Environmental DNA metabarcoding: Transforming how we survey animal and plant communities

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High‐throughput sequencing (“HTS”) platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed “environmental DNA” or “eDNA”). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called “eDNA metabarcoding” and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education.     

Trans-Golgi Network—An Intersection of Trafficking Cell Wall Components

The cell wall, a crucial cell compartment, is composed of a network of polysaccharides and proteins, providing structural support and protection from external stimuli. While the cell wall structure and biosynthesis have been extensively studied, very little is known about the transport of polysaccharides and other components into the developing cell wall. This review focuses on endomembrane trafficking pathways involved in cell wall deposition. Cellulose synthase complexes are assembled in the Golgi, and are transported in vesicles to the plasma membrane. Non-cellulosic polysaccharides are synthesized in the Golgi apparatus, whereas cellulose is produced by enzyme complexes at the plasma membrane. Polysaccharides and enzymes that are involved in cell wall modification and assembly are transported by distinct vesicle types to their destinations; however, the precise mechanisms involved in selection, sorting and delivery remain to be identified. The endomembrane system orchestrates the delivery of Golgi-derived and possibly endocytic vesicles carrying cell wall and cell membrane components to the newly-formed cell plate. However, the nature of these vesicles, their membrane compositions, and the timing of their delivery are largely unknown. Emerging technologies such as chemical genomics and proteomics are promising avenues to gain insight into the trafficking of cell wall components.     

Phosphorylation-dependent mitotic SUMOylation drives nuclear envelope–chromatin interactions

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin–nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.     

Two redundant ubiquitin‐dependent pathways of BRCA1 localization to DNA damage sites

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin‐dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80‐ and RING‐dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding‐deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80–BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.     

Novel monosaccharides as potent inhibitors of cell proliferation

The effects of several novel monosaccharides upon thymidine incorporation into both normal and tumour cells were investigated. The monosaccharide 2-deoxy-3-[1-(R)-(ethoxycarbonyl)ethyl]-α-D -allo-pyranose had the most inhibitory effect on proliferation, with the (S)-enantiomer having less inhibitory effects. The chiral centre at carbon-7 was found to be an important part of the molecule, as 2-deoxy-3-[methoxycarbonyl methyl]-α-D -allo-pyranose had greatly decreased anti-proliferative properties in comparison with the parent compound. In addition, the 2-deoxy structure at carbon-2 was also found to be important, as 3-[1-(S)-(ethoxycarbonyl)ethyl]-α-D -allo-hexopyranose had greatly decreased inhibitory properties in comparison with the parent compound. The results indicate that these novel monosaccharides possess potent anti-proliferative properties, related to their chiral carbon-7 and 2-deoxy carbon-2 structure and suggest that further substitutions of the functional group at carbon-7 may improve these properties and possibly produce inhibitor selectivity for tumour cells in preference to normal cells. © 1997 John Wiley & Sons, Ltd.     

A mutant-based analysis of the establishment of Nod-independent symbiosis in the legume Aeschynomene evenia

Intensive research on nitrogen-fixing symbiosis in two model legumes has uncovered the molecular mechanisms, whereby rhizobial Nod factors activate a plant symbiotic signaling pathway that controls infection and nodule organogenesis. In contrast, the so-called Nod-independent symbiosis found between Aeschynomene evenia and photosynthetic bradyrhizobia, which does not involve Nod factor recognition nor infection thread formation, is less well known. To gain knowledge on how Nod-independent symbiosis is established, we conducted a phenotypic and molecular characterization of A. evenia lines carrying mutations in different nodulation genes. Besides investigating the effect of the mutations on rhizobial symbiosis, we examined their consequences on mycorrhizal symbiosis and in nonsymbiotic conditions. Analyzing allelic mutant series for AePOLLUX, Ca²⁺/calmodulin dependent kinase, AeCYCLOPS, nodulation signaling pathway 2 (AeNSP2), and nodule inception demonstrated that these genes intervene at several stages of intercellular infection and during bacterial accommodation. We provide evidence that AeNSP2 has an additional nitrogen-dependent regulatory function in the formation of axillary root hairs at lateral root bases, which are rhizobia-colonized infection sites. Our investigation of the recently discovered symbiotic actor cysteine-rich receptor-like kinase specified that it is not involved in mycorrhization; however, it is essential for both symbiotic signaling and early infection during nodulation. These findings provide important insights on the modus operandi of Nod-independent symbiosis and contribute to the general understanding of how rhizobial–legume symbioses are established by complementing the information acquired in model legumes.

Characterization of Aeschynomene evenia mutants altered in nodulation provides information on alternative mechanisms of rhizobium–legume symbiosis     

Dissociation and reconstitution of chromatin without appreciable degradation of the proteins.

When chromatin from Novikoff hepatoma ascites cells was dissociated in 3 M NaCl – 7 M urea either at pH 6 or 8, degradation of chromosomal proteins was observed in two-dimensional gel electrophoretic patterns. This degradation was not prevented by 50 mM NaHSO₃ but was prevented by 1 mM PMSF (phenylmethylsulfonyl fluoride). Reconstitution of the chromatin components dissociated in 3 M NaCl – 7 M ure ? 0.05 M sodium acetate (pH 6.0) containing 1 mM PMSF resulted in reassociation of DNA, histones and the major nonhistone proteins (B24, B26, B33, BE, BJ, C1, C6, CG, CH, CM, C14, CP, C18, CR, CS and C25). Two-dimensional gel electrophoresis showed that although the proportion of the nonhistone proteins to histones was lower in reconstituted than in native chromatin, the template activity of the reconstituted chromatin was similar to that of native chromatin.     

Pilot Scale Bioremediation of Creosote-Contaminated Soil—Efficacy of Enhanced Natural Attenuation and Bioaugmentation Strategies

In this study, the efficacy of bioremediation strategies (enhanced natural attenuation with nitrate and phosphate addition [ENA] and bioaugmentation) for the remediation of creosote-contaminated soil (7767 ± 1286 mg kg^?1 of the 16 EPA priority PAHs) was investigated at pilot scale. Bioaugmentation of creosote-contaminated soil with freshly grown or freeze dried Mycobacterium sp. strain 1B (a PAH degrading microorganism) was applied following bench scale studies that indicated that the indigenous soil microflora had a limited PAH metabolic activity. After 182 days, the total PAH concentration in creosote-contaminated soil was reduced from 7767 ± 1286 mg kg^?1 to 5579 ± 321 mg kg^?1, 2250 ± 71 mg kg^?1, 2050 ± 354 mg kg^?1 and 1950 ± 70 mg kg^?1 in natural attenuation (no additions) and ENA biopiles and biopiles augmented with freshly grown or freeze dried Mycobacterium sp. strain 1B respectively. In ENA and bioaugmentation biopiles, between 82% and 99% of three-ring compounds (acenaphthene, anthracene, fluorene, phenanthrene) were removed while four-ring PAH removal ranged from 33 to 81%. However, the extent of PAH degradation did not vary significantly between the ENA treatment and biopiles augmented with Mycobacterium sp. strain 1B. Four-ring PAH removal followed the order fluoranthene > pyrene > benz[a]anthracene > chrysene. The high residual concentration of some four-ring PAHs may be attributable to bioavailability issues rather than a lack of microbial catabolic activity. Comparable results between ENA and bioaugmentation at pilot scale were surprising given the limited degradative capacity of the microbial consortia enriched from the creosote-contaminated soil.