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1.
EMBO J ; 38(21): e103331, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31602659

RESUMO

Research that uses stem cell-based chimeras promises to advance our understanding of human developmental biology, as well as new medical interventions, such as generating transplantable human organs in livestock. However, along with these exciting research possibilities come moral concerns about the moral humanization of animals, especially when it comes to the potential effects of human cells in the brains of experimental animals. Recent work involving neurologically chimeric mice may suggest that such worries are reasonable. However, this overlooks the crucial social and neurological conditions for enabling the development of conscious self-awareness, the absence of which leaves us only with animal welfare to monitor and consider.


Assuntos
Quimera/metabolismo , Modelos Animais , Modelos Neurológicos , Pesquisa com Células-Tronco/ética , Bem-Estar do Animal , Animais , Humanos , Camundongos
2.
Nature ; 572(7768): 199-204, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31292543

RESUMO

The human liver is an essential multifunctional organ. The incidence of liver diseases is rising and there are limited treatment options. However, the cellular composition of the liver remains poorly understood. Here we performed single-cell RNA sequencing of about 10,000 cells from normal liver tissue from nine human donors to construct a human liver cell atlas. Our analysis identified previously unknown subtypes of endothelial cells, Kupffer cells, and hepatocytes, with transcriptome-wide zonation of some of these populations. We show that the EPCAM+ population is heterogeneous, comprising hepatocyte-biased and cholangiocyte populations as well as a TROP2int progenitor population with strong potential to form bipotent liver organoids. As a proof-of-principle, we used our atlas to unravel the phenotypic changes that occur in hepatocellular carcinoma cells and in human hepatocytes and liver endothelial cells engrafted into a mouse liver. Our human liver cell atlas provides a powerful resource to enable the discovery of previously unknown cell types in normal and diseased livers.


Assuntos
Células Epiteliais/citologia , Hepatócitos/citologia , Fígado/citologia , Células-Tronco/citologia , Adulto , Animais , Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular/metabolismo , Quimera/imunologia , Quimera/metabolismo , Células Endoteliais/citologia , Células Endoteliais/imunologia , Células Epiteliais/imunologia , Feminino , Regulação da Expressão Gênica , Hepatócitos/imunologia , Hepatócitos/metabolismo , Humanos , Fígado/imunologia , Masculino , Camundongos , Organoides/metabolismo , RNA Citoplasmático Pequeno/genética , Reprodutibilidade dos Testes , Células-Tronco/imunologia
3.
PLoS One ; 14(5): e0216939, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31141543

RESUMO

In the present two-year study, an attempt was made to estimate the grain yield, grain nutrient uptake, and oil quality of three commonly grown maize (Zea mays L.) hybrids fertilized with varied levels of nitrogen (N), phosphorus (P) and potassium (K). Results obtained from both the experimental years indicated that application of 125% of recommended dose of fertilizer (RDF) recorded maximum grain yield (10.37 t ha-1; 124% higher than control). When compared with 100% RDF, grain yield reduction with nutrient omission was 44% for N omission, 17% for P omission, and 27% for K omission. Nitrogen uptake was increased with increasing NPK levels up to 150% RDF that was statistically at par (p ≥ 0.01) with 125% RDF. Increasing trend in P and K uptake was observed with successive increase in NPK levels up to 125% RDF, above which it declined. The protein content was significantly higher in grains of var. P 3396 with 125% RDF. Nutrient management has significant (p ≤ 0.01) role in the grain oil content. Saturated fatty acids (palmitic, stearic and arachidic acid) content decreased, and unsaturated fatty acid (oleic, linoleic and linolenic acid) increased with increasing NPK levels. The average oleic acid desaturation and linoleic acid desaturation ratios were increased with increasing NPK levels up to 100 and 125% RDF, respectively. However, average monounsaturated fatty acids (MUFA): poly-unsaturated fatty acids (PUFA), saturated: unsaturated as well as linoleic: linolenic acid ratios were increased on receiving 75% RDF, and beyond that it showed decreasing trend. The omission of K had the highest inhibitory effect on corn oil quality followed by N and P omission.


Assuntos
Óleo de Milho/química , Grão Comestível/efeitos dos fármacos , Nitrogênio/farmacologia , Fósforo/farmacologia , Potássio/farmacologia , Zea mays/efeitos dos fármacos , Quimera/crescimento & desenvolvimento , Quimera/metabolismo , Óleo de Milho/metabolismo , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/classificação , Ácidos Graxos/isolamento & purificação , Ácidos Graxos Insaturados/biossíntese , Ácidos Graxos Insaturados/classificação , Ácidos Graxos Insaturados/isolamento & purificação , Fertilizantes/análise , Humanos , Nitrogênio/metabolismo , Nutrientes/metabolismo , Nutrientes/farmacologia , Fósforo/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/isolamento & purificação , Potássio/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-30898546

RESUMO

Hybrid Nile tilapia (Oreochromis niloticus, ♀) × blue tilapia (O. aureus, ♂) is a widely cultured tilapia variety due to its growth vigor compared to the parent species. As a peptide hormone, insulin-like growth factor 1 (IGF-1) plays a critical role in regulating somatic growth. The present study focuses on the expression characteristics of IGF-1 in hybrid tilapia. The cloned complete open reading frame of IGF-1 in hybrid tilapia is 549 bp in length, encoding a protein of 182 amino acids. The deduced protein is highly similar to that of Nile tilapia and blue tilapia. IGF-1 was found to be primarily expressed in the liver and muscle in the hybrid; lower expression levels were found in other tissues such as the intestine, spleen, and head-kidney. Increased mRNA expression was observed in the liver and muscle of the hybrid compared to Nile tilapia and blue tilapia, indicating a nonadditive expression pattern in the hybrid. An IGF-1 SNP site (397 site: C in Nile tilapia, G in blue tilapia) for differentiating the Nile tilapia or blue tilapia subgenome in hybrids was identified. Pyrosequencing analysis of the liver transcriptome indicated that most of the hybrids (9 of 10 individuals) predominantly expressed the G allele, demonstrating bias of the blue tilapia subgenome. The present study provides novel data indicating, for the first time, overall gene expression of IGF-1 and allele-specific expression in hybrid tilapia.


Assuntos
Alelos , Quimera , Ciclídeos , Proteínas de Peixes , Regulação da Expressão Gênica/fisiologia , Fator de Crescimento Insulin-Like I , Animais , Quimera/genética , Quimera/metabolismo , Ciclídeos/genética , Ciclídeos/metabolismo , Proteínas de Peixes/biossíntese , Proteínas de Peixes/genética , Fator de Crescimento Insulin-Like I/biossíntese , Fator de Crescimento Insulin-Like I/genética
5.
ACS Chem Biol ; 14(3): 361-368, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30721025

RESUMO

Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target/PROTAC/ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of bromodomain and extraterminal domain proteins Brd2, Brd3, and Brd4 to the von Hippel-Lindau E3 ligase (VHL). We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4BD2 or Brd2BD2 and VHL. Equivalent complexes with Brd3BD2 are destabilized due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of Brd2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.


Assuntos
Quimera/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Aminoácidos/metabolismo , Desenho de Drogas , Cinética , Ligação Proteica , Conformação Proteica , Proteólise , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
6.
Nature ; 566(7745): 490-495, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30787436

RESUMO

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.


Assuntos
Diferenciação Celular/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Gastrulação , Organogênese , Análise de Célula Única , Animais , Linhagem da Célula/genética , Quimera/embriologia , Quimera/genética , Quimera/metabolismo , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Endotélio/citologia , Endotélio/embriologia , Endotélio/metabolismo , Feminino , Gastrulação/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , Masculino , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Mutação/genética , Células Mieloides/citologia , Organogênese/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/citologia , Linha Primitiva/embriologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T/deficiência , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética
7.
Nucleic Acids Res ; 47(8): 4255-4271, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30788506

RESUMO

The piRNA pathway is an adaptive mechanism that maintains genome stability by repression of selfish genomic elements. In the male germline of Drosophila melanogaster repression of Stellate genes by piRNAs generated from Supressor of Stellate (Su(Ste)) locus is required for male fertility, but both Su(Ste) piRNAs and their targets are absent in other Drosophila species. We found that D. melanogaster genome contains multiple X-linked non-coding genomic repeats that have sequence similarity to the protein-coding host gene vasa. In the male germline, these vasa-related AT-chX repeats produce abundant piRNAs that are antisense to vasa; however, vasa mRNA escapes silencing due to imperfect complementarity to AT-chX piRNAs. Unexpectedly, we discovered AT-chX piRNAs target vasa of Drosophila mauritiana in the testes of interspecies hybrids. In the majority of hybrid flies, the testes were strongly reduced in size and germline content. A minority of hybrids maintained wild-type array of premeiotic germ cells in the testes, but in them harmful Stellate genes were derepressed due to the absence of Su(Ste) piRNAs, and meiotic failures were observed. Thus, the piRNA pathway contributes to reproductive isolation between D. melanogaster and closely related species, causing hybrid male sterility via misregulation of two different host protein factors.


Assuntos
Quimera/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila/genética , Inativação Gênica , Genoma de Inseto , Proteínas Quinases/genética , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Quimera/metabolismo , Cruzamentos Genéticos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Fertilidade , Infertilidade , Masculino , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Isolamento Reprodutivo , Alinhamento de Sequência , Espermatozoides/metabolismo , Espermatozoides/patologia , Testículo/anormalidades , Testículo/metabolismo
8.
Xenobiotica ; 49(6): 678-687, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29969338

RESUMO

The evaluation of drug-mediated cytochrome P450 (P450) induction using human hepatocytes is important for predicting drug interactions. In this study, we prepared hepatocytes from chimeric mice with humanised livers (Hu-Liver mice) and evaluated the expression and inducibility of P450s in these hepatocytes. Up to 95% of the Hu-Liver cells stained positive for human leukocyte antigen and the mean viability exceeded 85% (n = 10). Monolayer-cultured Hu-Liver cells displayed a similar morphology to cultures of the corresponding human hepatocytes used as transplantation donors. The mRNA expression levels in Hu-Liver cells of 16 P450 forms belonging to P450 subfamilies 1-4 correlated well with the expression levels of the same enzymes in human hepatocytes. The variations in individual P450 mRNA levels between Hu-Liver cells and the corresponding human hepatocytes were within five-fold for 13 P450 forms. The production of 6ß-hydroxytestosterone in Hu-Liver cells was significantly increased (p < .05) following treatment with the CYP3A inducer, rifampicin. Hu-Liver cells have characteristics similar to those of human hepatocytes in terms of mRNA expression levels and the inducibility of the various P450 forms. Thus, Hu-Liver cells can potentially be used for in vitro drug-mediated induction assays of human hepatic P450s.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/metabolismo , Animais , Células Cultivadas , Quimera/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Indução Enzimática , Humanos , Hidroxitestosteronas/metabolismo , Fígado/metabolismo , Camundongos , Microssomos Hepáticos/metabolismo
9.
Sci Rep ; 8(1): 16267, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30390041

RESUMO

Genetic diversity in cytoplasmic and nuclear genomes and their interaction affecting adaptive traits is an attractive research subject in plants. We addressed submergence stress response of wheat that has become increasingly important but remained largely uninvestigated. Our primary aim was to disclose cytoplasmic diversity using nucleus-cytoplasm (NC) hybrids possessing a series of heterologous cytoplasms in a common nuclear background. Effects of submergence on seedling emergence and growth from imbibed seeds were studied and compared with euplasmic lines. Marked phenotypic variabilities were observed among both lines, demonstrating divergent cytoplasmic and nuclear effects on submergence response. NC hybrids with cytoplasm of Aegilops mutica showed a less inhibition, indicative of their positive contribution to submergence tolerance, whereas cytoplasms of Aegilops umbellulata and related species caused a greater inhibition. Superoxide dismutase (SOD) activity showed a marked increase accompanied by retardation of seedling growth in a susceptible NC hybrid. The observation suggested that the elevated SOD activity was resulted from a high level of reactive oxygen species accumulated and remained in susceptible seedlings. Taken together, our results point to the usefulness of NC hybrids in further studies needed to clarify molecular mechanisms underlying the nucleus-cytoplasm interaction regulating submergence stress response in wheat.


Assuntos
Aegilops/genética , Quimera/genética , Citoplasma/genética , Estresse Fisiológico/genética , Triticum/genética , Adaptação Fisiológica/genética , Aegilops/metabolismo , Bioensaio , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quimera/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Plântula/citologia , Plântula/genética , Plântula/metabolismo , Sementes/citologia , Sementes/genética , Sementes/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Triticum/metabolismo
10.
Plant Physiol Biochem ; 133: 50-56, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30390431

RESUMO

Polyploidy plays an important role in plant breeding, inducing a wide range of effects on photosynthetic performance, cellular and photochemical aspects of photosynthesis. Although the impacts of polyploidization on photosynthesis have been examined in several genera, comparatively little is known about the photosynthetic responses to polyploidy in Lilium. In this study, diploid and colchicine-induced tetraploid Lilium FO hybrids were used to study the effects of polyploidization on cellular, photochemical and photosynthetic characteristics in Lilium. Polyploidization caused a significant change in leaf anatomical structure, chloroplast ultrastructure and photosynthetic pigment contents. Thicker epidermal and spongy tissue, more and thicker thylakoid lamellae and higher chlorophyll and carotenoid contents were observed in the tetraploid than in the diploid. More regularly and tightly arranged thylakoids were closely associated with more efficient light-harvesting machinery. And the tetraploid plants showed a higher net photosynthetic rate (Pn) and maximum net photosynthetic rate (Pmax) than the diploid plants, under both natural conditions and gradients of light intensity/CO2 concentration. In addition, the tetraploid had a significantly higher light saturation point (LSP), but a lower light compensation point (LCP) than the diploid, indicating that tetraploid plants acquired light energy more efficiently. These results suggested that cellular and photochemical alterations caused by polyploidization improve the capacity for light absorption and conversion and further enhanced the photosynthetic performance in Lilium FO hybrids.


Assuntos
Quimera , Cloroplastos , Lilium , Fotossíntese , Tetraploidia , Quimera/genética , Quimera/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Lilium/genética , Lilium/metabolismo
12.
J Vis Exp ; (139)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30272645

RESUMO

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.


Assuntos
Hipercolesterolemia/diagnóstico , Hiperlipoproteinemia Tipo II/diagnóstico , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Quimera/metabolismo , Modelos Animais de Doenças , Humanos , Hipercolesterolemia/patologia , Hiperlipoproteinemia Tipo II/patologia , Camundongos , Mutação
13.
Theor Med Bioeth ; 39(4): 301-319, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30167942

RESUMO

This paper presents an account of how human spontaneous embryonic chimeras are formed. On the prevalent view in the philosophical literature, it is said that chimeras are the product of two embryos that fuse to form a new third embryo. We call this version of fusion synthesis. In contrast to synthesis, we present an alternative mechanism for chimera formation called incorporation, wherein one embryo incorporates the cells of a second embryo into its body. We argue that the incorporation thesis explains other types of chimera formation, which are better understood, and is more consistent than synthesis with what is known about embryological development. Incorporation also has different implications than synthesis and so avoids the philosophical puzzles that are often said to accompany embryonic chimera formation-puzzles which pose problems to the human embryo's persistence from fertilization to the fetal stage of human development.


Assuntos
Quimera/crescimento & desenvolvimento , Gêmeos/genética , Quimera/metabolismo , Humanos
14.
Proc Natl Acad Sci U S A ; 115(41): E9610-E9619, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30266792

RESUMO

Chromosomal rearrangements (e.g., fusions/fissions) have the potential to drive speciation. However, their accumulation in a population is generally viewed as unlikely, because chromosomal heterozygosity should lead to meiotic problems and aneuploid gametes. Canonical meiosis involves segregation of homologous chromosomes in meiosis I and sister chromatid segregation during meiosis II. In organisms with holocentric chromosomes, which are characterized by kinetic activity distributed along almost the entire chromosome length, this order may be inverted depending on their metaphase I orientation. Here we analyzed the evolutionary role of this intrinsic versatility of holocentric chromosomes, which is not available to monocentric ones, by studying F1 to F4 hybrids between two chromosomal races of the Wood White butterfly (Leptidea sinapis), separated by at least 24 chromosomal fusions/fissions. We found that these chromosomal rearrangements resulted in multiple meiotic multivalents, and, contrary to the theoretical prediction, the hybrids displayed relatively high reproductive fitness (42% of that of the control lines) and regular behavior of meiotic chromosomes. In the hybrids, we also discovered inverted meiosis, in which the first and critical stage of chromosome number reduction was replaced by the less risky stage of sister chromatid separation. We hypothesize that the ability to invert the order of the main meiotic events facilitates proper chromosome segregation and hence rescues fertility and viability in chromosomal hybrids, potentially promoting dynamic karyotype evolution and chromosomal speciation.


Assuntos
Borboletas , Quimera , Cromátides , Metáfase/fisiologia , Animais , Borboletas/genética , Borboletas/metabolismo , Quimera/genética , Quimera/metabolismo , Cromátides/genética , Cromátides/metabolismo , Cromossomos de Insetos/genética , Cromossomos de Insetos/metabolismo
15.
PLoS One ; 13(7): e0201583, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30063755

RESUMO

Tomatoes (Solanum lycopersicum L.) have been bred to exude higher amounts or different types of the specialized plant metabolites, acylsugars, from type IV trichomes. Acylsugars are known to deter several herbivorous insect pests, including the western flower thrips (WFT), Frankliniella occidentalis (Pergande); however, all previous studies investigated the effect of acylsugars on leaves, or acylsugar extracts obtained from leaves. In spite of the WFT predilection for flowers, there is a gap in knowledge about flower defenses against thrips damage. This is especially important in light of their capacity to acquire and inoculate viruses in the genus Orthotospovirus, such as Tomato spotted wilt orthotospovirus (TSWV), in flowers. Therefore, we turned our attention to assessing thrips oviposition differences on flowers of 14 entries, including 8 interspecific hybrids, 5 tomato lines bred for specific acylsugar-related characteristics (type IV trichome densities, acylsugar amount, sugar moiety and fatty acid profile), and a fresh market tomato hybrid, Mt. Spring, which only produces trace amounts of acylsugars. Our results show that the density of the acylsugar droplet bearing type IV trichomes is greatest on sepals, relative to other flower structures, and accordingly, WFT avoids oviposition on sepals in favor of trichome-sparse petals. In concordance with past studies, acylsugar amount was the most important acylsugar-related characteristic suppressing WFT oviposition. Certain acylsugar fatty acids, specifically i-C5, i-C9 and i-C11, were also significantly associated with changes in WFT oviposition. These results support continued breeding efforts to increase acylsugar amounts and explore modifications of fatty acid profile and their roles in deterring thrips oviposition. The finding that acylsugar production occurs and reduces thrips oviposition in tomato flowers will be important in efforts to use acylsugar-mediated resistance to reduce incidence of orthotospoviruses such as TSWV in tomato by deterring virus transmission and development of thrips vector populations in the crop.


Assuntos
Quimera/metabolismo , Ácidos Graxos/metabolismo , Flores , Lycopersicon esculentum , Melhoramento Vegetal , Açúcares/metabolismo , Tisanópteros , Animais , Metabolismo dos Carboidratos/fisiologia , Cruzamentos Genéticos , Flores/metabolismo , Herbivoria , Insetos/efeitos dos fármacos , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Oviposição/genética , Controle Biológico de Vetores , Doenças das Plantas/genética , Doenças das Plantas/prevenção & controle , Plantas Geneticamente Modificadas , Açúcares/análise , Açúcares/farmacologia , Tisanópteros/genética , Tisanópteros/metabolismo
16.
Drug Metab Dispos ; 46(11): 1734-1744, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30093418

RESUMO

Preclinical evaluation of drug candidates in experimental animal models is an essential step in drug development. Humanized mouse models have emerged as a promising alternative to traditional animal models. The purpose of this mini-review is to provide a brief survey of currently available mouse models for studying human xenobiotic metabolism. Here, we describe both genetic humanization and human liver chimeric mouse models, focusing on the advantages and limitations while outlining their key features and applications. Although this field of biomedical science is relatively young, these humanized mouse models have the potential to transform preclinical drug testing and eventually lead to a more cost-effective and rapid development of new therapies.


Assuntos
Quimera/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Inativação Metabólica/fisiologia , Fígado/metabolismo , Xenobióticos/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Modelos Animais
17.
Mol Cells ; 41(7): 631-638, 2018 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-29991673

RESUMO

Spermatogonial stem cells (SSCs) derived from mouse testis are unipotent in regard of spermatogenesis. Our previous study demonstrated that SSCs can be fully reprogrammed into pluripotent stem cells, so called germline-derived pluripotent stem cells (gPS cells), on feeder cells (mouse embryonic fibroblasts), which supports SSC proliferation and induction of pluripotency. Because of an uncontrollable microenvironment caused by interactions with feeder cells, feeder-based SSC reprogramming is not suitable for elucidation of the self-reprogramming mechanism by which SSCs are converted into pluripotent stem cells. Recently, we have established a Matrigel-based SSC expansion culture system that allows long-term SSC proliferation without mouse embryonic fibroblast support. In this study, we developed a new feeder-free SSC self-reprogramming protocol based on the Matrigel-based culture system. The gPS cells generated using a feeder-free reprogramming system showed pluripotency at the molecular and cellular levels. The differentiation potential of gPS cells was confirmed in vitro and in vivo. Our study shows for the first time that the induction of SSC pluripotency can be achieved without feeder cells. The newly developed feeder-free self-reprogramming system could be a useful tool to reveal the mechanism by which unipotent cells are self-reprogrammed into pluripotent stem cells.


Assuntos
Microambiente Celular , Reprogramação Celular/genética , Células Alimentadoras/citologia , Células-Tronco Pluripotentes/citologia , Espermatogônias/citologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Microambiente Celular/genética , Quimera/metabolismo , Análise Citogenética , Metilação de DNA/genética , Células Alimentadoras/metabolismo , Perfilação da Expressão Gênica , Impressão Genômica/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Pluripotentes/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Espermatogônias/metabolismo
18.
Arch Toxicol ; 92(6): 1953-1967, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29721588

RESUMO

The pharmacokinetics of diclofenac were investigated following single oral doses of 10 mg/kg to chimeric liver humanized and murinized FRG and C57BL/6 mice. In addition, the metabolism and excretion were investigated in chimeric liver humanized and murinized FRG mice. Diclofenac reached maximum blood concentrations of 2.43 ± 0.9 µg/mL (n = 3) at 0.25 h post-dose with an AUCinf of 3.67 µg h/mL and an effective half-life of 0.86 h (n = 2). In the murinized animals, maximum blood concentrations were determined as 3.86 ± 2.31 µg/mL at 0.25 h post-dose with an AUCinf of 4.94 ± 2.93 µg h/mL and a half-life of 0.52 ± 0.03 h (n = 3). In C57BL/6J mice, mean peak blood concentrations of 2.31 ± 0.53 µg/mL were seen 0.25 h post-dose with a mean AUCinf of 2.10 ± 0.49 µg h/mL and a half-life of 0.51 ± 0.49 h (n = 3). Analysis of blood indicated only trace quantities of drug-related material in chimeric humanized and murinized FRG mice. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles in humanized mice were different to those of both murinized and wild-type animals, e.g., a higher proportion of the dose was detected in the form of acyl glucuronide metabolites and much reduced amounts as taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57BL/6J mice and humans revealed a greater, though not complete, match between chimeric humanized mice and humans, such that the liver humanized FRG model may represent a model for assessing the biotransformation of such compounds in humans.


Assuntos
Anti-Inflamatórios não Esteroides/farmacocinética , Quimera/metabolismo , Diclofenaco/farmacocinética , Animais , Anti-Inflamatórios não Esteroides/sangue , Anti-Inflamatórios não Esteroides/urina , Área Sob a Curva , Bile/metabolismo , Biotransformação , Quimera/sangue , Quimera/urina , Diclofenaco/sangue , Diclofenaco/urina , Fezes/química , Meia-Vida , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade da Espécie
19.
PLoS One ; 13(5): e0197026, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29746547

RESUMO

Nutritional suitability of milk is not only related to gross composition, but is also strongly affected by the microheterogeniety of the protein fraction. Hence, to go further into the evaluation of the potential suitability of non-bovine milks in human/infant nutrition it is necessary to have a detailed characterization of their protein components. Combining proven proteomic approaches (SDS-PAGE, LC-MS/MS and LC-ESI-MS) and cDNA sequencing, we provide here in depth characterization of the milk protein fraction of dromedary and Bactrian camels, and their hybrids, from different regions of Kazakhstan. A total 391 functional groups of proteins were identified from 8 camel milk samples. A detailed characterization of 50 protein molecules, relating to genetic variants and isoforms arising from post-translational modifications and alternative splicing events, belonging to nine protein families (κ-, αs1-, αs2-, ß-; and γ-CN, WAP, α-LAC, PGRP, CSA/LPO) was achieved by LC-ESI-MS. The presence of two unknown proteins UP1 (22,939 Da) and UP2 (23,046 Da) was also reported as well as the existence of a ß-CN short isoform (946 Da lighter than the full-length ß-CN), arising very likely in both genetic variants (A and B) from proteolysis by plasmin. In addition, we report, for the first time to our knowledge, the occurrence of a αs2-CN phosphorylation isoform with 12P groups within two recognition motifs, suggesting thereby the existence of two kinase systems involved in the phosphorylation of caseins in the mammary gland. Finally, we demonstrate that genetic variants, which hitherto seemed to be species- specific (e.g. ß-CN A for Bactrian and ß-CN B for dromedary), are in fact present both in Camel dromedarius and C. bactrianus.


Assuntos
Camelus/metabolismo , Quimera/metabolismo , Proteínas do Leite/metabolismo , Proteômica , Animais , Camelus/genética , Quimera/genética , Feminino , Cazaquistão , Espectrometria de Massas
20.
Sci Rep ; 8(1): 7520, 2018 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-29760480

RESUMO

The pearl oyster is one of the rare animal models that support two distinct genomes, through the surgical graft process operated for culture pearl production. This grafted organism is assimilated to a chimera whose physiological functioning remains poorly known. The question of the energy expenditure comparison between chimera and non-chimera animals arises. To answer this question, grafted and non-grafted pearl oysters were evaluated for their energetic needs by the indirect calorimetry method. This method made it possible to measure the energy expenditure based on the respiration rate (RR) measurement, reflecting the basal metabolism. The results showed that the RR values for grafted and non-grafted pearl oysters were not significantly different (p < 0.05). The estimated cost of pearl calcification including CaCO3 and proteins synthesis was 0.237 ± 0.064 J h-1, representing 0.64% of the total energy expenditure of grafted pearl oysters. This study made it possible, for the first time, to see the energy cost of cultured pearl formation in P. margaritifera and the little impact in the energetic metabolism of the chimera organism.


Assuntos
Quimera/metabolismo , Nácar/metabolismo , Pinctada/genética , Animais , Aquicultura , Metabolismo Basal , Calcificação Fisiológica , Calorimetria , Metabolismo Energético , Pinctada/metabolismo , Transplante
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