Assessing DNA for fish identifications from reference collections: the good,bad and ugly shed light on formalin fixation and sequencing approaches

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.     

Incorporation of mineral nitrogen into the soil food web as affected by plant community composition

Although nitrogen (N) deposition is increasing globally, N availability still limits many organisms, such as microorganisms and mesofauna. However, little is known to which extent soil organisms rely on mineral‐derived N and whether plant community composition modifies its incorporation into soil food webs. More diverse plant communities more effectively compete with microorganisms for mineral N likely reducing the incorporation of mineral‐derived N into soil food webs. We set up a field experiment in experimental grasslands with different levels of plant species and functional group richness. We labeled soil with ¹⁵NH₄ ¹⁵NO₃ and analyzed the incorporation of mineral‐derived ¹⁵N into soil microorganisms and mesofauna over 3 months. Mineral‐derived N incorporation decreased over time in all investigated organisms. Plant species richness and presence of legumes reduced the uptake of mineral‐derived N into microorganisms. In parallel, the incorporation of mineral‐derived ¹⁵N into mesofauna species declined with time and decreased with increasing plant species richness in the secondary decomposer springtail Ceratophysella sp. Effects of both plant species richness and functional group richness on other mesofauna species varied with time. The presence of grasses increased the ¹⁵N incorporation into Ceratophysella sp., but decreased it in the primary decomposer oribatid mite Tectocepheus velatus sarekensis. The results highlight that mineral N is quickly channeled into soil animal food webs via microorganisms irrespective of plant diversity. The amount of mineral‐derived N incorporated into soil animals, and the plant community properties affecting this incorporation, differed markedly between soil animal taxa, reflecting species‐specific use of food resources. Our results highlight that plant diversity and community composition alter the competition for N in soil and change the transfer of N across trophic levels in soil food webs, potentially leading to changes in soil animal population dynamics and community composition. Sustaining high plant diversity may buffer detrimental effects of elevated N deposition on soil biota.     

Assessing the Environmental Impact of Water Consumption by Energy Crops Grown in Spain

The environmental impact of the water consumption of four typical crop rotations grown in Spain, including energy crops, was analyzed and compared against Spanish agricultural and natural reference situations. The life cycle assessment (LCA) methodology was used for the assessment of the potential environmental impact of blue water (withdrawal from water bodies) and green water (uptake of soil moisture) consumption. The latter has so far been disregarded in LCA. To account for green water, two approaches have been applied: the first accounts for the difference in green water demand of the crops and a reference situation. The second is a green water scarcity index, which measures the fraction of the soil‐water plant consumption to the available green water. Our results show that, if the aim is to minimize the environmental impacts of water consumption, the energy crop rotations assessed in this study were most suitable in basins in the northeast of Spain. In contrast, the energy crops grown in basins in the southeast of Spain were associated with the greatest environmental impacts. Further research into the integration of quantitative green water assessment in LCA is crucial in studies of systems with a high dependence on green water resources.     

Evaluation of Garden Crop Mercury Uptake and Potential Exposure from Consumption of Garden Crops Grown on Floodplain Soils

ABSTRACT

This study investigated Hg uptake from soil into garden crops to help assess the significance of human consumption of crops as a potential route of exposure to Hg. Locations for both a floodplain and a control garden were identified within the Augusta Forestry Center near Crimora, VA, USA, which is about 16 river-km downstream from the city of Waynesboro, along the South River. The floodplain garden had measured soil Hg concentrations ranging from 4.2 to 78 mg Hg kg^?1 dry weight basis in the surface to 15-cm deep layer. A total of 139 samples from the floodplain garden from 17 different crops were analyzed for Hg. All crop samples (except for nine) had less than 0.1 μg Hg g^?1 wet weight basis (ww). Many samples were less than the method detection limit (MDL) of 0.003 μg Hg g^?1 ww. Based on the measured Hg concentrations and several conservative assumptions (e.g., Hg assumed present when less than MDL; 100% consumption from the geographical area in which study was conducted; and 100% bioavailable Hg as methyl Hg), consumption of crops with these Hg levels is not expected to be a significant route of Hg exposure.     

Rapid Identification of Antifungal Compounds against Exserohilum rostratum Using High Throughput Drug Repurposing Screens

A recent large outbreak of fungal infections by Exserohilum rostratum from contaminated compounding solutions has highlighted the need to rapidly screen available pharmaceuticals that could be useful in therapy. The present study utilized two newly-developed high throughput assays to screen approved drugs and pharmaceutically active compounds for identification of potential antifungal agents. Several known drugs were found that have potent effects against E. rostratum including the triazole antifungal posaconazole. Posaconazole is likely to be effective against infections involving septic joints and may provide an alternative for refractory central nervous system infections. The anti-E. rostratum activities of several other drugs including bithionol (an anti-parasitic drug), tacrolimus (an immunosuppressive agent) and floxuridine (an antimetabolite) were also identified from the drug repurposing screens. In addition, activities of other potential antifungal agents against E. rostratum were excluded, which may avoid unnecessary therapeutic trials and reveals the limited therapeutic alternatives for this outbreak. In summary, this study has demonstrated that drug repurposing screens can be quickly conducted within a useful time-frame. This would allow clinical implementation of identified alternative therapeutics and should be considered as part of the initial public health response to new outbreaks or rapidly-emerging microbial pathogens.     

Rare Variants in PLXNA4 and Parkinson’s Disease

Approximately 20% of individuals with Parkinson’s disease (PD) report a positive family history. Yet, a large portion of causal and disease-modifying variants is still unknown. We used exome sequencing in two affected individuals from a family with late-onset familial PD followed by frequency assessment in 975 PD cases and 1014 ethnically-matched controls and linkage analysis to identify potentially causal variants. Based on the predicted penetrance and the frequencies, a variant in PLXNA4 proved to be the best candidate and PLXNA4 was screened for additional variants in 862 PD cases and 940 controls, revealing an excess of rare non-synonymous coding variants in PLXNA4 in individuals with PD. Although we cannot conclude that the variant in PLXNA4 is indeed the causative variant, these findings are interesting in the light of a surfacing role of axonal guidance mechanisms in neurodegenerative disorders but, at the same time, highlight the difficulties encountered in the study of rare variants identified by next-generation sequencing in diseases with autosomal dominant or complex patterns of inheritance.     

Pro-Inflammatory Adipokines as Predictors of Incident Cancers in a Chinese Cohort of Low Obesity Prevalence in Hong Kong

Background

Cytokines released from adipose tissues induce chronic low-grade inflammation, which may enhance cancer development. We investigated whether indices of obesity and circulating adipokine levels could predict incident cancer risk.

Materials and Methods

This longitudinal community-based study included subjects from the Hong Kong Cardiovascular Risk Factors Prevalence Study (CRISPS) study commenced in 1995-1996 (CRISP-1) with baseline assessments including indices of obesity. Subjects were reassessed in 2000-2004 (CRISPS-2) with measurement of serum levels of adipokines including interleukin-6 (IL-6), soluble tumor necrosis factor receptor 2 (sTNFR2; as a surrogate marker of tumor necrosis factor-α activity), leptin, lipocalin 2, adiponectin and adipocyte-fatty acid binding protein (A-FABP). Incident cancer cases were identified up to 31 December 2011.

Results

205 of 2893 subjects recruited at CRISPS-1 had developed incident cancers. More of the subjects who developed cancers were obese (22.1 vs 16.1%) or had central obesity (36.6 vs 24.5%) according to Asian cut-offs. Waist circumference (adjusted HR 1.02 [1.00-1.03] per cm; p=0.013), but not body mass index (adjusted HR 1.04 [1.00-1.08] per kg/m²; p=0.063), was a significant independent predictor for incident cancers after adjustment for age, sex and smoking status. 99 of 1899 subjects reassessed at CRISPS-2 had developed cancers. Subjects who developed cancers had significantly higher level of hsCRP, IL-6, sTNFR2 and lipocalin 2. After adjustment for conventional risk factors, only IL-6 (HR 1.51, 95% CI 1.18-1.95) and sTNFR2 (HR 3.27, 95%CI 1.65-6.47) predicted cancer development.

Conclusions

Our data supported the increased risk of malignancy by chronic low grade inflammation related to central obesity.