculo Esquelético/anormalidades , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/embriologia , Músculo Esquelético/ultraestrutura , Sistemas Neurossecretores/anormalidades , Sistemas Neurossecretores/efeitos dos fármacos , Sistemas Neurossecretores/embriologia , Sistemas Neurossecretores/ultraestrutura , Notocorda/anormalidades , Notocorda/efeitos dos fármacos , Notocorda/embriologia , Notocorda/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real , Peixe-Zebra/anormalidades , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
The nuclear receptors encompass a group of regulatory proteins involved in a number of physiological processes. The estrogen receptors (ERs), of which one alpha and one beta form exist in mammals function as transcription factors in response to 17beta-estradiol (E2). In zebrafish there are three gene products of estrogen receptors and they are denoted esr1 (ERalpha), esr2a (ERbeta2) and esr2b (ERbeta1). Total RNA of zebrafish early life stages (<3, 6, 12, 24, 48, 72, 96 and 120 hours post fertilization) and of adult fish (liver, intestine, eye, heart, brain, ovary, testis, gill, swim bladder and kidney) were isolated following in vivo exposures. Using specific primers for each of the three zebrafish ERs the expression levels were quantified using real time PCR methodology. It was shown that in absence of exposure all three estrogen receptors were expressed in adult fish. The levels of expression of two of these three ER genes, the esr1 and esr2a were altered in organs such as liver, intestine, brain and testis in response to ligand (E2, diethylstilbestrol or 4-nonylphenol). During embryogenesis two of the three receptor genes, esr1 and esr2b were expressed, and in presence of ligand the mRNA levels of these two genes increased. The conclusions are i) estrogen receptor genes are expressed during early development ii) altered expression of esr genes in response to ligand is dependent on the cellular context; iii) the estrogenic ligand 4-nonylphenol, a manufactured compound commonly found in sewage of water treatment plants, acts as an agonist of the estrogen receptor during development and has both agonist and antagonist properties in tissues of adult fish. This knowledge of esr gene function in development and in adult life will help to understand mechanisms of interfering mimicking endocrine chemicals in vivo.