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"S. Owen"
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To denoise or to cluster? That is not the question. Optimizing pipelines for COI metabarcoding and metaphylogeography
Background: The recent blooming of metabarcoding applications to biodiversity studies comes with some relevant methodological debates. One such issue concerns the treatment of reads by denoising or by clustering methods, which have been wrongly presented as alternatives. It has also been suggested that denoised sequence variants should replace clusters as the basic unit of metabarcoding analyses, missing the fact that sequence clusters are a proxy for species-level entities, the basic unit in biodiversity studies. We argue here that methods developed and tested for ribosomal markers have been uncritically applied to highly variable markers such as cytochrome oxidase I (COI) without conceptual or operational (e.g., parameter setting) adjustment. COI has a naturally high intraspecies variability that should be assessed and reported, as it is a source of highly valuable information. We contend that denoising and clustering are not alternatives. Rather, they are complementary and both should be used together in COI metabarcoding pipelines. Results: Using a COI dataset from benthic marine communities, we compared two denoising procedures (based on the UNOISE3 and the DADA2 algorithms), set suitable parameters for denoising and clustering, and applied these steps in diferent orders. Our results indicated that the UNOISE3 algorithm preserved a higher intra-cluster variability. We introduce the program DnoisE to implement the UNOISE3 algorithm taking into account the natural variability (measured as entropy) of each codon position in protein-coding genes. This correction increased the number of sequences retained by 88%. The order of the steps (denoising and clustering) had little infuence on the fnal outcome. Conclusions: We highlight the need for combining denoising and clustering, with adequate choice of stringency parameters, in COI metabarcoding. We present a program that uses the coding properties of this marker to improve the denoising step. We recommend researchers to report their results in terms of both denoised sequences (a proxy for haplotypes) and clusters formed (a proxy for species), and to avoid collapsing the sequences of the latter into a single representative. This will allow studies at the cluster (ideally equating species-level diversity) and at the intra-cluster level, and will ease additivity and comparability between studies.
Journal Article
المقالة العلمية في عصر الرقمنة
يوضح الكتاب كيف تشكل الدوريات التخصصية أهم قنوات الإتصال العلمي منذ منتصف القرن السابع عشر للميلاد والمقالات العلمية هى أهم مكونات الدوريات التخصصية ويرى كثيرون أن بإمكان الرقمنة إحداث ثورة في الاتصال العلمي، ويحاول هذا الكتاب التحقق من تداعيات الرقمنة بالنسبة للمقالات العلمية المحكمة التي تنشر في الدوريات الإلكترونية، ويتضمن هذا الكتاب الذى يتسم بالنظرة الثاقبة المتعمقة أساسا نظريا لدراسة تاريخ الإتصال العلمي بوجه عام والدوريات التخصصية والمقالات العلمية على وجه الخصوص فضلا عن مقومات الرقمنة وخصائص الوثائق الرقمية.
Book
DNA metabarcoding of littoral hard-bottom communities: high diversity and database gaps revealed by two molecular markers
Biodiversity assessment of marine hard-bottom communities is hindered by the high diversity and size-ranges of the organisms present. We developed a DNA metabarcoding protocol for biodiversity characterization of structurally complex natural marine hard-bottom communities. We used two molecular markers: the \"Leray fragment\" of mitochondrial cytochrome c oxidase (COI), for which a novel primer set was developed, and the V7 region of the nuclear small subunit ribosomal RNA (18S). Eight different shallow marine littoral communities from two National Parks in Spain (one in the Atlantic Ocean and another in the Mediterranean Sea) were studied. Samples were sieved into three size fractions from where DNA was extracted separately. Bayesian clustering was used for delimiting molecular operational taxonomic units (MOTUs) and custom reference databases were constructed for taxonomic assignment. Despite applying stringent filters, we found high values for MOTU richness (2,510 and 9,679 MOTUs with 18S and COI, respectively), suggesting that these communities host a large amount of yet undescribed eukaryotic biodiversity. Significant gaps are still found in sequence reference databases, which currently prevent the complete taxonomic assignment of the detected sequences. In our dataset, 85% of 18S MOTUs and 64% of COI MOTUs could be identified to phylum or lower taxonomic level. Nevertheless, those unassigned were mostly rare MOTUs with low numbers of reads, and assigned MOTUs comprised over 90% of the total sequence reads. The identification rate might be significantly improved in the future, as reference databases are further completed. Our results show that marine metabarcoding, currently applied mostly to plankton or sediments, can be adapted to structurally complex hard bottom samples. Thus, eukaryotic metabarcoding emerges as a robust, fast, objective and affordable method to comprehensively characterize the diversity of marine benthic communities dominated by macroscopic seaweeds and colonial or modular sessile metazoans. The 18S marker lacks species-level resolution and thus cannot be recommended to assess the detailed taxonomic composition of these communities. Our new universal primers for COI can potentially be used for biodiversity assessment with high taxonomic resolution in a wide array of marine, terrestrial or freshwater eukaryotic communities.
Journal Article
Molecular gut content analysis of different spider body parts
Molecular gut-content analysis has revolutionized the study of food webs and feeding inter- actions, allowing the detection of prey DNA within the gut of many organisms. However, suc- cessful prey detection is a challenging procedure in which many factors affect every step, starting from the DNA extraction process. Spiders are liquid feeders with branched gut diver- ticula extending into their legs and throughout the prosoma, thus digestion takes places in different parts of the body and simple gut dissection is not possible. In this study, we investi- gated differences in prey detectability in DNA extracts from different parts of the spider ́s body: legs, prosoma and opisthosoma, using prey-specific PCR and metabarcoding approaches. We performed feeding trials with the woodlouse hunter spider Dysdera ver- neaui Simon, 1883 (Dysderidae) to estimate the time at which prey DNA is detectable within the predator after feeding. Although we found that all parts of the spider body are suitable for gut-content analysis when using prey-specific PCR approach, results based on metabar- coding suggested the opisthosoma is optimal for detection of predation in spiders because it contained the highest concentration of prey DNA for longer post feeding periods. Other spi- ders may show different results compared to D. verneaui, but given similarities in the physi- ology and digestion in different families, it is reasonable to assume this to be common across species and this approach having broad utility across spiders.
Journal Article
Metabolic regulation of species-specific developmental rates
Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development
1
–
3
. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD
+
/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD
+
/NADH ratio in human cells by overexpression of the
Lactobacillus brevis
NADH oxidase
Lb
NOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.
An in vitro system that recapitulates temporal characteristics of embryonic development demonstrates that the different rates of mouse and human embryonic development stem from differences in metabolic rates and—further downstream—the global rate of protein synthesis.
Journal Article
A tunable library of substituted thiourea precursors to metal sulfide nanocrystals
Controlling the size of colloidal nanocrystals is essential to optimizing their performance in optoelectronic devices, catalysis, and imaging applications. Traditional synthetic methods control size by terminating the growth, an approach that limits the reaction yield and causes batch-to-batch variability. Herein we report a library of thioureas whose substitution pattern tunes their conversion reactivity over more than five orders of magnitude and demonstrate that faster thiourea conversion kinetics increases the extent of crystal nucleation. Tunable kinetics thereby allows the nanocrystal concentration to be adjusted and a desired crystal size to be prepared at full conversion. Controlled precursor reactivity and quantitative conversion improve the batch-to-batch consistency of the final nanocrystal size at industrially relevant reaction scales.
Journal Article
Regulation of Phytohormone biosynthesis and accumulation in Arabidopsis following treatment with commercial extract from the marine Macroalga Ascophyllum nodosum
Seaweeds and their extracts have been used for centuries in agriculture to improve plant growth and impart stress tolerance. There has been historical evidence that phytohormones present in seaweeds lead to these effects, but questions of this mode of action have always been raised. By quantifying phytohormones in seaweed extracts coupled with the use of phytohormone biosynthetic and insensitive mutants, we conclude that the phytohormone levels present within the extracts are insufficient to cause significant effects in plants when extracts are applied at recommended rates. However, components within seaweed extracts may modulate innate pathways for the biosynthesis of phytohormones in plants. Phytohormone profiles of plant tissue extracts were analyzed following root application of a commercial seaweed extract produced from Ascophyllum nodosum (ANE) to in vitro-grown Arabidopsis plants. We found an increase in total concentration of cytokinins (CKs), in particular, of trans-zeatin-type CKs, 24 and 96 h after ANE application, with an increase in cis-zeatintype CKs observed at 144 h. Concomitantly, increases in abscisic acid (ABA) and ABA catabolite levels were observed whereas auxin levels were reduced. Additionally, the profile of transcripts revealed that CK biosynthetic genes were upregulated, whereas the CK catabolic genes were repressed at 24 and 96 h following ANE application. Not surprisingly, the transcripts of ABA biosynthetic genes were increased whereas the auxin biosynthetic genes were repressed. These corroborated findings are the first to help explain the underlying physiological benefits derived from the application of ANE to plants.
Journal Article
Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell
Mitochondrial dysfunction is associated with activation of the integrated stress response (ISR) but the underlying triggers remain unclear. We systematically combined acute mitochondrial inhibitors with genetic tools for compartment-specific NADH oxidation to trace mechanisms linking different forms of mitochondrial dysfunction to the ISR in proliferating mouse myoblasts and in differentiated myotubes. In myoblasts, we find that impaired NADH oxidation upon electron transport chain (ETC) inhibition depletes asparagine, activating the ISR via the eIF2α kinase GCN2. In myotubes, however, impaired NADH oxidation following ETC inhibition neither depletes asparagine nor activates the ISR, reflecting an altered metabolic state. ATP synthase inhibition in myotubes triggers the ISR via a distinct mechanism related to mitochondrial inner-membrane hyperpolarization. Our work dispels the notion of a universal path linking mitochondrial dysfunction to the ISR, instead revealing multiple paths that depend both on the nature of the mitochondrial defect and on the metabolic state of the cell.
Journal Article