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1.
Sci Adv ; 8(3): eabj9466, 2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35044817

RESUMO

Diatoms are fast-growing and winning competitors in aquatic environments, possibly due to optimized growth performance. However, their life cycles are complex, heteromorphic, and not fully understood. Here, we report on the fine control of cell growth and physiology during the sexual phase of the marine diatom Pseudo-nitzschia multistriata. We found that mating, under nutrient replete conditions, induces a prolonged growth arrest in parental cells. Transcriptomic analyses revealed down-regulation of genes related to major metabolic functions from the early phases of mating. Single-cell photophysiology also pinpointed an inhibition of photosynthesis and storage lipids accumulated in the arrested population, especially in gametes and zygotes. Numerical simulations revealed that growth arrest affects the balance between parental cells and their siblings, possibly favoring the new generation. Thus, in addition to resources availability, life cycle traits contribute to shaping the species ecological niches and must be considered to describe and understand the structure of plankton communities.

2.
Molecules ; 26(24)2021 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-34946780

RESUMO

Microalgae have a great potential for the production of healthy food and feed supplements. Their ability to convert carbon into high-value compounds and to be cultured in large scale without interfering with crop cultivation makes these photosynthetic microorganisms promising for the sustainable production of lipids. In particular, microalgae represent an alternative source of polyunsaturated fatty acids (PUFAs), whose consumption is related to various health benefits for humans and animals. In recent years, several strategies to improve PUFAs' production in microalgae have been investigated. Such strategies include selecting the best performing species and strains and the optimization of culturing conditions, with special emphasis on the different cultivation systems and the effect of different abiotic factors on PUFAs' accumulation in microalgae. Moreover, developments and results obtained through the most modern genetic and metabolic engineering techniques are described, focusing on the strategies that lead to an increased lipid production or an altered PUFAs' profile. Additionally, we provide an overview of biotechnological applications of PUFAs derived from microalgae as safe and sustainable organisms, such as aquafeed and food ingredients, and of the main techniques (and their related issues) for PUFAs' extraction and purification from microalgal biomass.

3.
New Phytol ; 2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34533849

RESUMO

Because of their importance as chemical mediators, the presence of a rich and varied family of lipoxygenase (LOX) products, collectively named oxylipins, has been investigated thoroughly in diatoms, and the involvement of these products in important processes such as bloom regulation has been postulated. Nevertheless, little information is available on the enzymes and pathways operating in these protists. Exploiting transcriptome data, we identified and characterized a LOX gene, PaLOX, in Pseudo-nitzschia arenysensis, a marine diatom known to produce different species of oxylipins by stereo- and regio-selective oxidation of eicosapentaenoic acid (EPA) at C12 and C15. PaLOX RNA interference correlated with a decrease of the lipid-peroxidizing activity and oxylipin synthesis, as well as with a reduction of growth of P. arenysensis. In addition, sequence analysis and structure models of the C-terminal part of the predicted protein closely fitted with the data for established LOXs from other organisms. The presence in the genome of a single LOX gene, whose downregulation impairs both 12- and 15-oxylipins synthesis, together with the in silico 3D protein modelling suggest that PaLOX encodes for a 12/15S-LOX with a dual specificity, and provides additional support to the correlation between cell growth and oxylipin biosynthesis in diatoms.

4.
Harmful Algae ; 103: 101995, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33980435

RESUMO

The cosmopolitan, species-rich diatom genus Pseudo-nitzschia represents a good system for the study of speciation, evolution and diversity. Understanding elements linked to population dynamics and life cycle regulation for these species is of particular importance in view of their ability to produce the toxin domoic acid and cause harmful blooms. Pseudo-nitzschia multistriata, one of the toxic species that represents a model for the study of life cycle related questions, is the only diatom for which a sex determination mechanism has been described. Populations in the Gulf of Naples (Mediterranean Sea), can share four different allelic variants (A, M, B, N) of the mating type determination region, and one of them (A) is responsible for the determination of the mating type + (MT+), defined by the MT+ restricted expression of the gene MRP3. Here, we analysed the sex determination genomic region in three new strains isolated from the Gulf of Mexico and compared it to the alleles previously described in the Mediterranean strains. We first show that these geographically distant strains of P. multistriata belong to different populations but can interbreed. Next, we show that the two populations share an overall similar structure of the genomic locus although differences can be seen in the polymorphic regions upstream of MRP3. In strain P4-C1, we amplified and sequenced an allele (M) identical to one of those previously characterized in the Mediterranean strains. In the other two strains, P4-C2 and P4-C5, we identified three new alleles, which we named A2, B2 and N2. P4-C2 and P4-C5 are heterozygous and share the common allele A2 linked to the monoallelic expression of the MT+ specific sex determining gene MRP3. Our results expand information on the global distribution of P. multistriata and on the level of conservation of the sex determination region in different populations. The definition of the extent of intra- and inter-specific conservation of this region would be a relevant addition to our understanding of Pseudo-nitzschia diversity and evolution.


Assuntos
Diatomáceas , Alelos , Diatomáceas/genética , Golfo do México , Mar Mediterrâneo , Reprodução
5.
Open Biol ; 11(4): 200395, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33823659

RESUMO

Diatoms are one of the major and most diverse groups of phytoplankton, with chimeric genomes harbouring a combination of genes of bacterial, animal and plant origin. They have developed sophisticated mechanisms to face environmental variations. In marine environments, nutrients concentration shows significant temporal and spatial variability, influencing phytoplankton growth. Among nutrients, nitrogen, present at micromolar levels, is often a limiting resource. Here, we report a comprehensive characterization of the Nitrate Transporter 1/Peptide Transporter Family (NPF) in diatoms, diNPFs. NPFs are well characterized in many organisms where they recognize a broad range of substrates, ranging from short-chained di- and tri-peptides in bacteria, fungi and mammals to a wide variety of molecules including nitrate in higher plants. Scarce information is available for diNPFs. We integrated-omics, phylogenetic, structural and expression analyses, to infer information on their role in diatoms. diNPF genes diverged to produce two distinct clades with strong sequence and structural homology with either bacterial or plant NPFs, with different predicted sub-cellular localization, suggesting that the divergence resulted in functional diversification. Moreover, transcription analysis of diNPF genes under different laboratory and environmental growth conditions suggests that diNPF diversification led to genetic adaptations that might contribute to diatoms ability to flourish in diverse environmental conditions.

6.
Sci Rep ; 11(1): 1681, 2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33462289

RESUMO

Due to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar nature of their cell wall, that is made of silica and organic molecules and that hinders the application of standard methods for cell lysis required, for example, to extract organelles. In this study we present a protocol for intact nuclei isolation from diatoms that was successfully applied to three different species: two pennates, Pseudo-nitzschia multistriata and Phaeodactylum tricornutum, and one centric diatom species, Chaetoceros diadema. Intact nuclei were extracted by treatment with acidified NH4F solution combined to low intensity sonication pulses and separated from cell debris via FAC-sorting upon incubation with SYBR Green. Microscopy observations confirmed the integrity of isolated nuclei and high sensitivity DNA electrophoresis showed that genomic DNA extracted from isolated nuclei has low degree of fragmentation. This protocol has proved to be a flexible and versatile method to obtain intact nuclei preparations from different diatom species and it has the potential to speed up applications such as epigenetic explorations as well as single cell ("single nuclei") genomics, transcriptomics and proteomics in different diatom species.


Assuntos
Fracionamento Celular/métodos , Núcleo Celular/química , Diatomáceas/citologia , Fracionamento Celular/normas , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA/genética , DNA/metabolismo , Diatomáceas/genética , Diatomáceas/metabolismo , Microscopia Confocal , Frações Subcelulares/metabolismo
8.
Antioxidants (Basel) ; 9(8)2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32824292

RESUMO

Microalgae represent a promising resource for the production of beneficial natural compounds due to their richness in secondary metabolites and easy cultivation. Carotenoids feature among distinctive compounds of many microalgae, including diatoms, which owe their golden color to the xanthophyll fucoxanthin. Carotenoids have antioxidant, anti-obesity and anti-inflammatory properties, and there is a considerable market demand for these compounds. Here, with the aim to increase the carotenoid content in the model diatom Phaeodactylum tricornutum, we exploited genetic transformation to overexpress genes involved in the carotenoid biosynthetic pathway. We produced transgenic lines over-expressing simultaneously one, two or three carotenoid biosynthetic genes, and evaluated changes in pigment content with high-performance liquid chromatography. Two triple transformants over-expressing the genes Violaxanthin de-epoxidase (Vde), Vde-related (Vdr) and Zeaxanthin epoxidase 3 (Zep3) showed an accumulation of carotenoids, with an increase in the fucoxanthin content up to four fold. Vde, Vdr and Zep3 mRNA and protein levels in the triple transformants were coherently increased. The exact role of these enzymes in the diatom carotenoid biosynthetic pathway is not completely elucidated nevertheless our strategy successfully modulated the carotenoid metabolism leading to an accumulation of valuable compounds, leading the way toward improved utilization of microalgae in the field of antioxidants.

9.
Nat Commun ; 11(1): 3320, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620776

RESUMO

Benthic diatoms are the main primary producers in shallow freshwater and coastal environments, fulfilling important ecological functions such as nutrient cycling and sediment stabilization. However, little is known about their evolutionary adaptations to these highly structured but heterogeneous environments. Here, we report a reference genome for the marine biofilm-forming diatom Seminavis robusta, showing that gene family expansions are responsible for a quarter of all 36,254 protein-coding genes. Tandem duplications play a key role in extending the repertoire of specific gene functions, including light and oxygen sensing, which are probably central for its adaptation to benthic habitats. Genes differentially expressed during interactions with bacteria are strongly conserved in other benthic diatoms while many species-specific genes are strongly upregulated during sexual reproduction. Combined with re-sequencing data from 48 strains, our results offer insights into the genetic diversity and gene functions in benthic diatoms.


Assuntos
Adaptação Fisiológica/genética , Diatomáceas/genética , Ecossistema , Evolução Molecular , Genoma/genética , Diatomáceas/classificação , Diatomáceas/metabolismo , Água Doce , Tamanho do Genoma , Genômica/métodos , Polimorfismo de Nucleotídeo Único , Água do Mar , Especificidade da Espécie , Transcriptoma/genética
10.
Mar Drugs ; 18(6)2020 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-32545923

RESUMO

Pseudo-nitzschia multistriata is a planktonic marine diatom with a diplontic life cycle comprising a short sexual phase, during which gametes are produced following the encounter of two diploid cells of opposite mating type (MT). Gene expression studies have highlighted the presence of substantial changes occurring at the onset of sexual reproduction. Herein, we have hypothesized that the amount and nature of cellular metabolites varies along the mating process. To capture the metabolome of Pseudo-nitzschia multistriata at different harvesting times in an unbiased manner, we undertook an untargeted metabolomics approach based on liquid chromatography-tandem mass spectrometry. Using three different extraction steps, the method revealed pronounced differences in the metabolic profiles between control cells in the vegetative phase (MT+ and MT-) and mixed strains of opposite MTs (cross) undergoing sexual reproduction. Of the 2408 high-quality features obtained, 70 known metabolites could be identified based on in-house libraries and online databases; additional 46 features could be classified by molecular networking of tandem mass spectra. The reduction of phytol detected in the cross can be linked to the general downregulation of photosynthesis during sexual reproduction observed elsewhere. Moreover, the role of highly regulated compounds such as 7-dehydrodesmosterol, whose changes in abundance were the highest in the experiment, oleamide, ectoine, or trigonelline is discussed.


Assuntos
Diatomáceas/fisiologia , Reprodução/fisiologia , Animais , Metabolômica , Água do Mar
11.
Genes (Basel) ; 11(1)2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31861932

RESUMO

Transposable elements (TEs), activated as a response to unfavorable conditions, have been proposed to contribute to the generation of genetic and phenotypic diversity in diatoms. Here we explore the transcriptome of three warm water strains of the diatom Leptocylindrus aporus, and the possible involvement of TEs in their response to changing temperature conditions. At low temperature (13 °C) several stress response proteins were overexpressed, confirming low temperature to be unfavorable for L. aporus, while TE-related transcripts of the LTR retrotransposon superfamily were the most enriched transcripts. Their expression levels, as well as most of the stress-related proteins, were found to vary significantly among strains, and even within the same strains analysed at different times. The lack of overexpression after many months of culturing suggests a possible role of physiological plasticity in response to growth under controlled laboratory conditions. While further investigation on the possible central role of TEs in the diatom stress response is warranted, the strain-specific responses and possible role of in-culture evolution draw attention to the interplay between the high intraspecific variability and the physiological plasticity of diatoms, which can both contribute to the adaptation of a species to a wide range of conditions in the marine environment.


Assuntos
Elementos de DNA Transponíveis , Perfilação da Expressão Gênica/métodos , Estramenópilas/genética , Adaptação Fisiológica , Resposta ao Choque Frio , Evolução Molecular , Regulação da Expressão Gênica , Análise de Sequência de RNA
12.
BMC Genomics ; 20(1): 544, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277569

RESUMO

Following the publication of this article [1], the authors reported that the link to Additional file 11 linked to the wrong set of data. The correct supplementary data is provided in this Correction article (Additional file 11).

13.
Genes (Basel) ; 10(7)2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261777

RESUMO

Sexual reproduction plays a fundamental role in diatom life cycles. It contributes to increasing genetic diversity through meiotic recombination and also represents the phase where large-sized cells are produced to counteract the cell size reduction process that characterizes these microalgae. With the aim to identify genes linked to the sexual phase of the centric planktonic diatom Skeletonema marinoi, we carried out an RNA-seq experiment comparing the expression level of transcripts in sexualized cells with that of large cells not competent for sex. A set of genes involved in meiosis were found upregulated. Despite the fact that flagellate gametes were observed in the sample, we did not detect the expression of genes involved in the synthesis of flagella that were upregulated during sexual reproduction in another centric diatom. A comparison with the set of genes changing during the first phases of sexual reproduction of the pennate diatom Pseudo-nitzschia multistriata revealed the existence of commonalities, including the strong upregulation of genes with an unknown function that we named Sex Induced Genes (SIG). Our results further broadened the panel of genes that can be used as a marker for sexual reproduction of diatoms, crucial for the interpretation of metatranscriptomic datasets.


Assuntos
Diatomáceas/genética , Flagelos/genética , Expressão Gênica , Meiose/genética , RNA-Seq , Reprodução/genética , Reprodução/fisiologia , Comportamento Sexual/fisiologia , Transcriptoma/genética
14.
Mar Drugs ; 17(5)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067655

RESUMO

Over the last decade, genome sequences and other -omics datasets have been produced for a wide range of microalgae, and several others are on the way. Marine microalgae possess distinct and unique metabolic pathways, and can potentially produce specific secondary metabolites with biological activity (e.g., antipredator, allelopathic, antiproliferative, cytotoxic, anticancer, photoprotective, as well as anti-infective and antifouling activities). Because microalgae are very diverse, and adapted to a broad variety of environmental conditions, the chances to find novel and unexplored bioactive metabolites with properties of interest for biotechnological and biomedical applications are high. This review presents a comprehensive overview of the current efforts and of the available solutions to produce, explore and exploit -omics datasets, with the aim of identifying species and strains with the highest potential for the identification of novel marine natural products. In addition, funding efforts for the implementation of marine microalgal -omics resources and future perspectives are presented as well.


Assuntos
Microalgas/metabolismo , Produtos Biológicos , Biotecnologia , Descoberta de Drogas/métodos , Engenharia Genética , Genômica/métodos , Metabolômica/métodos , Proteômica/métodos , Transcriptoma
15.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(2): 181-190, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30521937

RESUMO

Diatoms are eukaryotic microalgae that play a pivotal role in biological and geochemical marine cycles. These microorganisms are at the basis of the trophic chain and their lipids are essential components (e.g. eicosapentaenoic acid, EPA) of aquatic food webs. Galactolipids are the primary lipid components of plastid membranes and form the largest lipid family of diatoms. As source of polyunsaturated fatty acids (PUFAs), these compounds are also involved in the synthesis of lipoxygenase (LOX) products such as non-volatile oxylipins and polyunsaturated aldehydes. Here, we report the first identification of two genes, namely PmLAH1 and PaLAH1, coding for lipolytic enzymes in two diatoms of the genus Pseudo-nitzschia. Functional and modeling studies evidence a patatin-like domain endowed with galactolipase and phospholipase activity at the C-terminus of both proteins. Homologues of Pseudo-nitzschia LAH1 genes were retrieved in other diatom species so far sequenced in agreement with conservation of the functional role of these proteins within the lineage.


Assuntos
Diatomáceas/metabolismo , Galactolipídeos/metabolismo , Hidrolases/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/metabolismo , Ácidos Graxos Insaturados/metabolismo , Galactolipídeos/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lipólise/fisiologia , Lipoxigenase/metabolismo , Microalgas/metabolismo , Oxilipinas/metabolismo
16.
PeerJ ; 6: e5507, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30310734

RESUMO

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, co-opted from a bacterial defense natural mechanism, is the cutting edge technology to carry out genome editing in a revolutionary fashion. It has been shown to work in many different model organisms, from human to microbes, including two diatom species, Phaeodactylum tricornutum and Thalassiosira pseudonana. Transforming P. tricornutum by bacterial conjugation, we have performed CRISPR/Cas9-based mutagenesis delivering the nuclease as an episome; this allowed for avoiding unwanted perturbations due to random integration in the genome and for excluding the Cas9 activity when it was no longer required, reducing the probability of obtaining off-target mutations, a major drawback of the technology. Since there are no reports on off-target occurrence at the genome level in microalgae, we performed whole-genome Illumina sequencing and found a number of different unspecific changes in both the wild type and mutant strains, while we did not observe any preferential mutation in the genomic regions in which off-targets were predicted. Our results confirm that the CRISPR/Cas9 technology can be efficiently applied to diatoms, showing that the choice of the conjugation method is advantageous for minimizing unwanted changes in the genome of P. tricornutum.

17.
ISME J ; 12(2): 463-472, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29160864

RESUMO

Genetic diversity is what selection acts on, thus shaping the adaptive potential of populations. We studied micro-evolutionary patterns of the key planktonic diatom Pseudo-nitzschia multistriata at a long-term sampling site over 2 consecutive years by genotyping isolates with 22 microsatellite markers. We show that both sex and vegetative growth interplay in shaping intraspecific diversity. We document a brief but massive demographic and clonal expansion driven by strains of the same mating type. The analysis of an extended data set (6 years) indicates that the genetic fingerprint of P. multistriata changed over time with a nonlinear pattern, with intermittent periods of weak and strong diversification related to the temporary predominance of clonal expansions over sexual recombination. These dynamics, rarely documented for phytoplankton, contribute to the understanding of bloom formation and of the mechanisms that drive microevolution in diatoms.


Assuntos
Diatomáceas/crescimento & desenvolvimento , Diatomáceas/genética , Evolução Biológica , Diatomáceas/isolamento & purificação , Variação Genética , Repetições de Microssatélites , Fitoplâncton/genética , Fitoplâncton/crescimento & desenvolvimento , Fitoplâncton/isolamento & purificação
18.
New Phytol ; 215(1): 140-156, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28429538

RESUMO

Microalgae play a major role as primary producers in aquatic ecosystems. Cell signalling regulates their interactions with the environment and other organisms, yet this process in phytoplankton is poorly defined. Using the marine planktonic diatom Pseudo-nitzschia multistriata, we investigated the cell response to cues released during sexual reproduction, an event that demands strong regulatory mechanisms and impacts on population dynamics. We sequenced the genome of P. multistriata and performed phylogenomic and transcriptomic analyses, which allowed the definition of gene gains and losses, horizontal gene transfers, conservation and evolutionary rate of sex-related genes. We also identified a small number of conserved noncoding elements. Sexual reproduction impacted on cell cycle progression and induced an asymmetric response of the opposite mating types. G protein-coupled receptors and cyclic guanosine monophosphate (cGMP) are implicated in the response to sexual cues, which overall entails a modulation of cell cycle, meiosis-related and nutrient transporter genes, suggesting a fine control of nutrient uptake even under nutrient-replete conditions. The controllable life cycle and the genome sequence of P. multistriata allow the reconstruction of changes occurring in diatoms in a key phase of their life cycle, providing hints on the evolution and putative function of their genes and empowering studies on sexual reproduction.


Assuntos
Evolução Biológica , Diatomáceas/fisiologia , Transporte Biológico/genética , Ciclo Celular , Diatomáceas/genética , Regulação da Expressão Gênica no Desenvolvimento , Filogenia , Dinâmica Populacional , Reprodução/genética , Transdução de Sinais
19.
Phys Rev E ; 94(2-1): 022418, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27627344

RESUMO

Chain formation in diatoms is relevant because of several aspects of their adaptation to the ecosystem. However, the tools to quantify the regulation of their assemblage and infer specific mechanisms in a laboratory setting are scarce. To address this problem, we define an approach based on a statistical physics model of chain growth and separation in combination with experimental evaluation of chain-length distributions. Applying this combined analysis to data from Chaetoceros decipiens and Phaeodactylum tricornutum, we find that cells of the first species control chain separation, likely through a cell-to-cell communication process, while the second species only modulates the separation rate. These results promote quantitative methods for characterizing chain formation in several chain-forming species and in diatoms in particular.


Assuntos
Diatomáceas/crescimento & desenvolvimento , Modelos Biológicos , Diatomáceas/citologia
20.
Protoplasma ; 253(6): 1421-1431, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26494151

RESUMO

Pseudo-nitzschia is a thoroughly studied pennate diatom genus for ecological and biological reasons. Many species in this genus, including Pseudo-nitzschia multistriata, can produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Physiological, phylogenetic and biological features of P. multistriata were studied extensively in the past. Life cycle stages, including the sexual phase, fundamental in diatoms to restore the maximum cell size and avoid miniaturization to death, have been well described for this species. P. multistriata is heterothallic; sexual reproduction is induced when strains of opposite mating type are mixed, and proceeds with cells producing two functionally anisogamous gametes each; however, detailed cytological information for this process is missing. By means of confocal laser scanning microscopy and nuclear staining, we followed the nuclear fate during meiosis, and using time-lapse cinematography, we timed every step of the sexual reproduction process from mate pairing to initial cell hatching. The present paper depicts cytological aspects during gametogenesis in P. multistriata, shedding light on the chloroplast behaviour during sexual reproduction, finely describing the timing of the sexual phases and providing reference data for further studies on the molecular control of this fundamental process.


Assuntos
Diatomáceas/citologia , Diatomáceas/fisiologia , Imagem com Lapso de Tempo/métodos , Cloroplastos/metabolismo , Gametogênese Vegetal , Reprodução , Fatores de Tempo
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