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1.
Int J Mol Sci ; 25(2)2024 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-38256061

RESUMO

The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Sistemas CRISPR-Cas , Células-Tronco Hematopoéticas , Alelos
2.
Plant J ; 110(5): 1493-1497, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35362151

RESUMO

Biosynthesis of plant cell walls requires UDP-glucose as the substrate for cellulose biosynthesis, and as an intermediate for the synthesis of other matrix polysaccharides. The sucrose cleaving enzyme sucrose synthase (SUS) is thought to have a central role in UDP-glucose biosynthesis, and a long-held and much debated hypothesis postulates that SUS is required to supply UDP-glucose to cellulose biosynthesis. To investigate the role of SUS in cellulose biosynthesis of Arabidopsis thaliana we characterized mutants in which four or all six Arabidopsis SUS genes were disrupted. These sus mutants showed no growth phenotypes, vascular tissue cell wall defects, or changes in cellulose content. Moreover, the UDP-glucose content of rosette leaves of the sextuple sus mutants was increased by approximately 20% compared with wild type. It can thus be concluded that cellulose biosynthesis is able to employ alternative UDP-glucose biosynthesis pathway(s), and thereby the model of SUS requirements for cellulose biosynthesis in Arabidopsis can be refuted.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Celulose/metabolismo , Glucose/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Sacarose/metabolismo , Uridina Difosfato Glucose/química , Uridina Difosfato Glucose/metabolismo
3.
Gastroenterology ; 162(4): 1171-1182.e3, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34914943

RESUMO

BACKGROUND & AIMS: The sucrase-isomaltase (SI) c.273_274delAG loss-of-function variant is common in Arctic populations and causes congenital sucrase-isomaltase deficiency, which is an inability to break down and absorb sucrose and isomaltose. Children with this condition experience gastrointestinal symptoms when dietary sucrose is introduced. We aimed to describe the health of adults with sucrase-isomaltase deficiency. METHODS: The association between c.273_274delAG and phenotypes related to metabolic health was assessed in 2 cohorts of Greenlandic adults (n = 4922 and n = 1629). A sucrase-isomaltase knockout (Sis-KO) mouse model was used to further elucidate the findings. RESULTS: Homozygous carriers of the variant had a markedly healthier metabolic profile than the remaining population, including lower body mass index (ß [standard error], -2.0 [0.5] kg/m2; P = 3.1 × 10-5), body weight (-4.8 [1.4] kg; P = 5.1 × 10-4), fat percentage (-3.3% [1.0%]; P = 3.7 × 10-4), fasting triglyceride (-0.27 [0.07] mmol/L; P = 2.3 × 10-6), and remnant cholesterol (-0.11 [0.03] mmol/L; P = 4.2 × 10-5). Further analyses suggested that this was likely mediated partly by higher circulating levels of acetate observed in homozygous carriers (ß [standard error], 0.056 [0.002] mmol/L; P = 2.1 × 10-26), and partly by reduced sucrose uptake, but not lower caloric intake. These findings were verified in Sis-KO mice, which, compared with wild-type mice, were leaner on a sucrose-containing diet, despite similar caloric intake, had significantly higher plasma acetate levels in response to a sucrose gavage, and had lower plasma glucose level in response to a sucrose-tolerance test. CONCLUSIONS: These results suggest that sucrase-isomaltase constitutes a promising drug target for improvement of metabolic health, and that the health benefits are mediated by reduced dietary sucrose uptake and possibly also by higher levels of circulating acetate.


Assuntos
Sacarose Alimentar , Complexo Sacarase-Isomaltase , Acetatos , Animais , Erros Inatos do Metabolismo dos Carboidratos , Sacarose Alimentar/efeitos adversos , Humanos , Camundongos , Oligo-1,6-Glucosidase , Complexo Sacarase-Isomaltase/deficiência , Complexo Sacarase-Isomaltase/genética , Complexo Sacarase-Isomaltase/metabolismo
4.
BMC Plant Biol ; 23(1): 108, 2023 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-36814198

RESUMO

BACKGROUND: Global warming raises serious concerns about the persistence of species and populations locally adapted to their environment, simply because of the shift it produces in their adaptive landscape. For instance, the phenological cycle of tree species may be strongly affected by higher winter temperatures and late frost in spring. Given the variety of ecosystem services they provide, the question of forest tree adaptation has received increasing attention in the scientific community and catalyzed research efforts in ecology, evolutionary biology and functional genomics to study their adaptive capacity to respond to such perturbations. RESULTS: In the present study, we used an elevation gradient in the Pyrenees Mountains to explore the gene expression network underlying dormancy regulation in natural populations of sessile oak stands sampled along an elevation cline and potentially adapted to different climatic conditions mainly driven by temperature. By performing analyses of gene expression in terminal buds we identified genes displaying significant dormancy, elevation or dormancy-by-elevation interaction effects. Our Results highlighted that low- and high-altitude populations have evolved different molecular strategies for minimizing late frost damage and maximizing the growth period, thereby increasing potentially their respective fitness in these contrasting environmental conditions. More particularly, population from high elevation overexpressed genes involved in the inhibition of cell elongation and delaying flowering time while genes involved in cell division and flowering, enabling buds to flush earlier were identified in population from low elevation. CONCLUSION: Our study made it possible to identify key dormancy-by-elevation responsive genes revealing that the stands analyzed in this study have evolved distinct molecular strategies to adapt their bud phenology in response to temperature.


Assuntos
Quercus , Quercus/genética , Ecossistema , Temperatura , Estações do Ano , Florestas , Árvores
5.
New Phytol ; 238(1): 297-312, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36600379

RESUMO

Wood is the most important repository of assimilated carbon in the biosphere, in the form of large polymers (cellulose, hemicelluloses including glucuronoxylan, and lignin) that interactively form a composite, together with soluble extractives including phenolic and aliphatic compounds. Molecular interactions among these compounds are not fully understood. We have targeted the expression of a fungal α-glucuronidase to the wood cell wall of aspen (Populus tremula L. × tremuloides Michx.) and Arabidopsis (Arabidopsis thaliana (L.) Heynh), to decrease contents of the 4-O-methyl glucuronopyranose acid (mGlcA) substituent of xylan, to elucidate mGlcA's functions. The enzyme affected the content of aliphatic insoluble cell wall components having composition similar to suberin, which required mGlcA for binding to cell walls. Such suberin-like compounds have been previously identified in decayed wood, but here, we show their presence in healthy wood of both hardwood and softwood species. By contrast, γ-ester bonds between mGlcA and lignin were insensitive to cell wall-localized α-glucuronidase, supporting the intracellular formation of these bonds. These findings challenge the current view of the wood cell wall composition and reveal a novel function of mGlcA substituent of xylan in fastening of suberin-like compounds to cell wall. They also suggest an intracellular initiation of lignin-carbohydrate complex assembly.


Assuntos
Arabidopsis , Populus , Madeira/química , Lignina/metabolismo , Xilanos/metabolismo , Ácido Glucurônico/análise , Ácido Glucurônico/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , Populus/metabolismo
6.
Plant Physiol ; 189(4): 1943-1960, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35604104

RESUMO

Leaf senescence can be induced by stress or aging, sometimes in a synergistic manner. It is generally acknowledged that the ability to withstand senescence-inducing conditions can provide plants with stress resilience. Although the signaling and transcriptional networks responsible for a delayed senescence phenotype, often referred to as a functional stay-green trait, have been actively investigated, very little is known about the subsequent metabolic adjustments conferring this aptitude to survival. First, using the individually darkened leaf (IDL) experimental setup, we compared IDLs of wild-type (WT) Arabidopsis (Arabidopsis thaliana) to several stay-green contexts, that is IDLs of two functional stay-green mutant lines, oresara1-2 (ore1-2) and an allele of phytochrome-interacting factor 5 (pif5), as well as to leaves from a WT plant entirely darkened (DP). We provide compelling evidence that arginine and ornithine, which accumulate in all stay-green contexts-likely due to the lack of induction of amino acids (AAs) transport-can delay the progression of senescence by fueling the Krebs cycle or the production of polyamines (PAs). Secondly, we show that the conversion of putrescine to spermidine (SPD) is controlled in an age-dependent manner. Thirdly, we demonstrate that SPD represses senescence via interference with ethylene signaling by stabilizing the ETHYLENE BINDING FACTOR1 and 2 (EBF1/2) complex. Taken together, our results identify arginine and ornithine as central metabolites influencing the stress- and age-dependent progression of leaf senescence. We propose that the regulatory loop between the pace of the AA export and the progression of leaf senescence provides the plant with a mechanism to fine-tune the induction of cell death in leaves, which, if triggered unnecessarily, can impede nutrient remobilization and thus plant growth and survival.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Arginina/metabolismo , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Ornitina/genética , Ornitina/metabolismo , Folhas de Planta/metabolismo , Senescência Vegetal , Fatores de Transcrição/metabolismo
7.
Int J Mol Sci ; 24(5)2023 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-36901964

RESUMO

Butyrate produced by the gut microbiota has beneficial effects on metabolism and inflammation. Butyrate-producing bacteria are supported by diets with a high fiber content, such as high-amylose maize starch (HAMS). We investigated the effects of HAMS- and butyrylated HAMS (HAMSB)-supplemented diets on glucose metabolism and inflammation in diabetic db/db mice. Mice fed HAMSB had 8-fold higher fecal butyrate concentration compared to control diet-fed mice. Weekly analysis of fasting blood glucose showed a significant reduction in HAMSB-fed mice when the area under the curve for all five weeks was analyzed. Following treatment, fasting glucose and insulin analysis showed increased homeostatic model assessment (HOMA) insulin sensitivity in the HAMSB-fed mice. Glucose-stimulated insulin release from isolated islets did not differ between the groups, while insulin content was increased by 36% in islets of the HAMSB-fed mice. Expression of insulin 2 was also significantly increased in islets of the HAMSB-fed mice, while no difference in expression of insulin 1, pancreatic and duodenal homeobox 1, MAF bZIP transcription factor A and urocortin 3 between the groups was observed. Hepatic triglycerides in the livers of the HAMSB-fed mice were significantly reduced. Finally, mRNA markers of inflammation in liver and adipose tissue were reduced in mice fed HAMSB. These findings suggest that HAMSB-supplemented diet improves glucose metabolism in the db/db mice, and reduces inflammation in insulin-sensitive tissues.


Assuntos
Butiratos , Amido , Ratos , Camundongos , Animais , Ratos Sprague-Dawley , Amilose/metabolismo , Inflamação , Fígado/metabolismo , Camundongos Endogâmicos , Insulina , Homeostase , Glucose , Camundongos Endogâmicos C57BL , Glicemia/metabolismo
8.
J Cell Mol Med ; 26(23): 5832-5845, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36377725

RESUMO

Metastatic disease is the leading cause of death in children suffering from medulloblastoma and a major treatment challenge. The evidence of leptomeningeal dissemination defines the most aggressive tumours and is associated with increased mortality; thus, inhibition of migration as a factor involved in the process of metastatic disease is fundamental for the treatment and prevention of metastatic dissemination. Targeting the small Rho GTPases Rac1 has been shown to effectively impair medulloblastoma cell migration in vitro. Yet clinically applicable selective Rac1 inhibitors are still lacking. In view of the pertinent oncogenic role of the PI3K signalling cascade and tyrosine kinase-mediated signalling pathways in medulloblastoma, we explored clinically available targeted therapeutics to this effect. Here, we show that Rac1 is expressed in both the cytoplasm and nucleus in the medulloblastoma cell lines Daoy and MEB-Med-8A representative of two high risk medulloblastoma entities. We demonstrate that activated Rac1 is subject to substantial downmodulation following administration of the clinically available inhibitor of the PI3K pathway Pictilisib (GDC-0941) and the multityrosine kinase inhibitors Pazopanib and Sorafenib. The application of those drugs was associated with reduced mobility of the medulloblastoma cells and alterations of the actin skeleton. Of note, PI3K inhibition reveals the strongest anti-migratory effect in Daoy cells. Thus, our in vitro observations provide new insights into different strategies of blocking Rac1 and inhibiting migration in medulloblastoma employing clinically available agents paving the way for confirmatory studies in in vivo models.


Assuntos
Neoplasias Cerebelares , Meduloblastoma , Proteínas rac1 de Ligação ao GTP , Humanos , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Cerebelares/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas rac1 de Ligação ao GTP/metabolismo , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico
9.
Diabetologia ; 64(9): 2077-2091, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34131782

RESUMO

AIMS/HYPOTHESIS: Increased levels of branched-chain amino acids (BCAAs) are associated with type 2 diabetes pathogenesis. However, most metabolomic studies are limited to an analysis of plasma metabolites under fasting conditions, rather than the dynamic shift in response to a metabolic challenge. Moreover, metabolomic profiles of peripheral tissues involved in glucose homeostasis are scarce and the transcriptomic regulation of genes involved in BCAA catabolism is partially unknown. This study aimed to identify differences in circulating and skeletal muscle BCAA levels in response to an OGTT in individuals with normal glucose tolerance (NGT) or type 2 diabetes. Additionally, transcription factors involved in the regulation of the BCAA gene set were identified. METHODS: Plasma and vastus lateralis muscle biopsies were obtained from individuals with NGT or type 2 diabetes before and after an OGTT. Plasma and quadriceps muscles were harvested from skeletal muscle-specific Ppargc1a knockout and transgenic mice. BCAA-related metabolites and genes were assessed by LC-MS/MS and quantitative RT-PCR, respectively. Small interfering RNA and adenovirus-mediated overexpression techniques were used in primary human skeletal muscle cells to study the role of PPARGC1A and ESRRA in the expression of the BCAA gene set. Radiolabelled leucine was used to analyse the impact of oestrogen-related receptor α (ERRα) knockdown on leucine oxidation. RESULTS: Impairments in BCAA catabolism in people with type 2 diabetes under fasting conditions were exacerbated after a glucose load. Branched-chain keto acids were reduced 37-56% after an OGTT in the NGT group, whereas no changes were detected in individuals with type 2 diabetes. These changes were concomitant with a stronger correlation with glucose homeostasis biomarkers and downregulated expression of branched-chain amino acid transaminase 2, branched-chain keto acid dehydrogenase complex subunits and 69% of downstream BCAA-related genes in skeletal muscle. In primary human myotubes overexpressing peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α, encoded by PPARGC1A), 61% of the analysed BCAA genes were upregulated, while 67% were downregulated in the quadriceps of skeletal muscle-specific Ppargc1a knockout mice. ESRRA (encoding ERRα) silencing completely abrogated the PGC-1α-induced upregulation of BCAA-related genes in primary human myotubes. CONCLUSIONS/INTERPRETATION: Metabolic inflexibility in type 2 diabetes impacts BCAA homeostasis and attenuates the decrease in circulating and skeletal muscle BCAA-related metabolites after a glucose challenge. Transcriptional regulation of BCAA genes in primary human myotubes via PGC-1α is ERRα-dependent.


Assuntos
Diabetes Mellitus Tipo 2 , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Cromatografia Líquida , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Humanos , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Receptores de Estrogênio , Espectrometria de Massas em Tandem , Receptor ERRalfa Relacionado ao Estrogênio
10.
Gene Ther ; 28(9): 477-493, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34276045

RESUMO

Inherited deficiency of the antiprotease alpha-1 antitrypsin (AAT) is associated with liver failure and early-onset emphysema. In mice, in vivo lentiviral transduction of alveolar macrophages (AMs) has been described to yield protective pulmonary AAT levels and ameliorate emphysema development. We here investigated the pulmonary transplantation of macrophages (PMT) transgenic for AAT as a potential therapy for AAT deficiency-associated lung pathology. Employing third-generation SIN-lentiviral vectors expressing the human AAT cDNA from the CAG or Cbx-EF1α promoter, we obtained high-level AAT secretion in a murine AM cell line as well as murine bone marrow-derived macrophages differentiated in vitro (AAT MΦ). Secreted AAT demonstrated a physiologic glycosylation pattern as well as elastase-inhibitory and anti-apoptotic properties. AAT MΦ preserved normal morphology, surface phenotype, and functionality. Furthermore, in vitro generated murine AAT MΦ successfully engrafted in AM-deficient Csf2rb-/- mice and converted into a CD11c+/Siglec-F+ AM phenotype as detected in bronchoalveolar lavage fluid and homogenized lung tissue 2 months after PMT. Moreover, human AAT was detected in the lung epithelial lining fluid of transplanted animals. Efficient AAT expression and secretion were also demonstrated for human AAT MΦ, confirming the applicability of our vectors in human cells.


Assuntos
Enfisema Pulmonar , Deficiência de alfa 1-Antitripsina , Animais , Animais Geneticamente Modificados , Humanos , Pulmão , Macrófagos , Camundongos , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/terapia
11.
J Proteome Res ; 19(1): 269-278, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31625748

RESUMO

Alum has been widely used as an adjuvant for human vaccines; however, the impact of Alum on host metabolism remains largely unknown. Herein, we applied mass spectrometry (MS) (liquid chromatography-MS)-based metabolic and lipid profiling to monitor the effects of the Alum adjuvant on mouse serum at 6, 24, 72, and 168 h post-vaccination. We propose a new strategy termed subclass identification and annotation for metabolomics for class-wise identification of untargeted metabolomics data generated from high-resolution MS. Using this approach, we identified and validated the levels of several lipids in mouse serum that were significantly altered following Alum administration. These lipids showed a biphasic response even 168 h after vaccination. The majority of the lipids were triglycerides (TAGs), where TAGs with long-chain unsaturated fatty acids (FAs) decreased at 24 h and TAGs with short-chain FAs decreased at 168 h. To our knowledge, this is the first report on the impact of human vaccine adjuvant Alum on the host metabolome, which may provide new insights into the mechanism of action of Alum.


Assuntos
Adjuvantes Imunológicos/farmacologia , Compostos de Alúmen/farmacologia , Metabolômica/métodos , Triglicerídeos/sangue , Animais , Antígenos de Bactérias/administração & dosagem , Cromatografia Líquida , Feminino , Imunização , Lipídeos/sangue , Espectrometria de Massas , Camundongos Endogâmicos , Reprodutibilidade dos Testes , Fatores de Tempo , Vacinas contra a Tuberculose/farmacologia
12.
Gene Ther ; 27(6): 297-306, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32051561

RESUMO

Induced pluripotent stem cells (iPSCs) from patients with genetic disorders are a valuable source for in vitro disease models, which enable drug testing and validation of gene and cell therapies. We generated iPSCs from a severe congenital neutropenia (SCN) patient, who presented with a nonsense mutation in the glucose-6-phosphatase catalytic subunit 3 (G6PC3) gene causing profound defects in granulopoiesis, associated with increased susceptibility of neutrophils to apoptosis. Generated SCN iPSC clones exhibited the capacity to differentiate into hematopoietic cells of the myeloid lineage and we identified two cytokine conditions, i.e., using granulocyte-colony stimulating factor or granulocyte-macrophage colony stimulating factor in combination with interleukin-3, to model the SCN phenotype in vitro. Reduced numbers of granulocytes were produced by SCN iPSCs compared with control iPSCs in both settings, which reflected the phenotype in patients. Interestingly, our model showed increased monocyte/macrophage production from the SCN iPSCs. Most importantly, lentiviral genetic correction of SCN iPSCs with a codon-optimized G6PC3 transgene restored granulopoiesis and reduced apoptosis of in vitro differentiated myeloid cells. Moreover, addition of vitamin B3 clearly induced granulocytic differentiation of SCN iPSCs and increased the number of neutrophils to levels comparable with those obtained from healthy control iPSCs. In summary, we established an iPSC-derived in vitro disease model, which will serve as a tool to test the potency of alternative treatment options for SCN patients, such as small molecules and gene therapeutic vectors.


Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Terapia Genética , Glucose-6-Fosfatase , Fator Estimulador de Colônias de Granulócitos , Humanos , Niacinamida
13.
New Phytol ; 228(5): 1559-1572, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32648607

RESUMO

Wood, or secondary xylem, is the product of xylogenesis, a developmental process that begins with the proliferation of cambial derivatives and ends with mature xylem fibers and vessels with lignified secondary cell walls. Fully mature xylem has undergone a series of cellular processes, including cell division, cell expansion, secondary wall formation, lignification and programmed cell death. A complex network of interactions between transcriptional regulators and signal transduction pathways controls wood formation. However, the role of metabolites during this developmental process has not been comprehensively characterized. To evaluate the role of metabolites during wood formation, we performed a high spatial resolution metabolomics study of the wood-forming zone of Populus tremula, including laser dissected aspen ray and fiber cells. We show that metabolites show specific patterns within the wood-forming zone, following the differentiation process from cell division to cell death. The data from profiled laser dissected aspen ray and fiber cells suggests that these two cell types host distinctly different metabolic processes. Furthermore, by integrating previously published transcriptomic and proteomic profiles generated from the same trees, we provide an integrative picture of molecular processes, for example, deamination of phenylalanine during lignification is of critical importance for nitrogen metabolism during wood formation.


Assuntos
Populus , Proteômica , Madeira , Câmbio , Regulação da Expressão Gênica de Plantas , Populus/genética , Xilema
14.
Plant Physiol ; 181(4): 1704-1720, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31551361

RESUMO

The plant cell wall plays an important role in damage-associated molecular pattern-induced resistance to pathogens and herbivorous insects. Our current understanding of cell wall-mediated resistance is largely based on the degree of pectin methylesterification. However, little is known about the role of pectin acetylesterification in plant immunity. This study describes how one pectin-modifying enzyme, PECTIN ACETYLESTERASE 9 (PAE9), affects the Arabidopsis (Arabidopsis thaliana) transcriptome, secondary metabolome, and aphid performance. Electro-penetration graphs showed that Myzus persicae aphids established phloem feeding earlier on pae9 mutants. Whole-genome transcriptome analysis revealed a set of 56 differentially expressed genes (DEGs) between uninfested pae9-2 mutants and wild-type plants. The majority of the DEGs were enriched for biotic stress responses and down-regulated in the pae9-2 mutant, including PAD3 and IGMT2, involved in camalexin and indole glucosinolate biosynthesis, respectively. Relative quantification of more than 100 secondary metabolites revealed decreased levels of several compounds, including camalexin and oxylipins, in two independent pae9 mutants. In addition, absolute quantification of phytohormones showed that jasmonic acid (JA), jasmonoyl-Ile, salicylic acid, abscisic acid, and indole-3-acetic acid were compromised due to PAE9 loss of function. After aphid infestation, however, pae9 mutants increased their levels of camalexin, glucosinolates, and JA, and no long-term effects were observed on aphid fitness. Overall, these data show that PAE9 is required for constitutive up-regulation of defense-related compounds, but that it is not required for aphid-induced defenses. The signatures of phenolic antioxidants, phytoprostanes, and oxidative stress-related transcripts indicate that the processes underlying PAE9 activity involve oxidation-reduction reactions.


Assuntos
Acetilesterase/metabolismo , Afídeos/fisiologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Herbivoria/fisiologia , Metaboloma/genética , Transcriptoma/genética , Animais , Arabidopsis/parasitologia , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Genes Reguladores , Glucosinolatos/metabolismo , Indóis/metabolismo , Mutação/genética , Estresse Oxidativo , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Metabolismo Secundário , Tiazóis/metabolismo , Fatores de Transcrição/metabolismo
15.
Haematologica ; 105(4): 1147-1157, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31289207

RESUMO

Hereditary pulmonary alveolar proteinosis due to GM-CSF receptor deficiency (herPAP) constitutes a life-threatening lung disease characterized by alveolar deposition of surfactant protein secondary to defective alveolar macrophage function. As current therapeutic options are primarily symptomatic, we have explored the potential of hematopoietic stem cell-based gene therapy. Using Csf2rb-/- mice, a model closely reflecting the human herPAP disease phenotype, we here demonstrate robust pulmonary engraftment of an alveolar macrophage population following intravenous transplantation of lentivirally corrected hematopoietic stem and progenitor cells. Engraftment was associated with marked improvement of critical herPAP disease parameters, including bronchoalveolar fluid protein, cholesterol and cytokine levels, pulmonary density on computed tomography scans, pulmonary deposition of Periodic Acid-Schiff+ material as well as respiratory mechanics. These effects were stable for at least nine months. With respect to engraftment and alveolar macrophage differentiation kinetics, we demonstrate the rapid development of CD11c+/SiglecF+ cells in the lungs from a CD11c-/SiglecF+ progenitor population within four weeks after transplantation. Based on these data, we suggest hematopoietic stem cell-based gene therapy as an effective and cause-directed treatment approach for herPAP.


Assuntos
Proteinose Alveolar Pulmonar , Animais , Modelos Animais de Doenças , Terapia Genética , Células-Tronco Hematopoéticas , Macrófagos Alveolares , Camundongos , Proteinose Alveolar Pulmonar/genética , Proteinose Alveolar Pulmonar/terapia
16.
Nature ; 514(7523): 450-4, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25274301

RESUMO

Bone-marrow transplantation is an effective cell therapy but requires myeloablation, which increases infection risk and mortality. Recent lineage-tracing studies documenting that resident macrophage populations self-maintain independently of haematological progenitors prompted us to consider organ-targeted, cell-specific therapy. Here, using granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor-ß-deficient (Csf2rb(-/-)) mice that develop a myeloid cell disorder identical to hereditary pulmonary alveolar proteinosis (hPAP) in children with CSF2RA or CSF2RB mutations, we show that pulmonary macrophage transplantation (PMT) of either wild-type or Csf2rb-gene-corrected macrophages without myeloablation was safe and well-tolerated and that one administration corrected the lung disease, secondary systemic manifestations and normalized disease-related biomarkers, and prevented disease-specific mortality. PMT-derived alveolar macrophages persisted for at least one year as did therapeutic effects. Our findings identify mechanisms regulating alveolar macrophage population size in health and disease, indicate that GM-CSF is required for phenotypic determination of alveolar macrophages, and support translation of PMT as the first specific therapy for children with hPAP.


Assuntos
Transplante de Células , Subunidade beta Comum dos Receptores de Citocinas/genética , Terapia Genética , Pulmão/citologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/transplante , Proteinose Alveolar Pulmonar/terapia , Animais , Separação Celular , Subunidade beta Comum dos Receptores de Citocinas/deficiência , Feminino , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proteinose Alveolar Pulmonar/genética , Proteinose Alveolar Pulmonar/patologia , Fatores de Tempo
17.
Mol Ther ; 27(9): 1597-1611, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31326401

RESUMO

Hereditary pulmonary alveolar proteinosis (PAP) is a genetic lung disease characterized by surfactant accumulation and respiratory failure arising from disruption of GM-CSF signaling. While mutations in either CSF2RA or CSF2RB (encoding GM-CSF receptor α or ß chains, respectively) can cause PAP, α chain mutations are responsible in most patients. Pulmonary macrophage transplantation (PMT) is a promising new cell therapy in development; however, no studies have evaluated this approach for hereditary PAP (hPAP) caused by Csf2ra mutations. Here, we report on the preclinical safety, tolerability, and efficacy of lentiviral-vector (LV)-mediated Csf2ra expression in macrophages and PMT of gene-corrected macrophages (gene-PMT therapy) in Csf2ra gene-ablated (Csf2ra-/-) mice. Gene-PMT therapy resulted in a stable transgene integration and correction of GM-CSF signaling and functions in Csf2ra-/- macrophages in vitro and in vivo and resulted in engraftment and long-term persistence of gene-corrected macrophages in alveoli; restoration of pulmonary surfactant homeostasis; correction of PAP-specific cytologic, histologic, and biomarker abnormalities; and reduced inflammation associated with disease progression in untreated mice. No adverse consequences of gene-PMT therapy in Csf2ra-/- mice were observed. Results demonstrate that gene-PMT therapy of hPAP in Csf2ra-/- mice was highly efficacious, durable, safe, and well tolerated.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Terapia Genética , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/transplante , Proteinose Alveolar Pulmonar/genética , Proteinose Alveolar Pulmonar/terapia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Animais , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos/métodos , Modelos Animais de Doenças , Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/genética , Imunofenotipagem , Lentivirus/genética , Camundongos , Camundongos Knockout , Proteinose Alveolar Pulmonar/diagnóstico , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de Sinais , Transdução Genética
18.
Nucleic Acids Res ; 46(11): e66, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29554314

RESUMO

Information about the intracellular concentration of dNTPs and NTPs is important for studies of the mechanisms of DNA replication and repair, but the low concentration of dNTPs and their chemical similarity to NTPs present a challenge for their measurement. Here, we describe a new rapid and sensitive method utilizing hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the simultaneous determination of dNTPs and NTPs in biological samples. The developed method showed linearity (R2 > 0.99) in wide concentration ranges and could accurately quantify dNTPs and NTPs at low pmol levels. The intra-day and inter-day precision were below 13%, and the relative recovery was between 92% and 108%. In comparison with other chromatographic methods, the current method has shorter analysis times and simpler sample pre-treatment steps, and it utilizes an ion-pair-free mobile phase that enhances mass-spectrometric detection. Using this method, we determined dNTP and NTP concentrations in actively dividing and quiescent mouse fibroblasts.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Desoxirribonucleotídeos/análise , Ribonucleotídeos/análise , Espectrometria de Massas em Tandem/métodos , Células 3T3 , Animais , Linhagem Celular , Interações Hidrofóbicas e Hidrofílicas , Camundongos
19.
Int J Mol Sci ; 21(7)2020 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-32260086

RESUMO

Induced pluripotent stem cells (iPSCs) offer great promise for the field of regenerative medicine, and iPSC-derived cells have already been applied in clinical practice. However, potential contamination of effector cells with residual pluripotent cells (e.g., teratoma-initiating cells) or effector cell-associated side effects may limit this approach. This also holds true for iPSC-derived hematopoietic cells. Given the therapeutic benefit of macrophages in different disease entities and the feasibility to derive macrophages from human iPSCs, we established human iPSCs harboring the inducible Caspase-9 (iCasp9) suicide safety switch utilizing transcription activator-like effector nuclease (TALEN)-based designer nuclease technology. Mono- or bi-allelic integration of the iCasp9 gene cassette into the AAVS1 locus showed no effect on the pluripotency of human iPSCs and did not interfere with their differentiation towards macrophages. In both, iCasp9-mono and iCasp9-bi-allelic clones, concentrations of 0.1 nM AP20187 were sufficient to induce apoptosis in more than 98% of iPSCs and their progeny-macrophages. Thus, here we provide evidence that the introduction of the iCasp9 suicide gene into the AAVS1 locus enables the effective clearance of human iPSCs and thereof derived macrophages.


Assuntos
Caspase 9/genética , Células-Tronco Pluripotentes Induzidas/citologia , Macrófagos/citologia , Caspase 9/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Genes Transgênicos Suicidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Macrófagos/metabolismo , Medicina Regenerativa , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia
20.
Plant Physiol ; 177(1): 132-150, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29523713

RESUMO

In plants, an individually darkened leaf initiates senescence much more rapidly than a leaf from a whole darkened plant. Combining transcriptomic and metabolomic approaches in Arabidopsis (Arabidopsis thaliana), we present an overview of the metabolic strategies that are employed in response to different darkening treatments. Under darkened plant conditions, the perception of carbon starvation drove a profound metabolic readjustment in which branched-chain amino acids and potentially monosaccharides released from cell wall loosening became important substrates for maintaining minimal ATP production. Concomitantly, the increased accumulation of amino acids with a high nitrogen-carbon ratio may provide a safety mechanism for the storage of metabolically derived cytotoxic ammonium and a pool of nitrogen for use upon returning to typical growth conditions. Conversely, in individually darkened leaf, the metabolic profiling that followed our 13C-enrichment assays revealed a temporal and differential exchange of metabolites, including sugars and amino acids, between the darkened leaf and the rest of the plant. This active transport could be the basis for a progressive metabolic shift in the substrates fueling mitochondrial activities, which are central to the catabolic reactions facilitating the retrieval of nutrients from the senescing leaf. We propose a model illustrating the specific metabolic strategies employed by leaves in response to these two darkening treatments, which support either rapid senescence or a strong capacity for survival.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Folhas de Planta/fisiologia , Proteínas de Arabidopsis/metabolismo , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Escuridão , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Luz , Metabolômica/métodos , Modelos Biológicos , Pigmentação , Folhas de Planta/metabolismo , Amido/metabolismo
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