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1.
Theranostics ; 11(17): 8430-8447, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34373751

RESUMO

Self-assembly of solid organs from single cells would greatly expand applicability of regenerative medicine. Stem/progenitor cells can self-organize into micro-sized organ units, termed organoids, partially modelling tissue function and regeneration. Here we demonstrated 3D self-assembly of adult and induced pluripotent stem cell (iPSC)-derived fibroblasts, keratinocytes and endothelial progenitors into both, planar human skin in vivo and a novel type of spheroid-shaped skin organoids in vitro, under the aegis of human platelet lysate. Methods: Primary endothelial colony forming cells (ECFCs), skin fibroblasts (FBs) and keratinocytes (KCs) were isolated from human tissues and polyclonally propagated under 2D xeno-free conditions. Human tissue-derived iPSCs were differentiated into endothelial cells (hiPSC-ECs), fibroblasts (hiPSC-FBs) and keratinocytes (hiPSC-KCs) according to efficiency-optimized protocols. Cell identity and purity were confirmed by flow cytometry and clonogenicity indicated their stem/progenitor potential. Triple cell type floating spheroids formation was promoted by human platelet-derived growth factors containing culture conditions, using nanoparticle cell labelling for monitoring the organization process. Planar human skin regeneration was assessed in full-thickness wounds of immune-deficient mice upon transplantation of hiPSC-derived single cell suspensions. Results: Organoids displayed a distinct architecture with surface-anchored keratinocytes surrounding a stromal core, and specific signaling patterns in response to inflammatory stimuli. FGF-7 mRNA transfection was required to accelerate keratinocyte long-term fitness. Stratified human skin also self-assembled within two weeks after either adult- or iPSC-derived skin cell-suspension liquid-transplantation, healing deep wounds of mice. Transplant vascularization significantly accelerated in the presence of co-transplanted endothelial progenitors. Mechanistically, extracellular vesicles mediated the multifactorial platelet-derived trophic effects. No tumorigenesis occurred upon xenografting. Conclusion: This illustrates the superordinate progenitor self-organization principle and permits novel rapid 3D skin-related pharmaceutical high-content testing opportunities with floating spheroid skin organoids. Multi-cell transplant self-organization facilitates development of iPSC-based organ regeneration strategies using cell suspension transplantation supported by human platelet factors.

2.
Adv Drug Deliv Rev ; 176: 113872, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34284058

RESUMO

Rigorous measures are required to cope with the advance of extracellular vesicle (EV) research, from 183 studies published in 2012 to 2,309 studies published in 2020. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines in 2014, updated in 2018, for assuring and improving EV research quality. We performed a systematic review using a text mining approach to assess adherence to MISEV criteria. A keyword search was conducted in 5,093 accessible publications over the period 2012-2020 and analyzed the methodology used for EV isolation and characterization. We found a significant improvement over the years particularly regarding EV characterization where recent papers used a higher number of methods and EV markers to check for quantity and purity. Interestingly, we also found that EV papers using more methods and EV markers were cited more frequently. Papers citing MISEV criteria were more prone to use a higher number of characterization methods. We therefore established a concise checklist summarizing MISEV criteria to support EV researchers towards reaching the highest standards in the field.

3.
Int J Mol Sci ; 22(5)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803482

RESUMO

Alzheimer's disease (AD) is the most common form of dementia. In particular, neuroinflammation, mediated by microglia cells but also through CD8+ T-cells, actively contributes to disease pathology. Leukotrienes are involved in neuroinflammation and in the pathological hallmarks of AD. In consequence, leukotriene signaling-more specifically, the leukotriene receptors-has been recognized as a potential drug target to ameliorate AD pathology. Here, we analyzed the effects of the leukotriene receptor antagonist montelukast (MTK) on hippocampal gene expression in 5xFAD mice, a commonly used transgenic AD mouse model. We identified glial activation and neuroinflammation as the main pathways modulated by MTK. The treatment increased the number of Tmem119+ microglia and downregulated genes related to AD-associated microglia and to lipid droplet-accumulating microglia, suggesting that the MTK treatment targets and modulates microglia phenotypes in the disease model compared to the vehicle. MTK treatment further reduced infiltration of CD8+T-cells into the brain parenchyma. Finally, MTK treatment resulted in improved cognitive functions. In summary, we provide a proof of concept for MTK to be a potential drug candidate for AD and provide novel modes of action via modulation of microglia and CD8+ T-cells. Of note, 5xFAD females showed a more severe pathology, and in consequence, MTK treatment had a more pronounced effect in the females compared to the males. The effects on neuroinflammation, i.e., microglia and CD8+ T-cells, as well as the effects on cognitive outcome, were dose-dependent, therefore arguing for the use of higher doses of MTK in AD clinical trials compared to the approved asthma dose.


Assuntos
Acetatos/farmacologia , Doença de Alzheimer/tratamento farmacológico , Encéfalo/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Cognição/efeitos dos fármacos , Ciclopropanos/farmacologia , Antagonistas de Leucotrienos/farmacologia , Quinolinas/farmacologia , Sulfetos/farmacologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/patologia , Linfócitos T CD8-Positivos/patologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Transgênicos
4.
Int J Mol Sci ; 22(8)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918735

RESUMO

Stem cells secrete paracrine factors including extracellular vesicles (EVs) which can mediate cellular communication and support the regeneration of injured tissues. Reduced oxygen (hypoxia) as a key regulator in development and regeneration may influence cellular communication via EVs. We asked whether hypoxic conditioning during human induced pluripotent stem cell (iPSC) culture effects their EV quantity, quality or EV-based angiogenic potential. We produced iPSC-EVs from large-scale culture-conditioned media at 1%, 5% and 18% air oxygen using tangential flow filtration (TFF), with or without subsequent concentration by ultracentrifugation (TUCF). EVs were quantified by tunable resistive pulse sensing (TRPS), characterized according to MISEV2018 guidelines, and analyzed for angiogenic potential. We observed superior EV recovery by TFF compared to TUCF. We confirmed hypoxia efficacy by HIF-1α stabilization and pimonidazole hypoxyprobe. EV quantity did not differ significantly at different oxygen conditions. Significantly elevated angiogenic potential was observed for iPSC-EVs derived from 1% oxygen culture by TFF or TUCF as compared to EVs obtained at higher oxygen or the corresponding EV-depleted soluble factor fractions. Data thus demonstrate that cell-culture oxygen conditions and mode of EV preparation affect iPSC-EV function. We conclude that selecting appropriate protocols will further improve production of particularly potent iPSC-EV-based therapeutics.


Assuntos
Vesículas Extracelulares/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neovascularização Fisiológica , Transporte Biológico , Biomarcadores , Hipóxia Celular , Autorrenovação Celular , Células Cultivadas , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Medicina Regenerativa/métodos
5.
Neurotherapeutics ; 18(3): 1963-1979, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33860461

RESUMO

Adult neurogenesis is a target for brain rejuvenation as well as regeneration in aging and disease. Numerous approaches showed efficacy to elevate neurogenesis in rodents, yet translation into therapies has not been achieved. Here, we introduce a novel human TGFß-RII (Transforming Growth Factor-Receptor Type II) specific LNA-antisense oligonucleotide ("locked nucleotide acid"-"NVP-13"), which reduces TGFß-RII expression and downstream receptor signaling in human neuronal precursor cells (ReNcell CX® cells) in vitro. After we injected cynomolgus non-human primates repeatedly i.th. with NVP-13 in a preclinical regulatory 13-week GLP-toxicity program, we could specifically downregulate TGFß-RII mRNA and protein in vivo. Subsequently, we observed a dose-dependent upregulation of the neurogenic niche activity within the hippocampus and subventricular zone: human neural progenitor cells showed significantly (up to threefold over control) enhanced differentiation and cell numbers. NVP-13 treatment modulated canonical and non-canonical TGFß pathways, such as MAPK and PI3K, as well as key transcription factors and epigenetic factors involved in stem cell maintenance, such as MEF2A and pFoxO3. The latter are also dysregulated in clinical neurodegeneration, such as amyotrophic lateral sclerosis. Here, we provide for the first time in vitro and in vivo evidence for a novel translatable approach to treat neurodegenerative disorders by modulating neurogenesis.

6.
BMC Biol ; 18(1): 157, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33121485

RESUMO

BACKGROUND: Insect pest control programs often use periods of insecticide treatment with intermittent breaks, to prevent fixing of mutations conferring insecticide resistance. Such mutations are typically costly in an insecticide-free environment, and their frequency is determined by the balance between insecticide treatment and cost of resistance. Ace, a key gene in neuronal signaling, is a prominent target of many insecticides and across several species, three amino acid replacements (I161V, G265A, and F330Y) provide resistance against several insecticides. Because temperature disturbs neuronal signaling homeostasis, we reasoned that the cost of insecticide resistance could be modulated by ambient temperature. RESULTS: Experimental evolution of a natural Drosophila simulans population at hot and cold temperature regimes uncovered a surprisingly strong effect of ambient temperature. In the cold temperature regime, the resistance mutations were strongly counter selected (s = - 0.055), but in a hot environment, the fitness costs of resistance mutations were reduced by almost 50% (s = - 0.031). We attribute this unexpected observation to the advantage of the reduced enzymatic activity of resistance mutations in hot environments. CONCLUSION: We show that fitness costs of insecticide resistance genes are temperature-dependent and suggest that the duration of insecticide-free periods need to be adjusted for different climatic regions to reflect these costs. We suggest that such environment-dependent fitness effects may be more common than previously assumed and pose a major challenge for modeling climate change.


Assuntos
Drosophila melanogaster/genética , Aptidão Genética , Resistência a Inseticidas/genética , Mutação , Temperatura , Animais , Drosophila melanogaster/efeitos dos fármacos , Inseticidas/farmacologia
7.
Sci Rep ; 9(1): 7258, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31076619

RESUMO

Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.


Assuntos
Expressão Gênica/fisiologia , Heparina/metabolismo , Células Estromais/metabolismo , Plaquetas/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Soro/metabolismo , Transdução de Sinais/fisiologia , Cordão Umbilical/metabolismo
8.
Insect Biochem Mol Biol ; 105: 60-68, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30660665

RESUMO

The biochemical and molecular mechanisms underlying insect cold acclimation prior to cold stress are relatively well explored, but the mechanisms linked to recovery and repair after cold stress have received much less attention. Here we focus on recovery from cold stress in the larvae of the vinegar fly (Drosophila melanogaster) that were exposed to two physiologically distinct cold stress situations: supercooling (S, survival > 95%) and freezing (F, survival < 10%), both at -5 °C. We analysed the metabolic and transcriptomic responses to cold stress via GC-MS/LC-MS and whole-genome microarrays, respectively. Both stresses (S and F) caused metabolic perturbations which were transient in supercooled larvae but deeper and irreversible in frozen larvae. Differential gene expression analysis revealed a clear disparity in responses to supercooling and freezing (less than 10% of DE genes overlapped between S and F larvae). Using GO term enrichment analysis and KEGG pathway mapping, we identified the stimulation of immune response pathways as a strong candidate mechanism for coping with supercooling. Supercooling caused complex transcriptional activation of innate immunity potential: from Lysozyme-mediated degradation of bacterial cell walls, recognition of pathogen signals, through phagocytosis and lysosomal degradation, Toll and Imd signaling, to upregulation of genes coding for different antimicrobial peptides. The transcriptomic response to freezing was instead dominated by degradation of macromolecules and death-related processes such as autophagy and apoptosis. Of the 45 upregulated DE genes overlapping in responses to supercooling and freezing, 26 were broadly ascribable to defense and repair functions.


Assuntos
Drosophila melanogaster/imunologia , Estresse Fisiológico/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Temperatura Baixa , Drosophila melanogaster/metabolismo , Feminino , Larva/imunologia , Larva/metabolismo
9.
Sci Rep ; 8(1): 4469, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29535355

RESUMO

Fecundity is probably the most frequently studied fitness component in Drosophila. Nevertheless, currently used methods to measure fecundity are not well-suited for large-scale experiments, with many populations being assayed in parallel. Here we present a standardized pipeline to measure fecundity in many Drosophila population samples with substantially reduced hand on times. Using a high-contrast medium for egg laying, we developed a Java plug-in for ImageJ to quantify the number of eggs by image processing. We show that our method is fast and provides reliable egg counts.


Assuntos
Drosophila melanogaster/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Algoritmos , Animais , Feminino , Fertilidade , Oviposição
10.
Proc Natl Acad Sci U S A ; 114(32): 8532-8537, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28720705

RESUMO

Insects often overcome unfavorable seasons in a hormonally regulated state of diapause during which their activity ceases, development is arrested, metabolic rate is suppressed, and tolerance of environmental stress is bolstered. Diapausing insects pass through a stereotypic succession of eco-physiological phases termed "diapause development." The phasing is varied in the literature, and the whole concept is sometimes criticized as being too artificial. Here we present the results of transcriptional profiling using custom microarrays representing 1,042 genes in the drosophilid fly, Chymomyza costata Fully grown, third-instar larvae programmed for diapause by a photoperiodic (short-day) signal were assayed as they traversed the diapause developmental program. When analyzing the gradual dynamics in the transcriptomic profile, we could readily distinguish distinct diapause developmental phases associated with induction/initiation, maintenance, cold acclimation, and termination by cold or by photoperiodic signal. Accordingly, each phase is characterized by a specific pattern of gene expression, supporting the physiological relevance of the concept of diapause phasing. Further, we have dissected in greater detail the changes in transcript levels of elements of several signaling pathways considered critical for diapause regulation. The phase of diapause termination is associated with enhanced transcript levels in several positive elements stimulating direct development (the 20-hydroxyecdysone pathway: Ecr, Shd, Broad; the Wnt pathway: basket, c-jun) that are countered by up-regulation in some negative elements (the insulin-signaling pathway: Ilp8, PI3k, Akt; the target of rapamycin pathway: Tsc2 and 4EBP; the Wnt pathway: shaggy). We speculate such up-regulations may represent the early steps linked to termination of diapause programming.


Assuntos
Diapausa de Inseto/genética , Drosophilidae/genética , Animais , Ritmo Circadiano/genética , Diapausa/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Insetos/genética , Insetos/genética , Larva/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fotoperíodo , Transcriptoma
11.
PLoS Negl Trop Dis ; 11(4): e0005526, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28379969

RESUMO

BACKGROUND: The capacity of Aedes mosquitoes to resist chemical insecticides threatens the control of major arbovirus diseases worldwide. Until alternative control tools are widely deployed, monitoring insecticide resistance levels and identifying resistance mechanisms in field mosquito populations is crucial for implementing appropriate management strategies. Metabolic resistance to pyrethroids is common in Aedes aegypti but the monitoring of the dynamics of resistant alleles is impeded by the lack of robust genomic markers. METHODOLOGY/PRINCIPAL FINDINGS: In an attempt to identify the genomic bases of metabolic resistance to deltamethrin, multiple resistant and susceptible populations originating from various continents were compared using both RNA-seq and a targeted DNA-seq approach focused on the upstream regions of detoxification genes. Multiple detoxification enzymes were over transcribed in resistant populations, frequently associated with an increase in their gene copy number. Targeted sequencing identified potential promoter variations associated with their over transcription. Non-synonymous variations affecting detoxification enzymes were also identified in resistant populations. CONCLUSION /SIGNIFICANCE: This study not only confirmed the role of gene copy number variations as a frequent cause of the over expression of detoxification enzymes associated with insecticide resistance in Aedes aegypti but also identified novel genomic resistance markers potentially associated with their cis-regulation and modifications of their protein structure conformation. As for gene transcription data, polymorphism patterns were frequently conserved within regions but differed among continents confirming the selection of different resistance factors worldwide. Overall, this study paves the way of the identification of a comprehensive set of genomic markers for monitoring the spatio-temporal dynamics of the variety of insecticide resistance mechanisms in Aedes aegypti.


Assuntos
Aedes/genética , Variações do Número de Cópias de DNA , Genômica , Resistência a Inseticidas/genética , Inseticidas/metabolismo , Nitrilas/metabolismo , Piretrinas/metabolismo , Aedes/efeitos dos fármacos , Aedes/metabolismo , Animais , Feminino , Perfilação da Expressão Gênica , Marcadores Genéticos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Inativação Metabólica , Inseticidas/farmacologia , Nitrilas/farmacologia , Piretrinas/farmacologia , Análise de Sequência de DNA , Análise de Sequência de RNA
12.
Malar J ; 15(1): 424, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27549778

RESUMO

BACKGROUND: Resistance to the carbamate insecticide bendiocarb is emerging in Anopheles gambiae populations from the city of Yaoundé in Cameroon. However, the molecular basis of this resistance remains uncharacterized. The present study objective is to investigate mechanisms promoting resistance to bendiocarb in An. gambiae populations from Yaoundé. METHODS: The level of susceptibility of An. gambiae s.l. to bendiocarb 0.1 % was assessed from 2010 to 2013 using bioassays. Mosquitoes resistant to bendiocarb, unexposed and susceptible mosquitoes were screened for the presence of the Ace-1(R) mutation using TaqMan assays. Microarray analyses were performed to assess the pattern of genes differentially expressed between resistant, unexposed and susceptible. RESULTS: Bendiocarb resistance was more prevalent in mosquitoes originating from cultivated sites compared to those from polluted and unpolluted sites. Both An. gambiae and Anopheles coluzzii were found to display resistance to bendiocarb. No G119S mutation was detected suggesting that resistance was mainly metabolic. Microarray analysis revealed the over-expression of several cytochrome P450 s genes including cyp6z3, cyp6z1, cyp12f2, cyp6m3 and cyp6p4. Gene ontology (GO) enrichment analysis supported the detoxification role of cytochrome P450 s with several GO terms associated with P450 activity significantly enriched in resistant samples. Other detoxification genes included UDP-glucosyl transferases, glutathione-S transferases and ABC transporters. CONCLUSION: The study highlights the probable implication of metabolic mechanisms in bendiocarb resistance in An. gambiae populations from Yaoundé and stresses the need for further studies leading to functional validation of detoxification genes involved in this resistance.


Assuntos
Anopheles/efeitos dos fármacos , Resistência a Inseticidas , Inseticidas/farmacologia , Fenilcarbamatos/farmacologia , Animais , Bioensaio , Camarões , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Perfilação da Expressão Gênica , Inativação Metabólica , Análise em Microsséries
13.
J Exp Biol ; 219(Pt 15): 2358-67, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27489218

RESUMO

The fruit fly Drosophila melanogaster is an insect of tropical origin. Its larval stage is evolutionarily adapted for rapid growth and development under warm conditions and shows high sensitivity to cold. In this study, we further developed an optimal acclimation and freezing protocol that significantly improves larval freeze tolerance (an ability to survive at -5°C when most of the freezable fraction of water is converted to ice). Using the optimal protocol, freeze survival to adult stage increased from 0.7% to 12.6% in the larvae fed standard diet (agar, sugar, yeast, cornmeal). Next, we fed the larvae diets augmented with 31 different amino compounds, administered in different concentrations, and observed their effects on larval metabolomic composition, viability, rate of development and freeze tolerance. While some diet additives were toxic, others showed positive effects on freeze tolerance. Statistical correlation revealed tight association between high freeze tolerance and high levels of amino compounds involved in arginine and proline metabolism. Proline- and arginine-augmented diets showed the highest potential, improving freeze survival to 42.1% and 50.6%, respectively. Two plausible mechanisms by which high concentrations of proline and arginine might stimulate high freeze tolerance are discussed: (i) proline, probably in combination with trehalose, could reduce partial unfolding of proteins and prevent membrane fusions in the larvae exposed to thermal stress (prior to freezing) or during freeze dehydration; (ii) both arginine and proline are exceptional among amino compounds in their ability to form supramolecular aggregates which probably bind partially unfolded proteins and inhibit their aggregation under increasing freeze dehydration.


Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Arginina/farmacologia , Drosophila melanogaster/fisiologia , Aditivos Alimentares/farmacologia , Congelamento , Prolina/farmacologia , Aclimatação/efeitos dos fármacos , Animais , Dieta , Drosophila melanogaster/efeitos dos fármacos , Larva , Metabolômica , Análise de Componente Principal
14.
Sci Rep ; 6: 32346, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27573891

RESUMO

The cryopreservation techniques proposed for embryos of the fruit fly Drosophila melanogaster are not yet ready for practical use. Alternative methods for long-term storage of D. melanogaster strains, although urgently needed, do not exist. Herein, we describe a narrow interval of low temperatures under which the larvae of D. melanogaster can be stored in quiescence for up to two months. The development of larvae was arrested at the pre-wandering stage under fluctuating thermal regime (FTR), which simultaneously resulted in diminishing the accumulation of indirect chill injuries. Our physiological, metabolomic, and transcriptomic analyses revealed that compared to larvae stored at constant low temperatures, the larvae stored under FTR conditions were able to decrease the rates of depletion of energy substrates, exploited brief warm episodes of FTR for homeostatic control of metabolite levels, and more efficiently exerted protection against oxidative damage.


Assuntos
Temperatura Baixa/efeitos adversos , Criobiologia , Drosophila melanogaster/fisiologia , Larva/fisiologia , Animais , Criopreservação , Drosophila melanogaster/genética , Larva/genética
15.
BMC Genomics ; 16: 720, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26391666

RESUMO

BACKGROUND: Diapause is a developmental alternative to direct ontogeny in many invertebrates. Its primary adaptive meaning is to secure survival over unfavourable seasons in a state of developmental arrest usually accompanied by metabolic suppression and enhanced tolerance to environmental stressors. During photoperiodically triggered diapause of insects, the ontogeny is centrally turned off under hormonal control, the molecular details of this transition being poorly understood. Using RNAseq technology, we characterized transcription profiles associated with photoperiodic diapause induction in the larvae of the drosophilid fly Chymomyza costata with the goal of identifying candidate genes and processes linked to upstream regulatory events that eventually lead to a complex phenotypic change. RESULTS: Short day photoperiod triggering diapause was associated to inhibition of 20-hydroxy ecdysone (20-HE) signalling during the photoperiod-sensitive stage of C. costata larval development. The mRNA levels of several key genes involved in 20-HE biosynthesis, perception, and signalling were significantly downregulated under short days. Hormonal change was translated into downregulation of a series of other transcripts with broad influence on gene expression, protein translation, alternative histone marking by methylation and alternative splicing. These changes probably resulted in blockade of direct development and deep restructuring of metabolic pathways indicated by differential expression of genes involved in cell cycle regulation, metabolism, detoxification, redox balance, protection against oxidative stress, cuticle formation and synthesis of larval storage proteins. This highly complex alteration of gene transcription was expressed already during first extended night, within the first four hours after the change of the photoperiodic signal from long days to short days. We validated our RNAseq differential gene expression results in an independent qRT-PCR experiment involving wild-type (photoperiodic) and NPD-mutant (non-photoperiodic) strains of C. costata. CONCLUSIONS: Our study revealed several strong candidate genes for follow-up functional studies. Candidate genes code for upstream regulators of a complex change of gene expression, which leads to phenotypic switch from direct ontogeny to larval diapause.


Assuntos
Drosophilidae/genética , Larva/genética , Transcrição Genética , Animais , Análise por Conglomerados , Drosophilidae/embriologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Transcriptoma
16.
Genome Res ; 25(9): 1347-59, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26206155

RESUMO

The capacity of mosquitoes to resist insecticides threatens the control of diseases such as dengue and malaria. Until alternative control tools are implemented, characterizing resistance mechanisms is crucial for managing resistance in natural populations. Insecticide biodegradation by detoxification enzymes is a common resistance mechanism; however, the genomic changes underlying this mechanism have rarely been identified, precluding individual resistance genotyping. In particular, the role of copy number variations (CNVs) and polymorphisms of detoxification enzymes have never been investigated at the genome level, although they can represent robust markers of metabolic resistance. In this context, we combined target enrichment with high-throughput sequencing for conducting the first comprehensive screening of gene amplifications and polymorphisms associated with insecticide resistance in mosquitoes. More than 760 candidate genes were captured and deep sequenced in several populations of the dengue mosquito Ae. aegypti displaying distinct genetic backgrounds and contrasted resistance levels to the insecticide deltamethrin. CNV analysis identified 41 gene amplifications associated with resistance, most affecting cytochrome P450s overtranscribed in resistant populations. Polymorphism analysis detected more than 30,000 variants and strong selection footprints in specific genomic regions. Combining Bayesian and allele frequency filtering approaches identified 55 nonsynonymous variants strongly associated with resistance. Both CNVs and polymorphisms were conserved within regions but differed across continents, confirming that genomic changes underlying metabolic resistance to insecticides are not universal. By identifying novel DNA markers of insecticide resistance, this study opens the way for tracking down metabolic changes developed by mosquitoes to resist insecticides within and among populations.


Assuntos
Aedes/efeitos dos fármacos , Aedes/genética , Genoma de Inseto , Genômica , Resistência a Inseticidas , Animais , Análise por Conglomerados , Amplificação de Genes , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Inseticidas/farmacologia , Dose Letal Mediana , Família Multigênica , Mutação , Nitrilas/farmacologia , Polimorfismo Genético , Piretrinas/farmacologia , Reprodutibilidade dos Testes , Transcrição Genética
17.
Biol Lett ; 10(12): 20140716, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25540155

RESUMO

Worldwide evolution of mosquito resistance to chemical insecticides represents a major challenge for public health, and the future of vector control largely relies on the development of biological insecticides that can be used in combination with chemicals (integrated management), with the expectation that populations already resistant to chemicals will not become readily resistant to biological insecticides. However, little is known about the metabolic pathways affected by selection with chemical or biological insecticides. Here we show that Aedes aegypti, a laboratory mosquito strain selected with a biological insecticide (Bacillus thuringiensis israelensis, Bti) evolved increased transcription of many genes coding for endopeptidases while most genes coding for detoxification enzymes were under-expressed. By contrast, in strains selected with chemicals, genes encoding detoxification enzymes were mostly over-expressed. In all the resistant strains, genes involved in immune response were under-transcribed, suggesting that basal immunity might be a general adjustment variable to compensate metabolic costs caused by insecticide selection. Bioassays generally showed no evidence for an increased susceptibility of selected strains towards the other insecticide type, and all chemical-resistant strains were as susceptible to Bti as the unselected parent strain, which is a good premise for sustainable integrated management of mosquito populations resistant to chemicals.


Assuntos
Aedes/genética , Perfilação da Expressão Gênica , Genes de Insetos , Inseticidas/farmacologia , Controle Biológico de Vetores , Animais
18.
Parasit Vectors ; 7: 480, 2014 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-25318645

RESUMO

BACKGROUND: Resistance of mosquitoes to insecticides is mainly attributed to their adaptation to vector control interventions. Although pesticides used in agriculture have been frequently mentioned as an additional force driving the selection of resistance, only a few studies were dedicated to validate this hypothesis and characterise the underlying mechanisms. While insecticide resistance is rising dramatically in Africa, deciphering how agriculture affects resistance is crucial for improving resistance management strategies. In this context, the multigenerational effect of agricultural pollutants on the selection of insecticide resistance was examined in Anopheles gambiae. METHODS: An urban Tanzanian An. gambiae population displaying a low resistance level was used as a parental strain for a selection experiment across 20 generations. At each generation larvae were selected with a mixture containing pesticides and herbicides classically used in agriculture in Africa. The resistance levels of adults to deltamethrin, DDT and bendiocarb were compared between the selected and non-selected strains across the selection process together with the frequency of kdr mutations. A microarray approach was used for pinpointing transcription level variations selected by the agricultural pesticide mixture at the adult stage. RESULTS: A gradual increase of adult resistance to all insecticides was observed across the selection process. The frequency of the L1014S kdr mutation rose from 1.6% to 12.5% after 20 generations of selection. Microarray analysis identified 90 transcripts over-transcribed in the selected strain as compared to the parental and the non-selected strains. Genes encoding cuticle proteins, detoxification enzymes, proteins linked to neurotransmitter activity and transcription regulators were mainly affected. RT-qPCR transcription profiling of candidate genes across multiple generations supported their link with insecticide resistance. CONCLUSIONS: This study confirms the potency of agriculture in selecting for insecticide resistance in malaria vectors. We demonstrated that the recurrent exposure of larvae to agricultural pollutants can select for resistance mechanisms to vector control insecticides at the adult stage. Our data suggest that in addition to selected target-site resistance mutations, agricultural pollutants may also favor cuticle, metabolic and synaptic transmission-based resistance mechanisms. These results emphasize the need for integrated resistance management strategies taking into account agriculture activities.


Assuntos
Anopheles/efeitos dos fármacos , Insetos Vetores/efeitos dos fármacos , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Malária/transmissão , Seleção Genética , Agricultura , Animais , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Genótipo , Insetos Vetores/parasitologia , Inseticidas/administração & dosagem , Análise Serial de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma
19.
Parasit Vectors ; 7: 409, 2014 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-25175167

RESUMO

BACKGROUND: Insecticide resistance in the mosquito vector is the one of the main obstacles against effective malaria control. In order to implement insecticide resistance management strategies, it is important to understand the genetic factors involved. In this context, we investigated the molecular basis of DDT resistance in the main malaria vector from Benin. METHODS: Anopheles gambiae mosquitoes were collected from four sites across Benin and identified to species/molecular form. Mosquitoes from Cotonou (M-form), Tori-Bossito (S-form) and Bohicon (S-form) were exposed to DDT 4% at a range of exposure times (30 min to 300 min). Another batch of mosquitoes from Cotonou and Malanville were exposed to DDT for 1 hour and the survivors 48 hours post exposure were used to quantify metabolic gene expression. Quantitative PCR assays were used to quantify mRNA levels of metabolic enzymes: GSTE2, GSTD3, CYP6P3 and CYP6M2. Expression (fold-change) was calculated using the ∆∆Ct method and compared to susceptible strains. Detection of target-site mutations (L1014F, L1014S and N1575Y) was performed using allelic discrimination TaqMan assays. RESULTS: DDT resistance was extremely high in all populations, regardless of molecular form, with no observed mortality after 300 min exposure. In both DDT-survivors and non-exposed mosquitoes, GSTE2 and GSTD3 were over-expressed in the M form at 4.4-fold and 3.5-fold in Cotonou and 1.5-fold and 2.5-fold in Malanville respectively, when compared to the susceptible strain. The CYP6M2 and CYP6P3 were over-expressed at 4.6-fold and 3.8-fold in Cotonou and 1.2-fold and 2.5-fold in Malanville respectively. In contrast, no differences in GSTE2 and CYP6M2 were observed between S form mosquitoes from Tori-Bossito and Bohicon compared to susceptible strain. The 1014 F allele was fixed in the S-form and at high frequency in the M-form (0.7-0.914). The frequency of 1575Y allele was 0.29-0.36 in the S-form and nil in the M-form. The 1014S allele was detected in the S form of An. gambiae in a 1014 F/1014S heterozygous specimen. CONCLUSION: Our results show that the kdr 1014 F, 1014S and 1575Y alleles are widespread in Benin and the expression of two candidate metabolic markers (GSTE2 and CYP6M2) are over-expressed specifically in the M-form.


Assuntos
Anopheles/efeitos dos fármacos , Anopheles/genética , DDT/farmacologia , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Animais , Benin , DNA Complementar , Regulação da Expressão Gênica , Genótipo , Mutação , RNA/genética , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
20.
PLoS Genet ; 10(3): e1004236, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24651294

RESUMO

Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassalé, Côte d'Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassalé. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassalé An. gambiae results from coupling of over-expressed target site allelic variants with heightened CYP6 P450 expression, which also provides resistance across contrasting insecticides. Mosquito populations displaying such a diverse basis of extreme and cross-resistance are likely to be unresponsive to standard insecticide resistance management practices.


Assuntos
Anopheles/genética , Sistema Enzimático do Citocromo P-450/genética , Resistência a Inseticidas/genética , Malária/transmissão , Acetilcolinesterase/genética , África Ocidental , Animais , Animais Geneticamente Modificados/genética , Carbamatos/farmacologia , Drosophila/efeitos dos fármacos , Drosophila/genética , Malária/genética , Fenótipo , Fenilcarbamatos/farmacologia , Piretrinas/farmacologia
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