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

The COVID-19 pandemic demonstrated the need for rapid molecular diagnostics. Vaccination programs can provide protection and facilitate the opening of society, but newly emergent and existing viral variants capable of evading the immune system endanger their efficacy. Effective surveillance for Variants of Concern (VOC) is therefore important. 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. Here we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to provide rapid and sensitive discrimination of SARS-CoV-2 VOCs that can be used at point of care, implemented in the pipelines of small or large testing facilities, and even determine the proportion of VOCs in pooled population-level wastewater samples. This technology complements sequencing efforts to allow facile and rapid identification of individuals infected with VOCs to help break infection chains. 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 and the rapid adaptability of the technique for new and emerging VOCs.

Modelling how plant cell-cycle progression leads to cell size regulation.

Populations of cells typically maintain a consistent size, despite cell division rarely being precisely symmetrical. Therefore, cells must possess a mechanism of "size control", whereby the cell volume at birth affects cell-cycle progression. While size control mechanisms have been elucidated in a number of other organisms, it is not yet clear how this mechanism functions in plants. Here, we present a mathematical model of the key interactions in the plant cell cycle. Model simulations reveal that the network of interactions exhibits limit-cycle solutions, with biological switches underpinning both the G1/S and G2/M cell-cycle transitions. Embedding this network model within growing cells, we test hypotheses as to how cell-cycle progression can depend on cell size. We investigate two different mechanisms at both the G1/S and G2/M transitions: (i) differential expression of cell-cycle activator and inhibitor proteins (with synthesis of inhibitor proteins being independent of cell size), and (ii) equal inheritance of inhibitor proteins after cell division. The model demonstrates that both these mechanisms can lead to larger daughter cells progressing through the cell cycle more rapidly, and can thus contribute to cell-size control. To test how these features enable size homeostasis over multiple generations, we then simulated these mechanisms in a cell-population model with multiple rounds of cell division. These simulations suggested that integration of size-control mechanisms at both G1/S and G2/M provides long-term cell-size homeostasis. We concluded that while both size independence and equal inheritance of inhibitor proteins can reduce variations in cell size across individual cell-cycle phases, combining size-control mechanisms at both G1/S and G2/M is essential to maintain size homeostasis over multiple generations. Thus, our study reveals how features of the cell-cycle network enable cell-cycle progression to depend on cell size, and provides a mechanistic understanding of how plant cell populations maintain consistent size over generations.

A higher spectral range of beetle bioluminescence with infraluciferin.

Coleopteran bioluminescence is unique in that beetle luciferases emit colors ranging between green (ca.550 nm) and red (ca.600 nm), including intermediate colors such as yellow and orange, allowing up to 3 simultaneous parameters to be resolved in vitro with natural luciferin (D-LH2). Here, we report a more than doubling of the maximum bioluminescence wavelength range using a single synthetic substrate, infraluciferin (iLH2). We report that different luciferases can emit colors ranging from visible green to near-infrared (nIR) with iLH2, including in human cells. iLH2 was designed for dual color far-red to nIR bioluminescence imaging (BLI) in small animals and has been utilized in different mouse models of cancer (including a metastatic hepatic model showing detailed hepatic morphology) and for robust dual parameter imaging in vivo (including in systemic hematological models). Here, we report the properties of different enzymes with iLH2: Lampyrid wild-type (WT) Photinus pyralis (Ppy) firefly luciferase, Ppy-based derivatives previously engineered to be thermostable with D-LH2, and also color-shifted Elaterid-based enzymes: blue-shifted Pyrearinus termitilluminans derivative Eluc (reported D-LH2 λmax = 538 nm) and red-shifted Pyrophorus plagiopthalamus derivative click beetle red (CBR) luciferase (D-LH2 λmax = 618 nm). As purified enzyme, in bacteria or in human cells, Eluc emitted green light (λmax = 536 nm) with DL-iLH2 whereas Ppy Fluc (λmax = 689 nm), x2 Fluc (λmax = 704 nm), x5 Fluc (λmax = 694 nm), x11 Fluc (λmax = 694 nm) and CBR (λmax = 721 nm) produced far-red to nIR peak wavelengths. Therefore, with iLH2, enzyme λmaxes can be separated by ca.185nm, giving almost non-overlapping spectra. This is the first report of single-substrate bioluminescence color emission ranging from visible green to nIR in cells and may help shed light on the color tuning mechanism of beetle luciferases. We also report on the reason for the improvement in activity of x11 Fluc with iLH2 and engineer an improved infraluciferase (iluc) based on this mutant.

Discovery of the Pseudomonas Polyyne Protegencin by a Phylogeny-Guided Study of Polyyne Biosynthetic Gene Cluster Diversity.

Natural products that possess alkyne or polyyne moieties have been isolated from a variety of biological sources and possess a broad a range of bioactivities. In bacteria, the basic biosynthesis of polyynes is known, but their biosynthetic gene cluster (BGC) distribution and evolutionary relationship to alkyne biosynthesis have not been addressed. Through comprehensive genomic and phylogenetic analyses, the distribution of alkyne biosynthesis gene cassettes throughout bacteria was explored, revealing evidence of multiple horizontal gene transfer events. After investigation of the evolutionary connection between alkyne and polyyne biosynthesis, a monophyletic clade was identified that possessed a conserved seven-gene cassette for polyyne biosynthesis that built upon the conserved three-gene cassette for alkyne biosynthesis. Further diversity mapping of the conserved polyyne gene cassette revealed a phylogenetic subclade for an uncharacterized polyyne BGC present in several Pseudomonas species, designated pgn. Pathway mutagenesis and high-resolution analytical chemistry showed the Pseudomonas protegens pgn BGC directed the biosynthesis of a novel polyyne, protegencin. Exploration of the biosynthetic logic behind polyyne production, through BGC mutagenesis and analytical chemistry, highlighted the essentiality of a triad of desaturase proteins and a thioesterase in both the P. protegens pgn and Trinickia caryophylli (formerly Burkholderia caryophylli) caryoynencin pathways. We have unified and expanded knowledge of polyyne diversity and uniquely demonstrated that alkyne and polyyne biosynthetic gene clusters are evolutionarily related and widely distributed within bacteria. The systematic mapping of conserved biosynthetic genes across the available bacterial genomic diversity proved to be a fruitful method for discovering new natural products and better understanding polyyne biosynthesis. IMPORTANCE Natural products bearing alkyne (triple carbon bond) or polyyne (multiple alternating single and triple carbon bonds) moieties exhibit a broad range of important biological activities. Polyyne metabolites have been implicated in important ecological roles such as cepacin mediating biological control of plant pathogens and caryoynencin protecting Lagriinae beetle eggs against pathogenic fungi. After further phylogenetic exploration of polyyne diversity, we identified a novel gene cluster in Pseudomonas bacteria with known biological control abilities and proved it was responsible for synthesizing a new polyyne metabolite, protegencin. The evolutionary analysis of polyyne pathways showed that multiple biosynthetic genes were conserved, and using mutagenesis, their essentiality was demonstrated. Our research provides a foundation for the future modification of polyyne metabolites and has identified a novel polyyne, protegencin, with potential bioactive roles of ecological and agricultural importance.

Rare-Earth Magnets Influence Movement Patterns of the Magnetically Sensitive Nudibranch Tritonia exsulans in Its Natural Habitat.

AbstractThe nudibranch Tritonia exsulans (previously Tritonia diomedea) is known to have behaviors and neurons that can be modified by perturbations of the Earth's magnetic field. There is no definitive evidence for how this magnetic sense is used in nature. Using an exploratory approach, we tested for possible effects of magnetic perturbations based on underwater video of crawling patterns in the slugs' natural habitat, with magnets of varying strength deployed on the substrate. For analysis, we used a paired comparison of tracks of animals between segments 25-50 cm distant from the magnets and segments of the same tracks 0-25 cm from the magnets, to determine whether any differences depended on the strength of the magnet. Most track measurements (length, displacement, velocity, and tortuosity) showed no such differences. However, effects were observed for the changes in track headings between successive points. These results showed that tracks had relatively higher heading variability when they moved closer to stronger magnets. We suggest that this supports a hypothesis that T. exsulans continuously uses a magnetic sense to help maintain straight-line navigation. Further specific testing of the hypothesis is now needed to verify this new possibility for how animals can benefit from a compass sense.

Bioluminescent detection of isothermal DNA amplification in microfluidic generated droplets and artificial cells.

Microfluidic droplet generation affords precise, low volume, high throughput opportunities for molecular diagnostics. Isothermal DNA amplification with bioluminescent detection is a fast, low-cost, highly specific molecular diagnostic technique that is triggerable by temperature. Combining loop-mediated isothermal nucleic acid amplification (LAMP) and bioluminescent assay in real time (BART), with droplet microfluidics, should enable high-throughput, low copy, sequence-specific DNA detection by simple light emission. Stable, uniform LAMP-BART droplets are generated with low cost equipment. The composition and scale of these droplets are controllable and the bioluminescent output during DNA amplification can be imaged and quantified. Furthermore these droplets are readily incorporated into encapsulated droplet interface bilayers (eDIBs), or artificial cells, and the bioluminescence tracked in real time for accurate quantification off chip. Microfluidic LAMP-BART droplets with high stability and uniformity of scale coupled with high throughput and low cost generation are suited to digital DNA quantification at low template concentrations and volumes, where multiple measurement partitions are required. The triggerable reaction in the core of eDIBs can be used to study the interrelationship of the droplets with the environment and also used for more complex chemical processing via a self-contained network of droplets, paving the way for smart soft-matter diagnostics.

Genomic Assemblies of Members of Burkholderia and Related Genera as a Resource for Natural Product Discovery.

The genomes of 450 members of Burkholderiaceae, isolated from clinical and environmental sources, were sequenced and assembled as a resource for genome mining. Genomic analysis of the collection has enabled the identification of multiple metabolites and their biosynthetic gene clusters, including the antibiotics gladiolin, icosalide A, enacyloxin, and cepacin A.

Full Dynamic Range Quantification using Loop-mediated Amplification (LAMP) by Combining Analysis of Amplification Timing and Variance between Replicates at Low Copy Number.

Quantification of nucleic acid targets at low copy number is problematic with the limit of detection at 95 percent confidence predicted to be 3 molecules or higher for quantitative PCR. Conversely the accuracy of digital PCR is diminished at higher concentrations of template approaching 100 percent positive partitions, with the Poisson distribution showing that an average of only 3 molecules per partition represents an amplification frequency of greater than 95 percent. Therefore a full range of template concentrations cannot be quantified accurately with these methods alone without dilution. Here we report the development of quantification metrics for use with loop-mediated amplification (LAMP) as a bridge between concentrated and dilute template concentrations. The basis for this is that real-time monitoring of LAMP reactions either by bioluminescent reporting (BART) or by fluorescent dye binding shows increasing variation in timings between replicates at low copy number due to the LAMP amplification mechanism. This effect increases with decreasing copy number, closely associated with the amplification frequency. The use of an artificial template showed that the increasing variation is not linked to the use of displacement primers during the initiation of amplification and is therefore a fundamental feature of the LAMP initiation event. Quantification between 1 and 10 copies of a template was successfully achieved with a number of methods with a low number of replicates with the strongest correlation to timing variance. These ultra-quantification methods for LAMP amplification either singularly or in combination have potential in a full dynamic range quantification strategy based on LAMP, in a closed tube, undiluted sample molecular diagnostic.

Health Care Provider Knowledge and Perceptions of FDA-Approved and Do-It-Yourself Automated Insulin Delivery.

BACKGROUND: Automated insulin delivery (AID) technology may reduce variability in blood glucose, resulting in lower risk for hypoglycemia and associated complications, and by extension improve quality of life. While clinical trials, research, and patient experience have consistently demonstrated the value of AID, this technology is still inaccessible to many patients. Patient-driven innovation has resulted in alternative do-it-yourself (DIY) solutions to available off-the-shelf AID devices. METHOD: This two-phase cross-sectional observational study addressed health care provider (HCP) perceptions of AID as well as the perceived need for, development of, and evaluation of an AID fact sheet comparing the most commonly used Federal Drug Administration approved AID and DIY AID devices. RESULTS: Negative attitudes toward the use of DIY AID were low. The majority of HCPs saw their lack of knowledge about how DIY AID work to be the greatest barrier to answering patient questions about what is available (74.4%). Additionally, the majority of HCPs (64.5%) indicated they were either "likely" or "very likely" to use the fact sheet when answering patient questions about AID options. CONCLUSION: Increased awareness and utilization of AID technology offer hope to further reduce the burden of diabetes, but there is a need to bridge the knowledge gap about DIY AID. A fact sheet provides a way to facilitate discussions of this emerging technology between HCPs and patients. Next steps could investigate additional ways to put needed information in the hands of HCPs.

Double or Nothing? Cell Division and Cell Size Control.

Size is a fundamental property that must be tightly regulated to ensure that cells and tissues function efficiently. Dynamic size control allows unicellular organisms to adapt to environmental changes, but cell size is also integral to multicellular development, affecting tissue size and structure. Despite clear evidence for homeostatic cell size maintenance, we are only now beginning to understand cell size regulation in the actively dividing meristematic tissues of higher plants. We discuss here how coupled advances in live cell imaging and modelling are uncovering dynamic mechanisms for size control mediated at the cellular level. We argue that integrated models of cell growth and division will be necessary to predict cell size and fully understand multicellular growth and development.

Lack of specificity associated with using molecular beacons in loop mediated amplification assays.

BACKGROUND: Loop mediated isothermal amplification of nucleic acid templates is a rapid, sensitive and specific method suitable for molecular diagnostics. However the complexity of primer design and the number of primers involved can lead to false positives from non-specific primer interactions. Standard methods of LAMP detection utilise the increasing concentrations of DNA or inorganic pyrophosphate and therefore lack specificity for identifying the desired LAMP amplification. Molecular beacons used in PCR reactions are target specific and may enhance specificity with LAMP. RESULTS: We present a potential molecular beacon approach to LAMP detection targeting the single stranded region between loops, and test this for LAMP molecular beacons targeting the 35S promoter and NOS terminator sequences commonly used in GM crops. From these studies we show that molecular beacons used in LAMP, despite providing a change in fluorescent intensity with amplification, appear not to anneal to specific target sequences and therefore target specificity is not a benefit of this method. However, molecular beacons demonstrate a change in fluorescence which is indicative of LAMP amplification products. We identify the LAMP loop structure as likely to be responsible for this change in signal. CONCLUSIONS: Molecular beacons can be used to detect LAMP amplification but do not provide sequence specificity. The method can be used to determine effectively LAMP amplification from other primer-driven events, but does not discriminate between different LAMP amplicons. It is therefore unsuitable for multiplex LAMP reactions due to non-specific detection of LAMP amplification.

Reduced False Positives and Improved Reporting of Loop-Mediated Isothermal Amplification using Quenched Fluorescent Primers.

Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as an alternative to PCR based methods. There are numerous reported techniques to detect the LAMP amplification including turbidity, bioluminescence and intercalating fluorescent dyes. In this report we show that quenched fluorescent labels on various LAMP primers can be used to quantify and detect target DNA molecules down to single copy numbers. By selecting different fluorophores, this method can be simply multiplexed. Moreover this highly specific LAMP detection technique can reduce the incidence of false positives originating from mispriming events. Attribution of these events to particular primers will help inform and improve LAMP primer design.

Genome mining identifies cepacin as a plant-protective metabolite of the biopesticidal bacterium Burkholderia ambifaria.

Beneficial microorganisms are widely used in agriculture for control of plant pathogens, but a lack of efficacy and safety information has limited the exploitation of multiple promising biopesticides. We applied phylogeny-led genome mining, metabolite analyses and biological control assays to define the efficacy of Burkholderia ambifaria, a naturally beneficial bacterium with proven biocontrol properties but potential pathogenic risk. A panel of 64 B. ambifaria strains demonstrated significant antimicrobial activity against priority plant pathogens. Genome sequencing, specialized metabolite biosynthetic gene cluster mining and metabolite analysis revealed an armoury of known and unknown pathways within B. ambifaria. The biosynthetic gene cluster responsible for the production of the metabolite cepacin was identified and directly shown to mediate protection of germinating crops against Pythium damping-off disease. B. ambifaria maintained biopesticidal protection and overall fitness in the soil after deletion of its third replicon, a non-essential plasmid associated with virulence in Burkholderia cepacia complex bacteria. Removal of the third replicon reduced B. ambifaria persistence in a murine respiratory infection model. Here, we show that by using interdisciplinary phylogenomic, metabolomic and functional approaches, the mode of action of natural biological control agents related to pathogens can be systematically established to facilitate their future exploitation.

Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART).

Loop-mediated amplification (LAMP) has been widely used to amplify and hence detect nucleic acid target sequences from various pathogens, viruses and genetic modifications. Two distinct types of primer are required for LAMP; hairpin-forming LAMP and displacement. High specificity arises from this use of multiple primers, but without optimal conditions for LAMP, sensitivity can be poor. We confirm here the importance of LAMP primer design, concentrations and ratios for efficient LAMP amplification. We further show that displacement primers are non-essential to the LAMP reaction at certain concentrations providing accelerating loop primers are present. We investigate various methods to quantify DNA extracts from GM maize certified reference materials to calculate the target copy numbers of template presented to the LAMP reaction, and show that LAMP can amplify transgenic promoter/terminator sequences in DNA extracted from various maize GM events using primers designed to target the 35S promoter (35Sp) or NOS terminator (NOSt) sequences, detection with both bioluminescence in real-time (BART) and fluorescent methods. With prior denaturation and HPLC grade LAMP primers single copy detection was achieved, showing that optimised LAMP conditions can be combined with BART for single copy targets, with simple and cost efficient light detection electronics over fluorescent alternatives.

Coordination of meristem and boundary functions by transcription factors in the SHOOT MERISTEMLESS regulatory network.

The Arabidopsis homeodomain transcription factor SHOOT MERISTEMLESS (STM) is crucial for shoot apical meristem (SAM) function, yet the components and structure of the STM gene regulatory network (GRN) are largely unknown. Here, we show that transcriptional regulators are overrepresented among STM-regulated genes and, using these as GRN components in Bayesian network analysis, we infer STM GRN associations and reveal regulatory relationships between STM and factors involved in multiple aspects of SAM function. These include hormone regulation, TCP-mediated control of cell differentiation, AIL/PLT-mediated regulation of pluripotency and phyllotaxis, and specification of meristem-organ boundary zones via CUC1. We demonstrate a direct positive transcriptional feedback loop between STM and CUC1, despite their distinct expression patterns in the meristem and organ boundary, respectively. Our further finding that STM activates expression of the CUC1-targeting microRNA miR164c combined with mathematical modelling provides a potential solution for this apparent contradiction, demonstrating that these proposed regulatory interactions coupled with STM mobility could be sufficient to provide a mechanism for CUC1 localisation at the meristem-organ boundary. Our findings highlight the central role for the STM GRN in coordinating SAM functions.

Lineage- and stage-specific expressed CYCD7;1 coordinates the single symmetric division that creates stomatal guard cells.

Plants, with cells fixed in place by rigid walls, often utilize spatial and temporally distinct cell division programs to organize and maintain organs. This leads to the question of how developmental regulators interact with the cell cycle machinery to link cell division events with particular developmental trajectories. In Arabidopsis leaves, the development of stomata, two-celled epidermal valves that mediate plant-atmosphere gas exchange, relies on a series of oriented stem cell-like asymmetric divisions followed by a single symmetric division. The stomatal lineage is embedded in a tissue in which other cells transition from proliferation to postmitotic differentiation earlier, necessitating stomatal lineage-specific factors to prolong competence to divide. We show that the D-type cyclin, CYCD7;1, is specifically expressed just prior to the symmetric guard cell-forming division, and that it is limiting for this division. Further, we find that CYCD7;1 is capable of promoting divisions in multiple contexts, likely through RBR1-dependent promotion of the G1/S transition, but that CYCD7;1 is regulated at the transcriptional level by cell type-specific transcription factors that confine its expression to the appropriate developmental window.

ΔFlucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant.

The bright bioluminescence catalyzed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bio imaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within six loop structures of the bright and thermostable ×11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi-rational ΔFluc library and isolated significantly brighter enzymes after finding ×11 Fluc activity was largely tolerant to deletions. Targeting an "omega-loop" motif (T352-G360) significantly enhanced activity, altered kinetics, reduced Km for D-luciferin, altered emission colors, and altered substrate specificity for redshifted analog DL-infraluciferin. Experimental and in silico analyses suggested remodeling of the Ω-loop impacts on active site hydrophobicity to increase light yields. This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox.

Genome-wide chromatin mapping with size resolution reveals a dynamic sub-nucleosomal landscape in Arabidopsis.

All eukaryotic genomes are packaged as chromatin, with DNA interlaced with both regularly patterned nucleosomes and sub-nucleosomal-sized protein structures such as mobile and labile transcription factors (TF) and initiation complexes, together forming a dynamic chromatin landscape. Whilst details of nucleosome position in Arabidopsis have been previously analysed, there is less understanding of their relationship to more dynamic sub-nucleosomal particles (subNSPs) defined as protected regions shorter than the ~150bp typical of nucleosomes. The genome-wide profile of these subNSPs has not been previously analysed in plants and this study investigates the relationship of dynamic bound particles with transcriptional control. Here we combine differential micrococcal nuclease (MNase) digestion and a modified paired-end sequencing protocol to reveal the chromatin structure landscape of Arabidopsis cells across a wide particle size range. Linking this data to RNAseq expression analysis provides detailed insight into the relationship of identified DNA-bound particles with transcriptional activity. The use of differential digestion reveals sensitive positions, including a labile -1 nucleosome positioned upstream of the transcription start site (TSS) of active genes. We investigated the response of the chromatin landscape to changes in environmental conditions using light and dark growth, given the large transcriptional changes resulting from this simple alteration. The resulting shifts in the suites of expressed and repressed genes show little correspondence to changes in nucleosome positioning, but led to significant alterations in the profile of subNSPs upstream of TSS both globally and locally. We examined previously mapped positions for the TFs PIF3, PIF4 and CCA1, which regulate light responses, and found that changes in subNSPs co-localized with these binding sites. This small particle structure is detected only under low levels of MNase digestion and is lost on more complete digestion of chromatin to nucleosomes. We conclude that wide-spectrum analysis of the Arabidopsis genome by differential MNase digestion allows detection of sensitive features hereto obscured, and the comparisons between genome-wide subNSP profiles reveals dynamic changes in their distribution, particularly at distinct genomic locations (i.e. 5'UTRs). The method here employed allows insight into the complex influence of genetic and extrinsic factors in modifying the sub-nucleosomal landscape in association with transcriptional changes.