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1.
Cell Prolif ; 53(10): e12863, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32871045

RESUMO

OBJECTIVES: Immunodeficient mice injected with human cancer cell lines have been used for human oncology studies and anti-cancer drug trials for several decades. However, rodents are not ideal species for modelling human cancer because rodents are physiologically dissimilar to humans. Therefore, anti-tumour drugs tested effective in rodents have a failure rate of 90% or higher in phase III clinical trials. Pigs are similar to humans in size, anatomy, physiology and drug metabolism rate, rendering them a desirable pre-clinical animal model for assessing anti-cancer drugs. However, xenogeneic immune rejection is a major barrier to the use of pigs as hosts for human tumours. Interleukin (IL)-2 receptor γ (IL2RG), a common signalling subunit for multiple immune cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, is required for proper lymphoid development. MATERIALS AND METHODS: IL2RG-/Y pigs were generated by CRISPR/Cas9 technology, and examined for immunodeficiency and ability to support human oncogenesis. RESULTS: Compared to age-matched wild-type pigs, IL2RG-/Y pigs exhibited a severely impaired immune system as shown by lymphopenia, lymphoid organ atrophy, poor immunoglobulin function, and T- and NK-cell deficiency. Human melanoma Mel888 cells generated tumours in IL2RG-/Y pigs but not in wild-type littermates. The human tumours grew faster in IL2RG-/Y pigs than in nude mice. CONCLUSIONS: Our results indicate that these pigs are promising hosts for modelling human cancer in vivo, which may aid in the discovery and development of anti-cancer drugs.


Assuntos
Sistemas CRISPR-Cas/genética , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Neoplasias Cutâneas/patologia , Animais , Animais Geneticamente Modificados/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Edição de Genes , Humanos , Sistema Imunitário/metabolismo , Subunidade gama Comum de Receptores de Interleucina/antagonistas & inibidores , Subunidade gama Comum de Receptores de Interleucina/genética , Linfopenia/patologia , Melanoma/metabolismo , Melanoma/patologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/mortalidade , Taxa de Sobrevida , Suínos , Porco Miniatura , Transplante Heterólogo
2.
PLoS One ; 15(7): e0236788, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735629

RESUMO

This study aimed to establish and reproduce transgenic pigs expressing human growth hormone (hGH) in their milk. We also aimed to purify hGH from the milk, to characterize the purified protein, and to assess the potential of our model for mass production of therapeutic proteins using transgenic techniques. Using ~15.5 L transgenic pig milk, we obtained proteins with ≥ 99% purity after three pre-treatments and five column chromatography steps. To confirm the biosimilarity of our milk-derived purified recombinant hGH (CGH942) with commercially available somatropin (Genotropin), we performed spectroscopy, structural, and biological analyses. We observed no difference between the purified protein and Genotropin samples. Furthermore, rat models were used to assess growth promotion potential. Our results indicate that CGH942 promotes growth, by increasing bone development and body weight. Toxicity assessments revealed no abnormal findings after 4 weeks of continuous administration and 2 weeks of recovery. The no-observed-adverse-effect level for both males and females was determined to be 0.6 mg/kg/day. Thus, no toxicological differences were observed between commercially available somatropin and CGH942 obtained from transgenic pig milk. In conclusion, we describe a transgenic technique using pigs, providing a new platform to produce human therapeutic proteins.


Assuntos
Animais Geneticamente Modificados/metabolismo , Hormônio do Crescimento Humano , Proteínas Recombinantes , Animais , Cromatografia de Afinidade , Feminino , Técnicas de Transferência de Genes , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/metabolismo , Humanos , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Suínos
3.
PLoS One ; 15(8): e0237775, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813739

RESUMO

Nile tilapia, Oreochromis niloticus is the third most commonly farmed finfish species in the world, accounting for nearly 5% of global aquaculture production. In the past few decades much of the success of this species has been attributed to the development and distribution of Genetically Improved Farmed Tilapia (GIFT). Despite the increasing availability of GIFT, the productivity of small-scale farming remains highly variable, particularly in developing nations. Commercial fish-feed pellets can increase fish farm productivity; however, many small-scale farmers rely on other means of feeding fish due to the high cost and limited availability of commercial fish feed pellets. Therefore, understanding how locally-sourced feeds affect the production of GIFT is an important step towards improving feeding practices, particularly for farmers with low financial capital. This study used stable isotope analysis (SIA) and 16S rRNA gene sequencing to compare the effects of a locally-sourced vegetable-based diet and commercial pellet-based diets on the relative condition, nutrient assimilation patterns and gastrointestinal microbiota of GIFT. GIFT fed a locally-sourced diet were smaller, and in a significantly poorer condition than those fed with commercial fish feeds. SIA showed no differences in dietary carbon between the two diets; however, δ13C, poor fish condition and the abundance of specific bacterial taxa (of such as Fusobacteria) were correlated. SIA revealed that GIFT fed locally-sourced diets that predominantly consisted of vegetables were significantly enriched in δ15N despite a perceived lack of dietary protein. This enrichment suggests that GIFT fed a locally-sourced diet may be supplementing their diet via cannibalism, a behaviour representative of poor farming practice. Overall this study highlights the need to increase the availability of suitable GIFT feeds in developing nations. The development a low-cost feed alternative could improve the success of small-scale GIFT farmers in PNG, increasing both food and income security within the region.


Assuntos
Ração Animal , Animais Geneticamente Modificados/metabolismo , Aquicultura/métodos , Ciclídeos/metabolismo , Microbioma Gastrointestinal/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/microbiologia , Aquicultura/economia , Aquicultura/organização & administração , Canibalismo , Ciclídeos/genética , Ciclídeos/microbiologia , DNA Bacteriano/isolamento & purificação , Suplementos Nutricionais/economia , Eficiência Organizacional/economia , Fazendas/economia , Fazendas/organização & administração , New South Wales , Nutrientes/metabolismo , RNA Ribossômico 16S/genética
4.
Sci Rep ; 10(1): 12378, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32704046

RESUMO

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne pathogen causing a febrile illness in humans, which can progress to hemorrhagic manifestations, multi-organ failure, and death. Current mouse models of CCHFV infection reliably succumb to virus challenge but vary in their ability to reflect signs of disease similar to humans. In this study, we established a signal transducer and activator of transcription 2 (STAT2) knockout hamster model to expand the repertoire of animal models of CCHFV pathogenesis that can be used for therapeutic development. These hamsters demonstrated a systemic and lethal disease in response to infection. Hallmarks of human disease were observed including petechial rash, blood coagulation dysfunction, and various biochemistry and blood cell count abnormalities. Furthermore, we also demonstrated the utility of this model for anti-CCHFV therapeutic evaluation. The STAT2 knock-out hamster model of CCHFV infection may provide some further insights into clinical disease, viral pathogenesis, and pave the way for testing of potential drug and vaccine candidates.


Assuntos
Animais Geneticamente Modificados , Modelos Animais de Doenças , Vírus da Febre Hemorrágica da Crimeia-Congo/metabolismo , Febre Hemorrágica da Crimeia , Fator de Transcrição STAT2/deficiência , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Animais Geneticamente Modificados/virologia , Linhagem Celular , Cricetinae , Feminino , Técnicas de Inativação de Genes , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Febre Hemorrágica da Crimeia/genética , Febre Hemorrágica da Crimeia/metabolismo , Febre Hemorrágica da Crimeia/patologia , Masculino , Fator de Transcrição STAT2/metabolismo
5.
PLoS One ; 15(6): e0232308, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32530962

RESUMO

Zebrafish have the ability to regenerate damaged cells and tissues by activating quiescent stem and progenitor cells or reprogramming differentiated cells into regeneration-competent precursors. Proliferation among the cells that will functionally restore injured tissues is a fundamental biological process underlying regeneration. Midkine-a is a cytokine growth factor, whose expression is strongly induced by injury in a variety of tissues across a range of vertebrate classes. Using a zebrafish Midkine-a loss of function mutant, we evaluated regeneration of caudal fin, extraocular muscle and retinal neurons to investigate the function of Midkine-a during epimorphic regeneration. In wildtype zebrafish, injury among these tissues induces robust proliferation and rapid regeneration. In Midkine-a mutants, the initial proliferation in each of these tissues is significantly diminished or absent. Regeneration of the caudal fin and extraocular muscle is delayed; regeneration of the retina is nearly completely absent. These data demonstrate that Midkine-a is universally required in the signaling pathways that convert tissue injury into the initial burst of cell proliferation. Further, these data highlight differences in the molecular mechanisms that regulate epimorphic regeneration in zebrafish.


Assuntos
Midkina/metabolismo , Regeneração/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Nadadeiras de Animais/fisiologia , Animais , Animais Geneticamente Modificados/metabolismo , Diferenciação Celular , Proliferação de Células , Midkina/genética , Mutagênese , Neuroglia/citologia , Neuroglia/metabolismo , Músculos Oculomotores/fisiologia , Neurônios Retinianos/fisiologia , Proteínas de Peixe-Zebra/genética
6.
Am J Physiol Cell Physiol ; 319(2): C359-C370, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32520610

RESUMO

SLC4A11 is the only member of the SLC4 family that transports protons rather than bicarbonate. SLC4A11 is expressed in corneal endothelial cells, and its mutation causes corneal endothelial dystrophy, although the mechanism of pathogenesis is unknown. We previously demonstrated that the magnitude of the H+ conductance (Gm) mediated by SLC4A11 is increased by rises in intracellular as well as extracellular pH (pHi and pHe). To better understand this feature and whether it is altered in disease, we studied the pH dependence of wild-type and mutant mouse Slc4a11 expressed in Xenopus oocytes. Using voltage-clamp circuitry in conjunction with a H+-selective microelectrode and a microinjector loaded with NaHCO3, we caused incremental rises in oocyte pHi and measured the effect on Gm. We find that the rise of Gm has a steeper pHi dependence at pHe =8.50 than at pHe =7.50. Data gathered at pHe =8.50 can be fit to the Hill equation enabling the calculation of a pK value that reports pHi dependence. We find that mutation of lysine residues that are close to the first transmembrane span (TM1) causes an alkaline shift in pK. Furthermore, two corneal-dystrophy-causing mutations close to the extracellular end of TM1, E399K and T401K (E368K and T370K in mouse), cause an acidic shift in pK, while a third mutation in the fourth intracellular loop, R804H (R774H in mouse), causes an alkaline shift in pK. This is the first description of determinants of SLC4A11 pH dependence and the first indication that a shift in pH dependence could modify disease expressivity in some cases of corneal dystrophy.


Assuntos
Proteínas de Transporte de Ânions/genética , Transporte Biológico/genética , Distrofias Hereditárias da Córnea/genética , Lisina/genética , Simportadores/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Bicarbonatos/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Modelos Animais de Doenças , Epitélio Posterior/metabolismo , Epitélio Posterior/patologia , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/genética , Lisina/metabolismo , Camundongos , Mutação/genética , Oócitos/metabolismo , Oócitos/patologia , Sódio , Xenopus/genética
7.
Gene ; 754: 144854, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32525045

RESUMO

Alzheimer's disease (AD) is one of the most common forms of neurodegenerative diseases. Aggregation of Aß42 and hyperphosphorylated tau are two major hallmarks of AD. Whether different forms of tau (soluble or hyperphosphorylated) or Aß are the main culprit in the events observed in AD is still under investigation. Here, we examined the effect of wild-type, prone to hyperphosphorylation and hyperphosphorylated tau, and also Aß42 peptide on the brain antioxidant defense system and two mitochondrial genes, Marf (homologous to human MFN2) and Drp1 involved in mitochondrial dynamics in transgenic Drosophila melanogaster. AD is an age associated disease. Therefore, the activity of antioxidant agents, CAT, SOD, and GSH levels and the mRNA levels of Marf and Drp1 were assessed in different time points of the flies lifespan. Reduction in cognitive function and antioxidant activity was observed in all transgenic flies at any time point. The most and the least effect on the eye phenotype was exerted by hyperphosphorylated tau and Aß42, respectively. In addition, the most remarkable alteration in Marf and Drp1 mRNA levels was observed in transgenic flies expressing hyperphosphorylated tau when pan neuronal expression of transgenes was applied. However, when the disease causing gene expression was confined to the mushroom body, Marf and Drp1 mRNA levels alteration was more prominent in tauWT and tauE14 transgenic flies, respectively. In conclusion, in spite of antioxidant deficiency caused by different types of tau and Aß42, it seems that tau exerts more toxic effect on the eye phenotype and mitochondrial genes regulation (Marf and Drp1). Moreover, different mechanisms seem to be involved in mitochondrial genes dysregulation when Aß or various forms of tau are expressed.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Animais Geneticamente Modificados/metabolismo , Drosophila melanogaster/metabolismo , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , RNA Mensageiro/metabolismo , Proteínas tau/metabolismo , Animais , Animais Geneticamente Modificados/genética , Encéfalo/metabolismo , Drosophila melanogaster/genética , Dinaminas/genética , GTP Fosfo-Hidrolases/genética , Regulação da Expressão Gênica , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Fosforilação , RNA Mensageiro/genética , Proteínas tau/genética
8.
Ecotoxicol Environ Saf ; 201: 110808, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32516676

RESUMO

Cyprodinil is a broad-spectrum pyrimidine amine fungicide that has been reportedly used worldwide. However, toxicity studies of cyprodinil on aquatic organisms, specifically zebrafish (Danio rerio), are lacking. In our present study, we predicted cyprodinil binding to the aryl hydrocarbon receptor (AhR) by using molecular docking simulation. Then, we used recombinant HepG2 cells and Tg(cyp1a1-12DRE:egfp) transgenic zebrafish to further assess the AhR agonistic activity of cyprodinil. Besides, the significant upregulation of cyp1a1 further verified that statement. Moreover, we found that zebrafish exposure to cyprodinil induced developmental toxicity in the larvae, particularly during cardiac development. The expression levels of cardiac development-related genes, namely tbx5, nkx2.5, gata4, and tnnt2, were markedly altered, which might cause the adverse effects of cyprodinil on cardiac function and development. In summary, we found that cyprodinil, as an AhR agonist, induced development toxicity in zebrafish larvae, especially on cardiac. Data here can assess the potential effects on organisms in the aquatic environment and promote the regulation and safe use of cyprodinil.


Assuntos
Animais Geneticamente Modificados/metabolismo , Larva/efeitos dos fármacos , Pirimidinas/toxicidade , Receptores de Hidrocarboneto Arílico/agonistas , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Fungicidas Industriais/metabolismo , Coração/efeitos dos fármacos , Coração/embriologia , Células Hep G2 , Humanos , Larva/genética , Larva/metabolismo , Simulação de Acoplamento Molecular , Organogênese/efeitos dos fármacos , Organogênese/genética , Ligação Proteica , Peixe-Zebra/genética
9.
Cell ; 181(3): 487-491, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32234518

RESUMO

This year's Gairdner Foundation Award for Biomedical Research goes to Roel Nusse for his pioneering work on the Wnt signaling pathway and its many roles in development, cancer, and stem cells.


Assuntos
Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Bibliografias como Assunto , Comunicação Celular , Drosophila , Proteínas de Drosophila/metabolismo , Feminino , Humanos , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Proteína Wnt1/metabolismo
10.
Insect Biochem Mol Biol ; 120: 103360, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32126276

RESUMO

Ammonia is one of the principal kairomones originating from human and other animal emanations and in that context, plays an essential role in the host-seeking behaviors of the malaria vector mosquito Anopheles gambiae. Nevertheless, despite its importance in directing host-seeking, the mechanisms underlying ammonia detection in the mosquito olfactory system remains largely unknown. In addition to ongoing efforts to identify and characterize the molecular receptors that underlie ammonia sensitivity, previous studies have revealed a prominent role for ammonium transporters (Amt) in modulating antennal and behavioral responses in Drosophila melanogaster and An. gambiae. In the former, localization of DmAmt in antennal sensilla to auxiliary cells surrounding the ammonia sensory neurons led to the hypothesis that its role was to clear excess ammonium ions in the sensillar lymph. In the latter, RT-PCR and heterologous expression have been used to examine the expression and functional characteristics of the An. gambiae ammonium transporter, AgAmt. We now employ advanced transgenic tools to comprehensively examine AgAmt spatial localization across the peripheral chemosensory appendages in larvae and adult female An. gambiae. In the larval antennae, AgAmt appears localized in both neuronal and auxiliary cells. In contrast to D. melanogaster, in the adult antennae, AgAmt-derived signals are observed in both non-neuronal auxiliary cells and in sensory neurons in ammonia-responsive basiconic and coeloconic sensilla. In the maxillary palps, labella, and tarsi, AgAmt appears restricted to sensory neurons. We have also characterized the responses to ammonia of adult antennal coeloconic sensilla and maxillary palp capitate pegs revealing a correlation between sensillar AgAmt expression and ammonia sensitivity. Taken together, these data suggest that AgAmt may play heterogeneous roles in the adult and larval chemosensory apparatus and potentially broad utility as a supra-receptor target in mosquito control.


Assuntos
Compostos de Amônio/metabolismo , Anopheles/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Insetos/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Anopheles/crescimento & desenvolvimento , Anopheles/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Malária , Mosquitos Vetores/genética , Mosquitos Vetores/crescimento & desenvolvimento , Mosquitos Vetores/metabolismo , Sensilas/metabolismo
11.
J Immunol ; 204(7): 1929-1942, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32111733

RESUMO

The bacteria LPS is one of the leading endotoxins responsible for sepsis; its sensing pathway-induced pyroptosis plays an important role in innate immunity. However, excessive pyroptosis might cause immunological diseases, even multiple organ failure and death by undefined mechanisms. Given that the development of acute kidney injury (AKI) in patients with sepsis causes significant morbidity and mortality, the mechanism of pyroptosis in regulating septic AKI remains unknown. In this study, we establish a zebrafish crispant in vivo analysis model and reveal that both caspy2 and gasdermin Eb (GSDMEb) contribute to lethal LPS-induced septic shock. Meanwhile, the in vitro analysis reveals that caspy2 activation can specifically cleave GSDMEb to release its N terminus to mediate pyroptosis, which functions as GSDMD in mammals. Interestingly, we establish an in vivo propidium iodide-staining method and reveal that the caspy2-GSDMEb signaling cascade is essential for enhancing renal tubular damage during lethal LPS-induced septic shock, whereas administration of the zebrafish-specific GSDMEb-derived peptide inhibitor Ac-FEID-CMK can attenuate mortality and septic AKI in vivo. Moreover, we confirm that either caspase-11 or GSDMD deficiency decreases both inflammatory cytokines and kidney dysfunction enzyme release and prolongs survival in a murine model of septic shock. Taken together, these findings demonstrate an evolutionary executor for pyroptosis in zebrafish and reveal that the pyroptosis of renal tubular cells is a major cause of septic AKI, and also provide an ideal in vivo screening model for potential antisepsis therapeutic strategies.


Assuntos
Lesão Renal Aguda/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Piroptose/fisiologia , Sepse/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Caspases/metabolismo , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Túbulos Renais/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Insuficiência de Múltiplos Órgãos/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
12.
Insect Mol Biol ; 29(4): 353-362, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32086963

RESUMO

RNA interference (RNAi), one of the strategies that organisms use to defend against invading viruses, is an important tool for functional genomic analysis. In insects, the efficacy of RNAi varies amongst taxa. Lepidopteran insects are, in large part, recalcitrant to RNAi. The overall goal of this study is to overcome such insensitivity in lepidopterans to RNAi. We hypothesize that over-expression of core RNAi machinery enzymes can improve RNAi efficacy in traditionally recalcitrant species. A transgenic Bombyx mori strain, Baculovirus Immediate-Early Gene, ie1, promoter driven expression of silkworm Dicer2 coding sequence (IE1-BmDicer2), which over-expresses BmDicer2, was generated by piggyBac transposon-mediated transgenesis. Two indexes, the ratio of animals that showed a silencing phenotype and the duration of silencing, were used to evaluate silencing efficiency. Significant knockdown of target gene expression was observed at 48 h postinjection at both the transcriptional and translational levels. Furthermore, we coexpressed B. mori Argonaute 2 BmAgo2)and BmDicer 2 and found that 22% of the animals (n = 18) showed an obvious silencing effect even at 72 h, suggesting that coexpression of these two RNAi core machinery enzymes further increased the susceptibility of B. mori to injected double-stranded RNAs. This study offers a new strategy for functional genomics research in RNAi-refractory insect taxa in general and for lepidopterans in particular.


Assuntos
Bombyx/genética , Regulação da Expressão Gênica , Larva/genética , Interferência de RNA , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo
13.
PLoS One ; 15(2): e0228348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32032373

RESUMO

The synaptic vesicle protein, synaptotagmin, is the principle Ca2+ sensor for synaptic transmission. Ca2+ influx into active nerve terminals is translated into neurotransmitter release by Ca2+ binding to synaptotagmin's tandem C2 domains, triggering the fast, synchronous fusion of multiple synaptic vesicles. Two hydrophobic residues, shown to mediate Ca2+-dependent membrane insertion of these C2 domains, are required for this process. Previous research suggested that one of its tandem C2 domains (C2B) is critical for fusion, while the other domain (C2A) plays only a facilitatory role. However, the function of the two hydrophobic residues in C2A have not been adequately tested in vivo. Here we show that these two hydrophobic residues are absolutely required for synaptotagmin to trigger vesicle fusion. Using in vivo electrophysiological recording at the Drosophila larval neuromuscular junction, we found that mutation of these two key C2A hydrophobic residues almost completely abolished neurotransmitter release. Significantly, mutation of both hydrophobic residues resulted in more severe deficits than those seen in synaptotagmin null mutants. Thus, we report the most severe phenotype of a C2A mutation to date, demonstrating that the C2A domain is absolutely essential for synaptotagmin's function as the electrostatic switch.


Assuntos
Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Transmissão Sináptica , Sinaptotagminas/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Potenciais Pós-Sinápticos Excitadores , Humanos , Larva/metabolismo , Larva/fisiologia , Mutagênese Sítio-Dirigida , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Alinhamento de Sequência , Sinaptotagminas/química , Sinaptotagminas/genética
14.
Sci Rep ; 10(1): 72, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919453

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of end-stage renal failure in humans and results from germline mutations in PKD1 or PKD2. Despite the recent approval of tolvaptan, safer and more effective alternative drugs are clearly needed to slow disease progression. As a first step in drug discovery, we conducted an unbiased chemical screen on zebrafish pkd2 mutant embryos using two publicly available compound libraries (Spectrum, PKIS) totalling 2,367 compounds to identify novel treatments for ADPKD. Using dorsal tail curvature as the assay readout, three major chemical classes (steroids, coumarins, flavonoids) were identified from the Spectrum library as the most promising candidates to be tested on human PKD1 cystic cells. Amongst these were an androgen, 5α-androstane 3,17-dione, detected as the strongest enhancer of the pkd2 phenotype but whose effect was found to be independent of the canonical androgen receptor pathway. From the PKIS library, we identified several ALK5 kinase inhibitors as strong suppressors of the pkd2 tail phenotype and in vitro cyst expansion. In summary, our results identify ALK5 and non-canonical androgen receptors as potential therapeutic targets for further evaluation in drug development for ADPKD.


Assuntos
Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Canais de Cátion TRPP/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/metabolismo , Apoptose/efeitos dos fármacos , Cães , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Células Madin Darby de Rim Canino , Fenótipo , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptores Androgênicos/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Canais de Cátion TRPP/deficiência , Canais de Cátion TRPP/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/metabolismo
15.
Aquat Toxicol ; 220: 105403, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31927064

RESUMO

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (TG 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 µg/L). In addition to "classical" endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that 172.6 µg/L PCZ significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 µg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals.


Assuntos
Animais Geneticamente Modificados , Aromatase/genética , Disruptores Endócrinos/toxicidade , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/metabolismo , Feminino , Corantes Fluorescentes , Proteínas de Fluorescência Verde/genética , Guias como Assunto , Organização para a Cooperação e Desenvolvimento Econômico , Ovário/efeitos dos fármacos , Ovário/metabolismo , Regiões Promotoras Genéticas , Reprodutibilidade dos Testes , Vitelogeninas/metabolismo , Peixe-Zebra/metabolismo
16.
FASEB J ; 34(2): 3021-3036, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31909520

RESUMO

Spinal muscular atrophy is a rare and fatal neuromuscular disorder caused by the loss of alpha motor neurons. The affected individuals have mutated the ubiquitously expressed SMN1 gene resulting in the loss or reduction in the survival motor neuron (SMN) protein levels. However, an almost identical paralog exists in humans: SMN2. Pharmacological activation of SMN2 exon 7 inclusion by small molecules or modified antisense oligonucleotides is a valid approach to treat SMA. Here we describe an in vivo SMN2 minigene reporter system in Drosophila motor neurons that serves as a cost-effective, feasible, and stringent primary screening model for identifying chemicals capable of crossing the conserved Drosophila blood-brain barrier and modulating exon 7 inclusion. The model was used for the screening of 1100 drugs from the Prestwick Chemical Library, resulting in 2.45% hit rate. The most promising candidate drugs were validated in patient-derived fibroblasts where they proved to increase SMN protein levels. Among them, moxifloxacin modulated SMN2 splicing by promoting exon 7 inclusion. The recovery of SMN protein levels was confirmed by increased colocalization of nuclear gems with Cajal Bodies. Thus, a Drosophila-based drug screen allowed the discovery of an FDA-approved small molecule with the potential to become a novel therapy for SMA.


Assuntos
Animais Geneticamente Modificados , Barreira Hematoencefálica , Éxons , Genes Reporter , Moxifloxacina/farmacologia , Atrofia Muscular Espinal , Processamento Alternativo/efeitos dos fármacos , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Modelos Animais de Doenças , Drosophila melanogaster , Avaliação Pré-Clínica de Medicamentos , Humanos , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
17.
Insect Biochem Mol Biol ; 119: 103325, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31978586

RESUMO

RNA activation (RNAa) is a newly emerging area of research in which dsRNA targeting promoter regions can induce the expression of the target gene. Although still in its infancy, it is already having significant impacts in several research areas in particular as cancer therapeutics. So far, the scope of RNAa has been limited to mammals and Caenorhabditis elegans with no indication of its prevalence in insects. In this study, we aimed to demonstrate the presence of RNAa in the insect dengue vector Aedes aegypti. Furthermore, we looked to uncover some details surrounding the involvement of host factors in order to present this as a new technique for insect research. The outcomes of this study provide new opportunities to further research into arthropod-borne diseases and insect biology in the same way as RNA interference.


Assuntos
Aedes/genética , Mosquitos Vetores/genética , RNA de Cadeia Dupla/genética , Ativação Transcricional , Aedes/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Mosquitos Vetores/metabolismo , RNA de Cadeia Dupla/metabolismo
18.
Insect Biochem Mol Biol ; 119: 103324, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31978587

RESUMO

Polycistronic expression systems in insects can be used for applications such as recombinant protein production in cells, enhanced transgenesis methods, and the development of novel pest-control strategies based on the sterile insect technique (SIT). Here we tested the performance of four picornaviral 2A self-cleaving peptides (TaV-2A, DrosCV-2A, FMDV 2A1/31 and FMDV 2A1/32) for the co-expression and differential subcellular targeting of two fluorescent marker proteins in cell lines (Anastrepha suspensa AsE01 and Drosophila melanogaster S2 cells). We found that all four 2A peptides showed comparable activity in cell lines, leading to the production of independent upstream and downstream proteins that were directed to the nucleus or membrane by a C-terminal nuclear localization signal (NLS) on the upstream protein and a poly-lysine/CAAX membrane anchor on the downstream protein. TaV-2A and DrosCV-2A were inserted into piggyBac constructs to create transgenic D. suzukii strains, confirming efficient ribosomal skipping in vivo. Interestingly, we found that the EGFP-CAAX protein was distributed homogeneously in the membrane whereas the DsRed-CAAX protein formed clumps and aggregates that induced extensive membrane blebbing. Accordingly, only flies expressing the DsRed-NLS and EGFP-CAAX proteins could be bred to homozygosity whereas expression of EGFP-NLS and DsRed-CAAX was lethal in the homozygous state. Our results therefore demonstrate that the 2A constructs and two novel targeting motifs are functional in D. suzukii, and that the combination of EGFP-NLS and DsRed-CAAX shows dosage-dependent lethality. These molecular elements could be further used to improve expression systems in insects and generate novel pest control strains.


Assuntos
Drosophila/genética , Expressão Gênica , Proteínas de Insetos/genética , Picornaviridae/metabolismo , Tephritidae/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Linhagem Celular , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Insetos/metabolismo , Proteínas Luminescentes/química , Peptídeos , Tephritidae/metabolismo , Proteínas Virais/metabolismo
19.
Int J Mol Sci ; 21(2)2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31940967

RESUMO

Recent decades have seen groundbreaking advances in cancer research. Genetically engineered animal models, mainly in mice, have contributed to a better understanding of the underlying mechanisms involved in cancer. However, mice are not ideal for translating basic research into studies closer to the clinic. There is a need for complementary information provided by non-rodent species. Pigs are well suited for translational biomedical research as they share many similarities with humans such as body and organ size, aspects of anatomy, physiology and pathophysiology and can provide valuable means of developing and testing novel diagnostic and therapeutic procedures. Porcine oncology is a new field, but it is clear that replication of key oncogenic mutation in pigs can usefully mimic several human cancers. This review briefly outlines the technology used to generate genetically modified pigs, provides an overview of existing cancer models, their applications and how the field may develop in the near future.


Assuntos
Animais Geneticamente Modificados , Neoplasias Experimentais , Suínos , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Humanos , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/terapia , Suínos/genética , Suínos/metabolismo
20.
Insect Sci ; 27(4): 646-654, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31131541

RESUMO

MicroRNAs (miRNAs) are a class of short, non-coding transcripts that bind to 3'-untranslated regions to trigger messenger RNA degradation or translational inhibition. Here we explored how miRNAs regulate sex determination in Bombyx mori, a lepidopteran model insect. Genes known to be involved in sex determination, BmPSI, Bmdsx, and BmMasc, are predicted targets of the species-specific miR-2738. Using a dual luciferase reporter assay in HEK293T cells, we confirmed that miR-2738 suppressed transcription of BmPSI, Bmdsx, and BmMasc. The levels of BmPSI and BmMasc were significantly down-regulated in B. mori miR-2738 overexpression. In contrast, the genetic disruption of miR-2738 using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 transgenic system increased the levels of BmPSI and BmMasc transcripts, whereas splicing of Bmdsx was unaltered by miR-2738 depletion or overexpression. Taken together, this study implicates miR-2738 as a minor regulator of sex determination genes in the silkworm.


Assuntos
Bombyx/crescimento & desenvolvimento , Bombyx/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Processos de Determinação Sexual/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Sequência de Bases , Bombyx/metabolismo , Células HEK293 , Humanos , MicroRNAs/metabolismo
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