Divergence thresholds and divergent biodiversity estimates: can metabarcoding reliably describe zooplankton communities?

DNA metabarcoding is a promising method for describing communities and estimating biodiversity. This approach uses high‐throughput sequencing of targeted markers to identify species in a complex sample. By convention, sequences are clustered at a predefined sequence divergence threshold (often 3%) into operational taxonomic units (OTUs) that serve as a proxy for species. However, variable levels of interspecific marker variation across taxonomic groups make clustering sequences from a phylogenetically diverse dataset into OTUs at a uniform threshold problematic. In this study, we use mock zooplankton communities to evaluate the accuracy of species richness estimates when following conventional protocols to cluster hypervariable sequences of the V4 region of the small subunit ribosomal RNA gene (18S) into OTUs. By including individually tagged single specimens and “populations” of various species in our communities, we examine the impact of intra‐ and interspecific diversity on OTU clustering. Communities consisting of single individuals per species generated a correspondence of 59–84% between OTU number and species richness at a 3% divergence threshold. However, when multiple individuals per species were included, the correspondence between OTU number and species richness dropped to 31–63%. Our results suggest that intraspecific variation in this marker can often exceed 3%, such that a single species does not always correspond to one OTU. We advocate the need to apply group‐specific divergence thresholds when analyzing complex and taxonomically diverse communities, but also encourage the development of additional filtering steps that allow identification of artifactual rRNA gene sequences or pseudogenes that may generate spurious OTUs.     

The Antibiotic Dosage of Fastest Resistance Evolution: Gene Amplifications Underpinning the Inverted-U

To determine the dosage at which antibiotic resistance evolution is most rapid, we treated Escherichia coli in vitro, deploying the antibiotic erythromycin at dosages ranging from zero to high. Adaptation was fastest just below erythromycin’s minimal inhibitory concentration (MIC) and genotype-phenotype correlations determined from whole genome sequencing revealed the molecular basis: simultaneous selection for copy number variation in three resistance mechanisms which exhibited an “inverted-U” pattern of dose-dependence, as did several insertion sequences and an integron. Many genes did not conform to this pattern, however, reflecting changes in selection as dose increased: putative media adaptation polymorphisms at zero antibiotic dosage gave way to drug target (ribosomal RNA operon) amplification at mid dosages whereas prophage-mediated drug efflux amplifications dominated at the highest dosages. All treatments exhibited E. coli increases in the copy number of efflux operons acrAB and emrE at rates that correlated with increases in population density. For strains where the inverted-U was no longer observed following the genetic manipulation of acrAB, it could be recovered by prolonging the antibiotic treatment at subMIC dosages.     

Characterization of water and wildlife strains as a subgroup of Campylobacter jejuni using DNA microarrays

Campylobacter jejuni is the leading cause of human bacterial gastroenteritis worldwide, but source attribution of the organism is difficult. Previously, DNA microarrays were used to investigate isolate source, which suggested a non‐livestock source of infection. In this study we analysed the genome content of 162 clinical, livestock and water and wildlife (WW) associated isolates combined with the previous study. Isolates were grouped by genotypes into nine clusters (C1 to C9). Multilocus sequence typing (MLST) data demonstrated that livestock associated clonal complexes dominated clusters C1–C6. The majority of WW isolates were present in the C9 cluster. Analysis of previously reported genomic variable regions demonstrated that these regions were linked to specific clusters. Two novel variable regions were identified. A six gene multiplex PCR (mPCR) assay, designed to effectively differentiated strains into clusters, was validated with 30 isolates. A further five WW isolates were tested by mPCR and were assigned to the C7‐C9 group of clusters. The predictive mPCR test could be used to indicate if a clinical case has come from domesticated or WW sources. Our findings provide further evidence that WW C. jejuni subtypes show niche adaptation and may be important in causing human infection.     

The miR-30 MicroRNA Family Targets smoothened to Regulate Hedgehog Signalling in Zebrafish Early Muscle Development

The importance of microRNAs in development is now widely accepted. However, identifying the specific targets of individual microRNAs and understanding their biological significance remains a major challenge. We have used the zebrafish model system to evaluate the expression and function of microRNAs potentially involved in muscle development and study their interaction with predicted target genes. We altered expression of the miR-30 microRNA family and generated phenotypes that mimicked misregulation of the Hedgehog pathway. Inhibition of the miR-30 family increases activity of the pathway, resulting in elevated ptc1 expression and increased numbers of superficial slow-muscle fibres. We show that the transmembrane receptor smoothened is a target of this microRNA family. Our results indicate that fine coordination of smoothened activity by the miR-30 family allows the correct specification and differentiation of distinct muscle cell types during zebrafish embryonic development.     

Schistosoma japonicum Soluble Egg Antigens Attenuate Invasion in a First Trimester Human Placental Trophoblast Model

Background

Schistosomiasis affects nearly 40 million women of reproductive age, and is known to elicit a pro-inflammatory signature in the placenta. We have previously shown that antigens from schistosome eggs can elicit pro-inflammatory cytokine production from trophoblast cells specifically; however, the influence of these antigens on other characteristics of trophoblast function, particularly as it pertains to placentation in early gestation, is unknown. We therefore sought to determine the impact of schistosome antigens on key characteristics of first trimester trophoblast cells, including migration and invasion.

Methods

First trimester HTR8/SVneo trophoblast cells were co-cultured with plasma from pregnant women with and without schistosomiasis or schistosome soluble egg antigens (SEA) and measured cytokine, cellular migration, and invasion responses.

Results

Exposure of HTR8 cells to SEA resulted in a pro-inflammatory, anti-invasive signature, characterized by increased pro-inflammatory cytokines (IL-6, IL-8, MCP-1) and TIMP-1. Additionally, these cells displayed 62% decreased migration and 2.7-fold decreased invasion in vitro after treatment with SEA. These results are supported by increased IL-6 and IL-8 in the culture media of HTR8 cells exposed to plasma from Schistosoma japonica infected pregnant women.

Conclusions

Soluble egg antigens found in circulation during schistosome infection increase pro-inflammatory cytokine production and inhibit the mobility and invasive characteristics of the first trimester HTR8/SVneo trophoblast cell line. This is the first study to assess the impact of schistosome soluble egg antigens on the behavior of an extravillous trophoblast model and suggests that schistosomiasis in the pre-pregnancy period may adversely impact placentation and the subsequent health of the mother and newborn.     

Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms

RING‐in‐between‐RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub‐conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT‐type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING‐type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub‐binding site on HHARI RING2 important for its recruitment to RING1‐bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.     

A heavy-atom isotope effect and kinetic investigation of the hydrolysis of semicarbazide by urease from jack bean (Canavalia ensiformis)

A kinetic investigation of the hydrolysis of semicarbazide by urease gives a relatively flat log V/ K versus pH plot between pH 5 and 8. A log V m versus pH plot shows a shift of the optimum V m toward lower pH when compared to urea. These results are explained in terms of the binding of the outer N of the NHNH 2 group in semicarbazide to an active site residue with a relatively low p K a ( approximately 6). Heavy-atom isotope effects for both leaving groups have been determined. For the NHNH 2 side, (15) k obs = 1.0045, whereas for the NH 2 side, (15) k obs = 1.0010. This is evidence that the NHNH 2 group leaves prior to the NH 2 group. Using previously published data from the urease-catalyzed hydrolysis of formamide, the commitment factors for semicarbazide and urea hydrolysis are estimated to be 2.7 and 1.2, respectively. The carbonyl-C isotope effect ( (13) k obs) equals 1.0357, which is consistent with the transition state occurring during either formation or breakdown of the tetrahedral intermediate.     

Targeted subfield switchgrass integration could improve the farm economy,water quality,and bioenergy feedstock production

Progress on reducing nutrient loss from annual croplands has been hampered by perceived conflicts between short‐term profitability and long‐term stewardship, but these may be overcome through strategic integration of perennial crops. Perennial biomass crops like switchgrass can mitigate nitrate‐nitrogen (NO₃‐N) leaching, address bioenergy feedstock targets, and – as a lower‐cost management alternative to annual crops (i.e., corn, soybeans) – may also improve farm profitability. We analyzed publicly available environmental, agronomic, and economic data with two integrated models: a subfield agroecosystem management model, Landscape Environmental Assessment Framework (LEAF), and a process‐based biogeochemical model, DeNitrification‐DeComposition (DNDC). We constructed a factorial combination of profitability and NO₃‐N leaching thresholds and simulated targeted switchgrass integration into corn/soybean cropland in the agricultural state of Iowa, USA. For each combination, we modeled (i) area converted to switchgrass, (ii) switchgrass biomass production, and (iii) NO₃‐N leaching reduction. We spatially analyzed two scenarios: converting to switchgrass corn/soybean cropland losing >US$ 100 ha^?1 and leaching >50 kg ha^?1 (‘conservative’ scenario) or losing >US$ 0 ha^?1 and leaching >20 kg ha^?1 (‘nutrient reduction’ scenario). Compared to baseline, the ‘conservative’ scenario resulted in 12% of cropland converted to switchgrass, which produced 11 million Mg of biomass and reduced leached NO₃‐N 18% statewide. The ‘nutrient reduction’ scenario converted 37% of cropland to switchgrass, producing 34 million Mg biomass and reducing leached NO₃‐N 38% statewide. The opportunity to meet joint goals was greatest within watersheds with undulating topography and lower corn/soybean productivity. Our approach bridges the scales at which NO₃‐N loss and profitability are usually considered, and is informed by both mechanistic and empirical understanding. Though approximated, our analysis supports development of farm‐level tools that can identify locations where both farm profitability and water quality improvement can be achieved through the strategic integration of perennial vegetation.