A unique feature of Oxford Nanopore Technologies sequencers, adaptive sampling, allows precise DNA molecule selection from sequencing libraries. Here we present enhancements to our tool, readfish, enabling all features for the industrial scale PromethION sequencer, including standard and "barcode-aware" adaptive sampling. We demonstrate effective coverage enrichment and assessment of multiple human genomes for copy number and structural variation on a single PromethION flow cell.

The duckweeds feature global distributions and diverse applications in phytoremediation and nutrition, as well as use in fundamental studies of development. Existing collections have minimal environmental data linked to natural habitats. Thus, there is a lack of understanding of natural variation in the context of native habitats. Here, a novel collection of 124 duckweed accessions from 115 sites across the United Kingdom was characterized by genome sequencing and ionomics. In common nutrient-replete experimental conditions, all accessions hyperaccumulated P, K, Mg and Ca. Local but not large-scale associations were revealed between elemental composition of duckweed in common, replete conditions and native water profiles. Lemna minor was the most prevalent species in the UK, with a closely related hybrid L. japonica frequently found in waters with higher micronutrient concentrations. Invasive L. minuta was common in the southern and midland regions, but restricted in Scotland. Lemna accessions accumulated heavy metal contaminants typically together with macronutrients, suggesting phytoremediation potential, but some limitations as food. Furthermore, monitoring the ecological interactions between native, hybrid and invasive Lemna species should be ongoing in the interest of biodiversity.

Since the advent of long read sequencing, achieving longer read lengths has been a key goal for many users. Ultra-long read sets (N50 > 100 kb) produced from Oxford Nanopore sequencers have improved genome assemblies in recent years. However, despite progress in extraction protocols and library preparation methods, ultra-long sequencing remains challenging for many sample types. Here we compare various methods and introduce the FindingNemo protocol that: (1) optimises ultra-high molecular weight (UHMW) DNA extraction and library clean-up by using glass beads and Hexamminecobalt(III) chloride (CoHex), (2) can deliver high ultra-long sequencing yield of >20 Gb of reads from a single MinION flow cell or >100 Gb from PromethION devices (R9.4 to R10.4 pore variants), and (3) is scalable to using fewer input cells or lower DNA amounts, with extraction to sequencing possible in a single working day. By comparison, we show this protocol is superior to previous ones due to precise determination of input DNA quantity and quality by cell count, sample dilution and homogenization approaches ### Competing Interest Statement M.L., J.T, J.Q. and N.L were members of the MinION early access program and have received free flow cells and sequencing reagents in the past. All have received reimbursement for travel and accommodation to speak at events organised by ONT.

Whole-genome duplication (WGD) occurs in all kingdoms and impacts speciation, domestication, and cancer outcome. However, doubled DNA management can be challenging for nascent polyploids. The study of within-species polyploidy (autopolyploidy) permits focus on this DNA management aspect, decoupling it from the confounding effects of hybridization (in allopolyploid hybrids). How is autopolyploidy tolerated, and how do young polyploids stabilize? Here, we introduce a powerful model to address this: the genus Cochlearia, which has experienced many polyploidization events. We assess meiosis and other polyploid-relevant phenotypes, generate a chromosome-scale genome, and sequence 113 individuals from 33 ploidy-contrasting populations. We detect an obvious autopolyploidy-associated selection signal at kinetochore components and ion transporters. Modeling the selected alleles, we detail evidence of the kinetochore complex mediating adaptation to polyploidy. We compare candidates in independent autopolyploids across three genera separated by 40 million years, highlighting a common function at the process and gene levels, indicating evolutionary flexibility in response to polyploidy.

Transforming genomic and cellular landscapes in a single generation, whole genome duplication (WGD) brings fundamental challenges, but is also associated with diversification. How is WGD tolerated, and what processes commonly evolve to stabilize the resulting polyploid? Here we study this in Cochlearia spp., which have experienced multiple WGDs in the last 300,000 years. We first generate a chromosome-scale genome and sequence 113 individuals from 33 diploid, tetraploid, hexaploid, and outgroup populations. We detect the clearest post-WGD selection signatures in functionally interacting kinetochore components and ion transporters. We structurally model these derived selected alleles, identifying striking WGD-relevant functional variation, and then compare these results to independent recent post-WGD selection in Arabidopsis arenosa and Cardamine amara . Most prominent in these results is genetic evidence of at least four functionally interacting kinetochore complex subunits in adaptation to WGD at the centromere among our very top selective sweep outliers. In addition, some of the same biological processes evolve in all three WGDs, but specific genes recruited are flexible. This points to a polygenic basis for modifying systems that control the kinetochore, meiotic crossover number, DNA repair, ion homeostasis, and cell cycle. Given that DNA management (especially repair) is the most salient category with the strongest selection signal, we speculate that the generation rate of structural genomic variants may be altered by WGD in young polyploids, contributing to their occasionally spectacular adaptability observed across kingdoms. Significance Statement Whole-genome duplication (WGD) occurs in all kingdoms and is linked to adaptation, speciation, domestication, and even cancer outcome. But WGD is a shock to the system, and commonly disrupts cell division due to increased DNA management burden and transformed cell physiology. Nevertheless, the hopeful monster that survives WGD is special, occasionally experiencing runaway success. Why do some thrive but others die? Here we introduce a powerful new model, Cochlearia, which has benefitted from multiple WGDs, and we provide the first genetic evidence of rapid adaptation of functionally interacting components of the cell division machinery, the kinetochore. We also compare which processes and genes evolve to stabilize the new polyploid in three independent cases and highlight common mechanisms.

Water‐soluble antibiotics are largely excluded from therapy of intracellular infections, such as Shigella spp., Listeria spp., or Salmonella spp., due to their inability to permeate mammalian cell membrane. Here, the authors study if targeting offers an advantage to deliver killing doses of membrane‐impermeable antibiotics intracellularly to infected cells. Mannose‐decorated liposomes, loaded with gentamicin, are fabricated to target mannose receptor, a recognition system of (infected) macrophages. Designing a family of liposomes with varying surface presentation of mannose ligand, the authors show a clear dependence of cellular internalization on the ligand surface presentation. Significantly for the killing of intracellular bacteria, the study demonstrates internalization of mannosylated liposomes by the entire population of macrophages, both Salmonella‐infected and non‐infected, resulting in an efficient treatment of intracellular infection. This contrasts with non‐targeted liposomes, where internalization does not occur by a substantial subpopulation of infected cells. The study points to the significance of targeted delivery of antibiotics for treatment of intracellular infections.

In worldwide conditions of increasingly antibiotic-resistant hospital infections, it is important to research alternative therapies. Bdellovibrio bacteriovorus bacteria naturally prey on Gram-negative pathogens, including antibiotic-resistant strains and so B. bacteriovorus have been proposed as "living antibiotics" to combat antimicrobially-resistant pathogens. Predator-prey interactions are complex and can be altered by environmental components. To be effective B. bacteriovorus predation needs to work in human body fluids such as serum where predation dynamics may differ to that studied in laboratory media. Here we combine mathematical modelling and lab experimentation to investigate the predation of an important carbapenem-resistant human pathogen, Klebsiella pneumoniae, by B. bacteriovorus in human serum versus buffer. We show experimentally that B. bacteriovorus is able to reduce prey numbers in each environment, on different timescales. Our mathematical model captures the underlying dynamics of the experimentation, including an initial predation-delay at the predator-prey-serum interface. Our research shows differences between predation in buffer and serum and highlights both the potential and limitations of B. bacteriovorus acting therapeutically against K. pneumoniae in serum, informing future research into the medicinal behaviours and dosing of this living antibacterial.

Bdellovibrio bacteriovorus is a small deltaproteobacterial predator that has evolved to invade, reseal, kill, and digest other gram-negative bacteria in soils and water environments. It has a broad host range and kills many antibiotic-resistant, clinical pathogens in vitro, a potentially useful capability if it could be translated to a clinical setting. We review relevant mechanisms of B. bacteriovorus predation and the physiological properties that would influence its survival in a mammalian host. Bacterial pathogens increasingly display conventional antibiotic resistance by expressing and varying surface and soluble biomolecules. Predators coevolved alongside prey bacteria and so encode diverse predatory enzymes that are hard for pathogens to resist by simple mutation. Predators do not replicate outside pathogens and thus express few transport proteins and thus few surface epitopes for host immune recognition. We explain these features, relating them to the potential of predatory bacteria as cellular medicines.

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In neuromuscular systems dystroglycan provides a vital link between laminin in the extracellular matrix and dystrophin in the membrane cytoskeleton. The integrity of this link is maintained and regulated by post-translational modifications of dystroglycan that have effects both inside and outside the cell. Glycosylation of α-dystroglycan is crucial for its link to laminin and phosphorylation of β-dystroglycan on tyrosine regulates its association with intracellular binding partners. This short review focuses on some of the recent developments in our understanding of the role of these post-translational modification in regulating dystroglycan function, and how new knowledge of signalling through the laminin-dystroglycan axis is leading to hope for treatment for some neuromuscular diseases associated with this adhesion complex.

Loss of dystrophin protein due to mutations in the DMD gene causes Duchenne muscular dystrophy. Dystrophin loss also leads to the loss of the dystrophin glycoprotein complex (DGC) from the sarcolemma which contributes to the dystrophic phenotype. Tyrosine phosphorylation of dystroglycan has been identified as a possible signal to promote the proteasomal degradation of the DGC. In order to test the role of tyrosine phosphorylation of dystroglycan in the aetiology of DMD, we generated a knock-in mouse with a phenylalanine substitution at a key tyrosine phosphorylation site in dystroglycan, Y890. Dystroglycan knock-in mice (Dag1(Y890F/Y890F)) had no overt phenotype. In order to examine the consequence of blocking dystroglycan phosphorylation on the aetiology of dystrophin-deficient muscular dystrophy, the Y890F mice were crossed with mdx mice an established model of muscular dystrophy. Dag1(Y890F/Y890F)/mdx mice showed a significant improvement in several parameters of muscle pathophysiology associated with muscular dystrophy, including a reduction in centrally nucleated fibres, less Evans blue dye infiltration and lower serum creatine kinase levels. With the exception of dystrophin, other DGC components were restored to the sarcolemma including α-sarcoglycan, α-/β-dystroglycan and sarcospan. Furthermore, Dag1(Y890F/Y890F)/mdx showed a significant resistance to muscle damage and force loss following repeated eccentric contractions when compared with mdx mice. While the Y890F substitution may prevent dystroglycan from proteasomal degradation, an increase in sarcolemmal plectin appeared to confer protection on Dag1(Y890F/Y890F)/mdx mouse muscle. This new model confirms dystroglycan phosphorylation as an important pathway in the aetiology of DMD and provides novel targets for therapeutic intervention.

Dystroglycan is a ubiquitously expressed heterodimeric adhesion receptor. The extracellular alpha-subunit makes connections with a number of laminin G domain ligands including laminins, agrin and perlecan in the extracellular matrix and the transmembrane beta-subunit makes connections to the actin filament network via cytoskeletal linkers including dystrophin, utrophin, ezrin and plectin, depending on context. Originally discovered as part of the dystrophin glycoprotein complex of skeletal muscle, dystroglycan is an important adhesion molecule and signalling scaffold in a multitude of cell types and tissues and is involved in several diseases. Dystroglycan has emerged as a multifunctional adhesion platform with many interacting partners associating with its short unstructured cytoplasmic domain. Two particular hotspots are the cytoplasmic juxtamembrane region and at the very carboxy terminus of dystroglycan. Regions which between them have several overlapping functions: in the juxtamembrane region; a nuclear localisation signal, ezrin/radixin/moesin protein, rapsyn and ERK MAP Kinase binding function, and at the C terminus a regulatory tyrosine governing WW, SH2 and SH3 domain interactions. We will discuss the binding partners for these motifs and how their interactions and regulation can modulate the involvement of dystroglycan in a range of different adhesion structures and functions depending on context. Thus dystroglycan presents as a multifunctional scaffold involved in adhesion and adhesion-mediated signalling with its functions under exquisite spatio-temporal regulation.

Dystroglycan is a ubiquitously expressed cell adhesion protein. Its principal role has been determined as a component of the dystrophin-glycoprotein complex of muscle, where it constitutes a key component of the costameric cell adhesion system. To investigate more fundamental aspects of dystroglycan function in cell adhesion, we examined the role of dystroglycan in the dynamics and assembly of cellular adhesions in myoblasts. We show that β-dystroglycan is recruited to adhesion structures and, based on staining for vinculin, that overexpression or depletion of dystroglycan affects both size and number of fibrillar adhesions. Knockdown of dystroglycan increases the size and number of adhesions, whereas overexpression decreases the number of adhesions. Dystroglycan knockdown or overexpression affects the ability of cells to adhere to different substrates, and has effects on cell migration that are consistent with effects on the formation of fibrillar adhesions. Using an SH3 domain proteomic screen, we identified vinexin as a binding partner for dystroglycan. Furthermore, we show that dystroglycan can interact indirectly with vinculin by binding to the vinculin-binding protein vinexin, and that this interaction has a role in dystroglycan-mediated cell adhesion and spreading. For the first time, we also demonstrate unequivocally that β-dystroglycan is a resident of focal adhesions.

Dystroglycan is an integral member of the skeletal muscle dystrophin glycoprotein complex, which links dystrophin to proteins in the extracellular matrix. Recently, a group of human muscular dystrophy disorders have been demonstrated to result from defective glycosylation of the α-dystroglycan subunit. Genetic studies of these diseases have identified six genes that encode proteins required for the synthesis of essential carbohydrate structures on dystroglycan. Here we highlight their known or postulated functions. This glycosylation pathway appears to be highly specific (dystroglycan is the only substrate identified thus far) and to be highly conserved during evolution.

Mutations in human genes encoding proteins involved in alpha-dystroglycan glycosylation result in dystroglycanopathies: severe congenital muscular dystrophy phenotypes often accompanied by CNS abnormalities and ocular defects. We have identified the zebrafish orthologues of the seven known genes in this pathway and examined their expression during embryonic development. Zebrafish Large, POMT1, POMT2, POMGnT1, Fukutin, and FKRP show in situ hybridization patterns similar to those of dystroglycan, with broad expression throughout early development. By 30 h postfertilization (hpf), transcripts of all these genes are most prominent in the CNS, eye, and muscle, tissues that are predominantly affected in the dystroglycanopathies. In contrast, Large2 expression is more restricted and by 30 hpf is confined to the lens, cerebellum, and pronephric duct. We show that the monoclonal antibody IIH6, which recognizes a glycoform of dystroglycan, also detects the zebrafish protein. Injection of morpholino oligonucleotides against zebrafish Large2 resulted in loss of IIH6 immunostaining. These data indicate that the dystroglycan glycosylation pathway is conserved in zebrafish and suggest this organism is likely to be a useful model system for functional studies.

The Large(myd) mouse has a loss-of-function mutation in the putative glycosyltransferase gene Large. Mutations in the human homolog (LARGE) have been described in a form of congenital muscular dystrophy (MDC1D). Other genes (POMT1, POMGnT1, fukutin, and FKRP) that encode known or putative glycosylation enzymes are also causally associated with human congenital muscular dystrophies. All these diseases are associated with hypoglycosylation of the membrane protein alpha-dystroglycan (alpha-DG) and consequent loss of extracellular ligand binding. Hence, they are termed dystroglycanopathies. A paralogous gene for LARGE (LARGE2 or GYLTL1B) may also have a role in DG glycosylation. Using database interrogation and reverse-transcriptase polymerase chain reaction (RT-PCR), we identified vertebrate orthologs of each of these LARGE genes in many vertebrates, including human, mouse, dog, chicken, zebrafish, and pufferfish. However, within invertebrate genomes, we were able to identify only single homologs. We suggest that vertebrate LARGE orthologs be referred to as LARGE1. RT-PCR, dot-blot, and northern analysis indicated that LARGE2 has a more restricted tissue-expression profile than LARGE1. Using epitope-tagged proteins, we show that both LARGE1 and LARGE2 localize to the Golgi apparatus. The high similarity between the LARGE paralogs suggests that LARGE2 may also act on DG. Overexpression of LARGE2 in mouse C2C12 myoblasts results in increased glycosylation of alpha-DG accompanied by an increase in laminin binding. Thus, there may be functional redundancy between LARGE1 and LARGE2. Consistent with this idea, we show that alpha-DG is still fully glycosylated in kidney (a tissue that expresses a high level of LARGE2 mRNA) of Large(myd) mutant mice.

The myodystrophy (Large(myd)) mouse has a spontaneous loss of function mutation in a putative glycosyltransferase gene (Large). Mutations in the human gene (LARGE) have been described in congenital muscular dystrophy type 1D (MDC1D). Mutations in four other genes that encode known or putative glycosylation enzymes (POMT1, POMGnT1, fukutin and FKRP) are also associated with muscular dystrophy. In all these diseases hypoglycosylation of alpha-dystroglycan, and consequent loss of ligand binding, is a common pathomechanism. Currently, the Large(myd) mouse is the principal animal model for studying the underlying molecular mechanisms of this group of disorders. Over-expression of LARGE in cells from patients with mutations in POMT1 or POMGnT1 results in hyperglycosylation of alpha-dystroglycan and restoration of laminin binding. Thus, LARGE is a potential therapeutic target. Here, we define the intronic deletion breakpoints of the Large(myd) mutation and describe a simple, PCR-based diagnostic assay, facilitating the study of this important animal model.