Accumulation of Environmental Radioactivity on the Surface of a High Arctic Ice Cap (Flade Isblink, NE Greenland).

Under climatic warming, glaciers are becoming a secondary source of atmospheric contaminants originally released into the environment decades ago. This phenomenon has been well-documented for glaciers near emission sources. However, less is known about polar ice sheets and ice caps. Radionuclides are one of the contaminants that can be remobilised through ice melting and accumulate in cryoconite material on the surface of glaciers. To understand the cycling of radionuclides in polar glacial contexts, we evaluate the radioactivity of cryoconite samples from Flade Isblink, a High Arctic ice cap in northeast Greenland. The measured radioactivity is among the highest reported across the High Arctic and the highest from Greenland. The high variability observed among the samples is explained by considering the different macroscopic features of single cryoconite deposits. The radioactivity source is compatible with the stratospheric reservoir established during atmospheric nuclear tests and with weapons-grade fissile fuel, likely originating from Novaya Zemlya proving grounds. This study shows that the ability of cryoconite to accumulate radioactivity in remote areas is undisputed, highlighting the need for a deeper understanding of the remobilisation of radioactive species in polar glacial contexts.

Plutonium isotopes can be used to model soil erosion in Kenya.

Climate change poses an immediate threat to tropical soils with changes in rainfall patterns resulting in accelerated land degradation processes. To ensure the future sustainability of arable land, it is essential to improve our understanding of the factors that influence soil erosion processes. This work aimed to evaluate patterns of soil erosion using the activity of plutonium isotopes (Pu) at sites with different land use and clearance scale in the Winam Gulf catchment of Lake Victoria in Kenya. Erosion rates were modelled at potential erosive sites using the MODERN model to understand small-scale erosion processes and the effect of different management practices. The lowest soil redistribution rates for arable land were 0.10 Mg ha-1 yr-1 showing overall deposition, resulting from community-led bottom-up mitigation practices. In contrast erosion rates of 8.93 Mg ha-1 yr-1 were found in areas where steep terraces have been formed. This demonstrates the significance of community-led participation in effectively managing land degradation processes. Another key factor identified in the acceleration of soil erosion rates was the clearance of land with an increased rate of erosion over three years reported (0.45 to 0.82 Mg ha-1 yr-1) underlining the importance vegetation cover plays in limiting soil erosion processes. This novel application of fallout plutonium as a tracer, highlights its potential to inform the understanding of how soil erosion processes respond to land management, which will better support implementation of effective mitigation strategies.

Anthropogenic and climatic impacts on historic sediment, carbon, and phosphorus accumulation rates using 210Pbex and 137Cs in a sub-watershed linked to Zarivar Lake, Iran.

To estimate a watershed's response to climate change, it is crucial to understand how human activities and climatic extremes have interacted over time. Over the last century, the Zarivar Lake watershed, Iran, has been subjected to various anthropogenic activates, including deforestation and inappropriate land-management practices alongside the implementation of conservation measures like check dams. To understand the effects of these changes on the magnitude of sediment, organic carbon (OC), and phosphorus supplies in a small sub-watershed connected to the lake over the last century, a lake sediment core was dated using 210Pbex and 137Cs as geochronometers. The average mass accumulation rate (MAR), organic carbon accumulation rates (OCAR), and particulate phosphorus accumulation rates (PPAR) of the sediment core were determined to be 6498 ± 2475, 205 ± 85, and 8.9 ± 3.3 g m-2 year-1, respectively. Between the late 1970s and early 1980s, accumulation rates were significantly higher than their averages at 7940 ± 3120, 220 ± 60, and 12.0 ± 2.8 g m-2 year-1 respectively. During this period, the watershed underwent extensive deforestation (12%) on steep slopes, coinciding with higher mean annual precipitations (more than double). Conversely, after 2009, when check dams were installed in the sub-watershed, the sediment load to the lake became negligible. The results of this research indicate that anthropogenic activities had a pronounced effect on MAR, OCAR, and PPAR, causing them to fluctuate from negligible amounts to values twice the averages over the last century, amplified by climatic factors. These results imply that implementing climate-smart watershed management strategies, such as constructing additional check dams and terraces, reinforcing restrictions on deforestation, and minimum tillage practices, can facilitate protection of lacustrine ecosystems under accelerating climate change conditions.

High-Throughput CRISPR-Cas13 SARS-CoV-2 Test.

BACKGROUND: The ability to control the spread of COVID-19 continues to be hampered by a lack of rapid, scalable, and easily deployable diagnostic solutions. METHODS: We developed a diagnostic method based on CRISPR (clustered regularly interspaced short palindromic repeats) that can deliver sensitive, specific, and high-throughput detection of Sudden Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). The assay utilizes SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the qualitative detection of SARS-CoV-2 RNA and may be performed directly on a swab or saliva sample without nucleic acid extraction. The assay uses a 384-well format and provides results in <1 hour. RESULTS: Assay performance was evaluated with 105 (55 negative, 50 positive) remnant SARS-CoV-2 specimens previously tested using Food and Drug Administration emergency use authorized assays and retested with a modified version of the Centers for Disease Control and Prevention (CDC) quantitative PCR with reverse transcription (RT-qPCR) assay. When combined with magnetic bead-based extraction, the high-throughput SHERLOCK SARS-CoV-2 assay was 100% concordant (n = 60) with the CDC RT-qPCR. When used with direct sample addition the high-throughput assay was also 100% concordant with the CDC RT-qPCR direct method (n = 45). With direct saliva sample addition, the negative and positive percentage agreements were 100% (15/15, 95% CI: 81.8-100%) and 88% (15/17, 95% CI: 63.6-98.5%), respectively, compared with results from a collaborating clinical laboratory. CONCLUSIONS: This high-throughput assay identifies SARS-CoV-2 from patient samples with or without nucleic acid extraction with high concordance to RT-qPCR methods. This test enables high complexity laboratories to rapidly increase their testing capacities with simple equipment.

Assessment of arsenic status and distribution in Usangu agro-ecosystem-Tanzania.

This study was conducted to assess arsenic (As) status and distribution in Usangu agroecosystem-Tanzania, including three land use. About 198 soil samples were collected in ten irrigation schemes in three land uses. Total and bioavailable As were determined by acid digestion (Aqua regia (AQ)) and Mehlich 3 method (M3) to estimate status, distribution and bioavailability. Arsenic concentration were variable among land use and irrigation schemes where total arsenic ranged 567.74-2909.84 µg/kg and bioavailable As ranged 26.17-712.37 µg/kg. About 12-16% of total arsenic were available for plant uptake. Approximately 86.53% of studied agricultural soils had total As concentration above Tanzania maximum allowable limit. Bioavailable As were lower compared to total As and were within the acceptable threshold. Total arsenic concentration were variable among schemes and higher values were observed in schemes which are highly intensified and mechanized. Thus, this study provides essential site specific preliminary baseline information for As status and distribution in agricultural soils to initiate monitoring and management strategies for increased land productivity and environmental safety.

Toxic metals in East African agro-ecosystems: Key risks for sustainable food production.

The dramatic increase in world population underpins current escalating food demand, which requires increased productivity in the available arable land through agricultural intensification. Agricultural intensification involves increased agrochemicals use to increase land productivity. Increased uses of agrochemicals pose environmental and ecological risks such as contamination and water eutrophication. Consequently, toxic metals accumulate in plant products, thus entering the food chain leading to health concerns. To achieve this study, secondary data from peer-reviewed papers, universities, and government authorities were collected from a public database using Tanzania as a case study. Data from Science Direct, Web of Science, and other internet sources were gathered using specific keywords such as nutrient saturation and losses, water eutrophication, potentially toxic metal (PTEs), and impact of toxic metals on soils, water, and food safety. The reported toxic metal concentrations in agro-ecosystem worldwide are linked to agricultural intensification, mining, and urbanization. Statistical analysis of secondary data collected from East African agro-ecosystem had wide range of toxic metals concentration such as; mercury (0.001-11.0 mg Hg/kg), copper (0.14-312 mg Cu/kg), cadmium (0.02-13.8 mg Cd/kg), zinc (0.27-19.30 mg Zn/kg), lead (0.75-51.7 mg Pb/kg) and chromium (19.14-34.9 mg Cr/kg). In some cases, metal concentrations were above the FAO/WHO maximum permissible limits for soil health. To achieve high agricultural productivity and environmental safety, key research-informed policy needs are proposed: (i) development of regulatory guidelines for agrochemicals uses, (ii) establishment of agro-environmental quality indicators for soils and water assessment to monitor agro-ecosystem quality changes, and (iii) adoption of best farming practices such as split fertilization, cover cropping, reduced tillage, drip irrigation to ensure crop productivity and agro-ecosystem sustainability. Therefore, robust and representative evaluation of current soil contamination status, sources, and processes leading to pollution are paramount. To achieve safe and sustainable food production, management of potential toxic metal in agro-ecosystems is vital.

Transport and Redistribution of Radiocesium in Fukushima Fallout through Rivers.

The Fukushima Daiichi Nuclear Power Plant (FDNPP) accident released the most significant quantity of radiocesium into the environment since Chernobyl, and detailed measurements over the initial 5 years provide new insights into fluvial redistribution of radiocesium. We found that the high initial activity concentration of 137Cs-bearing suspended sediment in rivers was followed by a steep exponential decline (λ1) which extended to approximately 1 year after the accident, while the rate of initial decline in radiocesium activity concentration in water was an order of magnitude higher than rates measured after Chernobyl. Fluvial transport of 137Cs to the ocean from the Abukuma river totaled 12 TBq between June 2011 and August 2015 and almost all this radiocesium (96.5%) was transported in the particulate form. The primary sources of 137Cs were paddy fields, farmland, and urban areas [plaque-forming unit (PFU)], discharging 85% of the exported 137Cs from 38% of the watershed area. After 1 year, activity concentrations were lower and exhibited a more gradual secondary decline (λ2) which was associated with reduced radiocesium losses from PFU areas, while forest areas continue to represent more stable contaminant stores.

Fingerprinting changes in source contribution for evaluating soil response during an exceptional rainfall in Spanish pre-pyrenees.

In the Mediterranean region, floods are expected to increase as a result of climate change and knowledge of soil erosion hot spots during exceptional rainfalls is required to support mitigation measures. This study quantifies the main sediment sources during an exceptional rainfall event in 2012 (235 mm) at the outlet of two catchments located in NE Spain. To this purpose, suspended sediments were collected during the flood event, complemented with entrapped sediments in mat taken one year after the event. We used fingerprinting methodology and applied the FingerPro unmixing model to estimate the contribution from main sources. The selected tracers clearly distinguished agricultural, rangeland, subsoil and channel banks as the four potential sources in both catchments. In the Vandunchil catchment, the 8 time-integrated suspended sediment samples revealed changes in source contribution during the 2-h sampling sequence. There were relatively high contributions from rangeland, agriculture and subsoil at the beginning of the sampling, representing 30, 40 and 35% of the total source contributions, respectively. Our records captured the delivery of pulses of eroded surface soil transported by runoff with direct connectivity to the stream. The sequence was followed by a sharp increase in channel bank contribution (up to 90%) in comparison to the other sources, reflecting streambank erosion and landslide occurrence, which manifested during the flood. In contrast, in the La Reina catchment, agricultural soils contributed the most (65%) and, together with subsoils (32%), were the main sources. These results reflect the effect of the higher connectivity and slope gradient of these cultivated fields of the La Reina catchment in comparison with those of the Vandunchil catchment. We discuss the possibility of using different properties, such as radionuclides, geochemistry and magnetic measurements, as tracers to distinguish between potential sources during an exceptional event in upland Mediterranean catchments. Our results support the use of fingerprinting techniques to determine variations in source contribution and sediment provenance during flood events, as extreme rainfalls are main drivers of sediment mobilization and key factors in changing landscapes. This is essential in identifying vulnerable hot spots, in which early-stage interventions are needed, and for helping policy makers with management of soil and water resources.

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design.

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.

A regional assessment of the cost and effectiveness of mitigation measures for reducing nutrient losses to water and greenhouse gas emissions to air from pastoral farms.

Using a novel approach that links geospatial land resource information with individual farm-scale simulation, we conducted a regional assessment of nitrogen (N) and phosphorous (P) losses to water and greenhouse gas (GHG) emissions to air from the predominant mix of pastoral industries in Southland, New Zealand. An evaluation of the cost-effectiveness of several nutrient loss mitigation strategies applied at the farm-scale, set primarily for reducing N and P losses and grouped by capital cost and potential ease of adoption, followed an initial baseline assessment. Grouped nutrient loss mitigation strategies were applied on an additive basis on the assumption of full adoption, and were broadly identified as 'improved nutrient management' (M1), 'improved animal productivity' (M2), and 'restricted grazing' (M3). Estimated annual nitrate-N leaching losses occurring under representative baseline sheep and beef (cattle) farms, and representative baseline dairy farms for the region were 10 ± 2 and 32 ± 6 kg N/ha (mean ± standard deviation), respectively. Both sheep and beef and dairy farms were responsive to N leaching loss mitigation strategies in M1, at a low cost per kg N-loss mitigated. Only dairy farms were responsive to N leaching loss abatement from adopting M2, at no additional cost per kg N-loss mitigated. Dairy farms were also responsive to N leaching loss abatement from adopting M3, but this reduction came at a greater cost per kg N-loss mitigated. Only dairy farms were responsive to P-loss mitigation strategies, in particular by adopting M1. Only dairy farms were responsive to GHG abatement; greater abatement was achieved by the most intensified dairy farm system simulated. Overall, M1 provided for high levels of regional scale N- and P-loss abatement at a low cost per farm without affecting overall farm production, M2 provided additional N-loss abatement but only marginal P-loss abatement, whereas M3 provided the greatest N-loss abatement, but delivered no additional P abatement, and came at a large financial cost to farmers, sheep and beef farmers in particular. The modelling approach provides a farm-scale framework that can be extended to other regions to accommodate different farm production systems and performances, capturing the interactions between farm types, land use capabilities and production levels, as these influence nutrient losses and GHG emissions, and the effectiveness of mitigation strategies.

Organic carbon accumulation in British saltmarshes.

Saltmarshes are a crucial component of the coastal carbon (C) system and provide a natural climate regulation service through the accumulation and long-term storage of organic carbon (OC) in their soils. These coastal ecosystems are under growing pressure from a changing climate and increasing anthropogenic disturbance. To manage and protect these ecosystems for C and to allow their inclusion in emissions and natural-capital accounting, as well as carbon markets, accurate and reliable estimates of OC accumulation are required. However, globally, such data are rare or of varying quality. Here, we quantify sedimentation rates and OC densities for 21 saltmarshes in Great Britain (GB). We estimate that, on average, saltmarshes accumulate OC at a rate of 110.88 ± 43.12 g C m-2 yr-1. This is considerably less than widely applied global saltmarsh averages. It is therefore highly likely that the contribution of northern European saltmarshes to global saltmarsh OC accumulation has been significantly overestimated. Taking account of the climatic, geomorphological, oceanographic, and ecological characteristics of all GB saltmarshes and the areal extent of different saltmarsh zones, we estimate that the 451.65 km2 of GB saltmarsh accumulates 46,563 ± 4353 t of OC annually. These low OC accumulation rates underline the importance of the 5.20 ± 0.65 million tonnes of OC already stored in these vulnerable coastal ecosystems. Going forward the protection and preservation of the existing stores of OC in GB saltmarshes must be a priority for the UK as this will provide climate benefits through avoided emissions several times more significant than the annual accumulation of OC in these ecosystems.

Basin scale sources of siltation in a contaminated hydropower reservoir.

Siltation and the loss of hydropower reservoir capacity is a global challenge with a predicted 26 % loss of storage at the global scale by 2050. Like in many other Latin American contexts, soil erosion constitutes one of the most significant water pollution problems in Chile with serious siltation consequences downstream. Identifying the sources and drivers affecting hydropower siltation and water pollution is a critical need to inform adaptation and mitigation strategies especially in the context of changing climate regimes e.g. rainfall patterns. We investigated, at basin scale, the main sources of sediments delivered to one of the largest hydropower reservoirs in South America using a spatio-temporal geochemical fingerprinting approach. Mining activities contributed equivalent to 9 % of total recent sediment deposited in the hydropower lake with notable concentrations of sediment-associated pollutants e.g. Cu and Mo in bed sediment between the mine tributary and the reservoir sediment column. Agricultural sources represented ca. 60 % of sediment input wherein livestock production and agriculture promoted the input of phosphorus to the lake. Evaluation of the lake sediment column against the tributary network showed that the tributary associated with both dominant anthropogenic activities (mining and agriculture) contributed substantially more sediment, but sources varied through time: mining activities have reduced in proportional contribution since dam construction and proportional inputs from agriculture have increased in recent years, mainly promoted by recent conversion of steep lands from native vegetation to agriculture. Siltation of major hydropower basins presents a global challenge exemplified by the Rapel basin. The specific challenges faced here highlight the urgent need for co-design of evidence-led, context-specific solutions that address the interplay of drivers both within and without the basin and its communities, enhancing the social acceptability of sediment management strategies to support the sustainability of clean, hydropower energy production.

Distribution and storage of uranium, and its decay products, in floodplain sediments.

Industrialised rivers contain legacy contaminants stored in their sediments and floodplain soils which may inhibit attainment of environmental quality criteria. The River Fal catchment, SW England, is impacted by inputs from uranium mining and clay production and serves as an exemplar for understanding the consequences of medium-term process dynamics in contaminated basins. Radionuclides were determined, by gamma spectroscopy, in six cores from the river floodplain with the aim of quantifying the activities of 238U, and its decay products, and the bomb fallout radionuclides137Cs and 241Am. Activity concentrations of 238U implied inputs from mining, accentuated by flood events and historic industrial accidents, whereas 210Pb activities included a significant input of unsupported 210Pb linked to processed mine spoil. The radionuclide inventories did not decrease systematically downstream revealing evidence of attenuation of particulate radionuclides within the river floodplain sediment column. Storage of legacy contaminants in fluvial systems, at levels in excess of contemporary environmental quality guidelines, emphasises the challenges posed by changing climatic conditions. This scenario raises significant consequences for the management of uranium-contaminated, fertile riverine floodplains within Europe.

Non-operative adjuncts for the prevention of mastectomy skin flap necrosis: a systematic review and meta-analysis.

BACKGROUND: Native skin flap necrosis is a potentially devastating complication following skin-sparing or nipple-sparing mastectomy with a reported incidence of as high as 30%. Treatment depends on the depth and extent of tissue necrosis and can range from dressings to surgical debridement and further reconstruction. This can have implications on patient physical and psychological wellbeing as well as cost of treatment. This study aims to identify and appraise cost-effective non-surgical adjuncts for the prevention of native skin flap necrosis. METHODS: A systematic review was performed using the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement and structured around existing recommended guidelines. A search of MEDLINE, EMBASE, Cumulative Index to Nursing and Allied Health Literature and ClinicalTrials.gov was performed with the medical subject headings 'mastectomy' and 'flap necrosis'. After exclusion, 12 articles were selected for review and analysed. RESULTS: A total of 8439 mastectomies were performed on 7895 patients. Preventative non-surgical adjuncts that demonstrated statistically significant reduction in mastectomy flap necrosis included topical nitroglycerin ointment (P = 0.000), closed-Incision negative pressure wound therapy (P = 0.000), topical dimethylsulfoxide ointment (P = 0.03), oral cilostazol (P = 0.032), and local heat pre-conditioning (P = 0.047). CONCLUSIONS: This study identifies multiple adjuncts that may aid in preventing mastectomy skin flap necrosis, especially in high-risk patients. Further studies could aim to define standardized protocols and compare the various adjuncts in different circumstances.

Aquatic phosphorus behaviour within a UK Ramsar wetland: Impacts of seasonality and hydrology on algal growth and implications for management.

Fundamental to all life, phosphorus is an essential nutrient and, contrastingly, a significant threat to surface water biodiversity globally as one of the most common causes of eutrophication in surface waters worldwide. Freshwater wetland ditches affected by these conditions undergo a conversion from primarily submerged aquatic vegetation to algae or duckweed dominance, leading to anoxic conditions. However, macrophyte biomass harvesting in eutrophic water systems is a promising means of remediation and nutrient recycling. This study seasonally assesses spatial distribution and chemical fractionation of surface water phosphorus, as well as surface biomass abundance and total phosphorus content in the ditch systems at West Sedgemoor (Somerset, UK), a designated site of special scientific interest. Elevated phosphorus concentrations in the surface water were observed across the site, with all sites exceeding e hi the Common Standards Monitoring environmental quality standard value of 0.1 mg L-1 during summer and autumn Sites lacking hydrological flow connectivity with contaminated freshwater inputs, typically had lower surface water phosphorus concentrations than the rest of the moor, with localised hotspots associated with likely cattle related agricultural activity. Summer and autumn were determined as the dominant duckweed growth seasons, in which an estimated 39 kg of phosphorus could be removed via duckweed biomass harvesting, per harvest period.

Optimisation of plutonium separations using TEVA cartridges and ICP-MS/MS analysis for applicability to large-scale studies in tropical soils.

The analysis of plutonium (Pu) in soil samples can inform the understanding of soil erosion processes globally. However, there are specific challenges associated for analysis in tropical soils and so an optimal analytical methodology ensuring best sensitivity is critical. This method aimed to demonstrate the feasibility of sample preparation and analysis of Pu isotopes in African soils, considering the environmental and cost implications applicable to low-resource laboratories. The separation procedure builds upon previous work using TEVA columns, further demonstrating their usefulness for the reduction of uranium (U) interference in ICP-MS analysis with enhanced selectivity for Pu. Here several steps were optimised to enhance Pu recovery, reducing method blank concentration, and improving the separation efficiency through the determination of the elution profiles of U and Pu. The elimination of the complexing agent in the eluent, increased the spike recovery by improving matrix tolerance of the plasma, and simplified the separation procedure, improving throughput by 20%. The subsequent method was validated through the analysis of Certified Reference Material IAEA-384, where high accuracy and improved precision of measurement were demonstrated (measured value 114 ± 12 versus certified value 108 ± 13 Bq kg-1). Optimisation of the column separation, along with the analysis of the samples using O2 gas in ICP-MS/MS mode to mass shift Pu isotopes away from interfering molecular U ions provided a simple, robust, and cost-effective method with low achievable method detection limits of 0.18 pg kg-1 239+240Pu, applicable to the detection of ultra-trace fallout Pu in African soils.

Composition of deposited sediment and its temporal variation in a disturbed tropical catchment in the Kelantan river basin, Peninsular Malaysia.

Pristine tropical river systems are coming under increasing pressure from the development of economic resources such as forestry and mining for valuable elements. The Lebir catchment, north eastern Malaysia, is now under development as a result of unregulated tree felling and mining for essential and rare metals. Two sediment cores, one in the upstream reaches and the other from the downstream reaches, were taken from flood prone area of the Lebir River, Malaysia, and analysed for their elemental composition by XRF, specifically Al, Si, Fe, Ca, K, Mg, Mn, V, Cu, Ni, Pb, Cr, Zn, As, Th and U. Activities of fallout radionuclides, 137Cs and 210Pb were also determined to from a geochronological context. The elemental concentrations in the soils were assessed in terms of their enrichment factor and Si, Ca, K, Mg, Mn, V, Cu, Ni and Zn were found not to be enriched, whereas As, Th and U had elevated enrichment factors. The Th and U were particularly enriched in the downstream core indicating inputs from a tributary that drains a catchment with known deposits of Th and possibly U. The results suggest that the growth in economic development is fostering the transport of contaminants by the major rivers which, in turn, is contaminating the riverine floodplains. This points to the need for a more integrated and holistic approach to river basin management to maintain the environmental quality of these fragile aquatic systems.

Real-Time, Multiplexed SHERLOCK for in Vitro Diagnostics.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for simple, low-cost, and scalable diagnostics that can be widely deployed for rapid testing. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as a promising technology, but its implementation in clinical laboratories has been limited by the requirement of a separate amplification step prior to CRISPR-associated (Cas) enzyme-based detection. This article reports the discovery of two novel Cas12 enzymes (SLK9 and SLK5-2) that exhibit enzymatic activity at 60°C, which, when combined with loop-mediated isothermal amplification (LAMP), enable a real-time, single-step nucleic acid detection method [real-time SHERLOCK (real-time SLK)]. Real-time SLK was demonstrated to provide accurate results comparable to those from real-time quantitative RT-PCR in clinical samples, with 100% positive and 100% negative percent agreement. The method is further demonstrated to be compatible with direct testing (real-time SLK Direct) of samples from anterior nasal swabs, without the need for standard nucleic acid extraction. Lastly, SLK9 was combined with either Alicyclobacillus acidoterrestris AacCas12b or with SLK5-2 to generate a real-time, multiplexed CRISPR-based diagnostic assay for the simultaneous detection of SARS-CoV-2 and a human-based control in a single reaction, with sensitivity down to 5 copies/µL and a time to result of under 30 minutes.

Detection of viral RNAs at ambient temperature via reporter proteins produced through the target-splinted ligation of DNA probes.

Nucleic acid assays are not typically deployable in point-of-care settings because they require costly and sophisticated equipment for the control of the reaction temperature and for the detection of the signal. Here we report an instrument-free assay for the accurate and multiplexed detection of nucleic acids at ambient temperature. The assay, which we named INSPECTR (for internal splint-pairing expression-cassette translation reaction), leverages the target-specific splinted ligation of DNA probes to generate expression cassettes that can be flexibly designed for the cell-free synthesis of reporter proteins, with enzymatic reporters allowing for a linear detection range spanning four orders of magnitude and peptide reporters (which can be mapped to unique targets) enabling highly multiplexed visual detection. We used INSPECTR to detect a panel of five respiratory viral targets in a single reaction via a lateral-flow readout and ~4,000 copies of viral RNA via additional ambient-temperature rolling circle amplification of the expression cassette. Leveraging synthetic biology to simplify workflows for nucleic acid diagnostics may facilitate their broader applicability at the point of care.