Showing papers by "Peter Kille published in 2022"

Moving towards improved surveillance and earlier diagnosis of aquatic pathogens: From traditional methods to emerging technologies

Abstract: Abstract Early and accurate diagnosis is key to mitigating the impact of infectious diseases, along with efficient surveillance. This however is particularly challenging in aquatic environments due to hidden biodiversity and physical constraints. Traditional diagnostics, such as visual diagnosis and histopathology, are still widely used, but increasingly technological advances such as portable next generation sequencing (NGS) and artificial intelligence (AI) are being tested for early diagnosis. The most straightforward methodologies, based on visual diagnosis, rely on specialist knowledge and experience but provide a foundation for surveillance. Future computational remote sensing methods, such as AI image diagnosis and drone surveillance, will ultimately reduce labour costs whilst not compromising on sensitivity, but they require capital and infrastructural investment. Molecular techniques have advanced rapidly in the last 30 years, from standard PCR through loop‐mediated isothermal amplification (LAMP) to NGS approaches, providing a range of technologies that support the currently popular eDNA diagnosis. There is now vast potential for transformative change driven by developments in human diagnostics. Here we compare current surveillance and diagnostic technologies with those that could be used or developed for use in the aquatic environment, against three gold standard ideals of high sensitivity, specificity, rapid diagnosis, and cost‐effectiveness.

VarLOCK - sequencing independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance

Abstract: The COVID-19 pandemic continues to pose a threat to the general population. The ongoing vaccination programs provide protection to individuals and facilitate the opening of society and a return to normality. However, emergent and existing SARS-CoV-2 variants capable of evading the immune system endanger the efficacy of the vaccination strategy. To preserve the efficacy of SARS-CoV-2 vaccination globally, aggressive and effective surveillance for known and emerging SARS-CoV-2 Variants of Concern (VOC) is required. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. In this work, we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to allow rapid and sensitive discrimination of VOCs, that can be used at point of care and/or implemented in the pipelines of small or large testing facilities, and even determine proportion of VOCs in pooled population-level wastewater samples. This technology aims to complement the ongoing sequencing efforts to allow facile and, crucially, rapid identification of individuals infected with VOCs to help break infection chains. Here, we show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants. In addition, we show the rapid adaptability of the technique for new and emerging VOCs such as Omicron.

Accounting for Dilution of SARS-CoV-2 in Wastewater Samples Using Physico-Chemical Markers

Abstract: Most sewer networks collect domestic wastewater and a variable proportion of extraneous water, such as rainwater, through surface runoff and industrial discharges. Accounting for wastewater dilution is essential to properly quantify wastewater particle loads, whether these are molecular fragments of SARS-CoV-2, or other substances of interest such as illicit drugs or microplastics. This paper presents a novel method for obtaining real-time estimates of wastewater dilution and total daily volume through wastewater treatment works, namely when flow data is not available or unreliable. The approach considers the levels of several physico-chemical markers (ammonia, electrical conductivity, and orthophosphate) in the wastewater against their dry-weather levels. Using high-resolution data from the national Wastewater Surveillance Programme of Wales, we illustrate how the method is robust to spikes in markers and can recover peaks in wastewater flow measurements that may have been capped by hydraulic relief valves. We show the method proves effective in normalising SARS-CoV-2 viral loads in wastewater samples and discuss other applications for this method, looking at wastewater surveillance as a vital tool to monitor both human and environmental health.

Molecular insights into high-altitude adaption and acclimatisation of Aporrectodea caliginosa

Abstract: A megabase genome assembly for Aporrectodea caliginosa is presented with transcriptomic and SNP-based evidence for acclimatisation and adaption to extreme weather conditions found at high altitude. Here, we explore the high-altitude adaptions and acclimatisation of Aporrectodea caliginosa. Population diversity is assessed through mitochondrial barcoding, identifying closely related populations across the island of Pico (Azores). We present the first megabase N50 assembly size (1.2 Mbp) genome for A. caliginosa. High- and low-altitude populations were exposed experimentally to a range of oxygen and temperature conditions, simulating altitudinal conditions, and the transcriptomic responses explored. SNP densities are assessed to identify signatures of selective pressure and their link to differentially expressed genes. The high-altitude A. caliginosa population had lower differential expression and fewer co-expressed genes between conditions, indicating a more condition-refined epigenetic response. Genes identified as under adaptive pressure through Fst and nucleotide diversity in the high-altitude population clustered around the differentially expressed an upstream environmental response control gene, HMGB1. The high-altitude population of A. caliginosa indicated adaption and acclimatisation to high-altitude conditions and suggested resilience to extreme weather events. This mechanistic understanding could help offer a strategy in further identifying other species capable of maintaining soil fertility in extreme environments.

Impacts of Life-Time Exposure of Arsenic, Cadmium and Fluoranthene on the Earthworms’ L. rubellus Global DNA Methylation as Detected by msAFLP

Abstract: This study reports on the effects of long-term exposure to the metals arsenic (As), cadmium (Cd) and the polycyclic aromatic hydrocarbon fluoranthene on the survival, growth, development and DNA methylation status of the earthworm Lumbricus rubellus. Exposures to the three chemicals were conducted over their whole juvenile developmental period from egg to adult. Significant effects on one or more measured endpoints were found for all three chemicals. Arsenic had no effect on survival, but had a significant effect on growth rates at concentrations of 36 mg/kg or higher and also slowed the rate of maturation. Cadmium significantly reduced juvenile survival at 500 mg/kg, juvenile growth at 148 mg/kg and maturation rates at all tested concentrations. Fluoranthene had no effect on survival or the developmental period, but did significantly reduce growth rates at 800 mg/kg. Effects at these concentrations are consistent with the known effects of these three chemicals on earthworms from previous studies conducted mainly with Eisenia fetida. Both As and Cd had no effect on DNA methylation patterning in earthworms measured at the end of the exposure. Fluoranthene was shown, for the first time. to have an effect on a species’ DNA methylation levels. These results suggest that apical phenotypic changes for As and Cd are not necessarily associated with changes in DNA methylation profiles. However, exposure to the organic chemical fluoranthene influenced DNA methylation patterns, suggesting wider remodelling of the epigenome for this chemical.

Carbon and nitrogen storage and stability by mineral-organic association in physical fractions of anthropogenic dark earth and of reference soils in Amazonia

Abstract: The anthropogenic dark earths (ADEs) are being assumed in recent years as a model representing the result of sustainable soil management practices carried out by Pre-Columbian peoples. However, little is known about the role of mineral-organic associations in organic matter storage in those soils compared to the emphasis generally given to the role of pyrogenic structures. We quantified the changes of carbon and nitrogen and their distribution in physical fractions of ADEs in relation to the reference (adjacent) soil. Four ADEs sites having the different soil textural classes of sandy clay loam, sandy clay, clayey, and very clayey were selected in the Amazon region of Brazil. Soil samples were collected from the 0–10 cm layer and a subset of the sample was separated into large aggregates (>500 μm) and small aggregates (<500 μm). The ADEs stored on average 45% more total organic carbon (TOC) and 44% more total nitrogen (TN) than the reference soils. Of the incremental TOC and TN in ADE relative to the reference soil, the silt size fraction stored on average 92% of this TOC and 37% of this TN and had C:N ratios as high as 25, which may indicate the presence of pyrogenic material. The clay fraction stored a substantial share of 27–46% of the incremental TOC and 27–66% of the incremental TN. The C:N ratio in the clay size fraction of ADEs, on average 10.5, was lower or not different than in the clay fraction of reference soil (average of 11.1), indicating that the organic matter in the clay fraction even of ADEs was predominantly of microbial origin, and not pyrogenic. We therefore conclude that the clay fraction proved to be an important location to the accumulation and stabilization of TOC and TN in these Anthrosols, possibly by mineral-organic association mechanisms.

Aryl hydrocarbon receptor utilises cellular zinc signals to maintain the gut epithelial barrier

Abstract: Both zinc and plant-derived ligands of the aryl hydrocarbon receptor (AHR) are dietary components which regulate intestinal epithelial barrier function and protect against Inflammatory Bowel Disease (IBD)1,2. Here, we explore whether zinc and AHR pathway are linked using a mouse IBD model with follow-on studies on human and mouse ileum organoids. Our data demonstrate that AHR regulates cellular zinc uptake, and that zinc is an integral part of AHR signalling processes. We show that dietary supplementation in mice with the plant-derived AHR ligand precursor, indole-3-carbinol (I3C), offers a high level of protection against dextran sulfate sodium induced IBD while protection fails in mice with AHR deleted in the intestinal epithelium. AHR agonist treatment is also ineffective in mice with a nutritional zinc deficiency. Experiments in the human Caco-2 cell line and ileum organoids showed that AHR activation increases total cellular zinc and cytosolic free Zn2+ concentrations through transcriptional upregulation of several SLC39 zinc importers. As a consequence, genes for tight junction (TJ) proteins were upregulated in a zinc-dependent manner involving zinc inhibition of signalling to NF-κB and attenuated degradation of TJ proteins through zinc inhibition of calpain activity. Thus, our data indicate that AHR activation by plant-derived dietary ligands improves gut barrier function via zinc-dependent cellular pathways, suggesting that combined dietary supplementation with AHR ligands and zinc might be effective in preventing and treating inflammatory gut disorders.

Challenges to Implementing Environmental-DNA Monitoring in Namibia

Abstract: By identifying fragments of DNA in the environment, eDNA approaches present a promising tool for monitoring biodiversity in a cost-effective way. This is particularly pertinent for countries where traditional morphological monitoring has been sparse. The first step to realising the potential of eDNA is to develop methodologies that are adapted to local conditions. Here, we test field and laboratory eDNA protocols (aqueous and sediment samples) in a range of semi-arid ecosystems in Namibia. We successfully gathered eDNA data on a broad suite of organisms at multiple trophic levels (including algae, invertebrates and bacteria) but identified two key challenges to the implementation of eDNA methods in the region: 1) high turbidity requires a tailored sampling technique and 2) identification of taxa by eDNA methods is currently constrained by a lack of reference data. We hope this work will guide the deployment of eDNA biomonitoring in the arid ecosystems of Namibia and neighbouring countries.