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.

Flying electron spin control gates.

The control of "flying" (or moving) spin qubits is an important functionality for the manipulation and exchange of quantum information between remote locations on a chip. Typically, gates based on electric or magnetic fields provide the necessary perturbation for their control either globally or at well-defined locations. Here, we demonstrate the dynamic control of moving electron spins via contactless gates that move together with the spins. The concept is realized using electron spins trapped and transported by moving potential dots defined by a surface acoustic wave (SAW). The SAW strain at the electron trapping site, which is set by the SAW amplitude, acts as a contactless, tunable gate that controls the precession frequency of the flying spins via the spin-orbit interaction. We show that the degree of precession control in moving dots exceeds previously reported results for unconstrained transport by an order of magnitude and is well accounted for by a theoretical model for the strain contribution to the spin-orbit interaction. This flying spin gate permits the realization of an acoustically driven optical polarization modulator based on electron spin transport, a key element for on-chip spin information processing with a photonic interface.

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.

The Non-Anhydrous, Minimally Basic Synthesis of the Dopamine D2 Agonist [18F]MCL-524.

The dopamine D2 agonist MCL-524 is selective for the D2 receptor in the high-affinity state (D2high), and, therefore, the PET analogue, [18F]MCL-524, may facilitate the elucidation of the role of D2high in disorders such as schizophrenia. However, the previously reported synthesis of [18F]MCL-524 proved difficult to replicate and was lacking experimental details. We therefore developed a new synthesis of [18F]MCL-524 using a "non-anhydrous, minimally basic" (NAMB) approach. In this method, [18F]F- is eluted from a small (10-12 mg) trap-and-release column with tetraethylammonium tosylate (2.37 mg) in 7:3 MeCN:H2O (0.1 mL), rather than the basic carbonate or bicarbonate solution that is most often used for [18F]F- recovery. The tosylated precursor (1 mg) in 0.9 mL anhydrous acetonitrile was added directly to the eluate, without azeotropic drying, and the solution was heated (150 °C/15 min). The catechol was then deprotected with the Lewis acid In(OTf)3 (10 equiv.; 150 °C/20 min). In contrast to deprotection with protic acids, Lewis-acid-based deprotection facilitated the efficient removal of byproducts by HPLC and eliminated the need for SPE extraction prior to HPLC purification. Using the NAMB approach, [18F]MCL-524 was obtained in 5-9% RCY (decay-corrected, n = 3), confirming the utility of this improved method for the multistep synthesis of [18F]MCL-524 and suggesting that it may applicable to the synthesis of other 18F-labeled radiotracers.

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.

Mitochondrial transplantation enhances murine lung viability and recovery after ischemia-reperfusion injury.

The most common cause of acute lung injury is ischemia-reperfusion injury (IRI), during which mitochondrial damage occurs. We have previously demonstrated that mitochondrial transplantation is an efficacious therapy to replace or augment mitochondria damaged by IRI, allowing for enhanced muscle viability and function in cardiac tissue. Here, we investigate the efficacy of mitochondrial transplantation in a murine lung IRI model using male C57BL/6J mice. Transient ischemia was induced by applying a microvascular clamp on the left hilum for 2 h. Upon reperfusion mice received either vehicle or vehicle-containing mitochondria either by vascular delivery (Mito V) through the pulmonary artery or by aerosol delivery (Mito Neb) via the trachea (nebulization). Sham control mice underwent thoracotomy without hilar clamping and were ventilated for 2 h before returning to the cage. After 24 h recovery, lung mechanics were assessed and lungs were collected for analysis. Our results demonstrated that at 24 h of reperfusion, dynamic compliance and inspiratory capacity were significantly increased and resistance, tissue damping, elastance, and peak inspiratory pressure (Mito V only) were significantly decreased (P < 0.05) in Mito groups as compared with their respective vehicle groups. Neutrophil infiltration, interstitial edema, and apoptosis were significantly decreased (P < 0.05) in Mito groups as compared with vehicles. No significant differences in cytokines and chemokines between groups were shown. All lung mechanics results in Mito groups except peak inspiratory pressure in Mito Neb showed no significant differences (P > 0.05) as compared with Sham. These results conclude that mitochondrial transplantation by vascular delivery or nebulization improves lung mechanics and decreases lung tissue injury.

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.

Ligand discrimination between active and inactive activation loop conformations of Aurora-A kinase is unmodified by phosphorylation.

Structure-based drug design is commonly used to guide the development of potent and specific enzyme inhibitors. Many enzymes - such as protein kinases - adopt multiple conformations, and conformational interconversion is expected to impact on the design of small molecule inhibitors. We measured the dynamic equilibrium between DFG-in-like active and DFG-out-like inactive conformations of the activation loop of unphosphorylated Aurora-A alone, in the presence of the activator TPX2, and in the presence of kinase inhibitors. The unphosphorylated kinase had a shorter residence time of the activation loop in the active conformation and a shift in the position of equilibrium towards the inactive conformation compared with phosphorylated kinase for all conditions measured. Ligand binding was associated with a change in the position of conformational equilibrium which was specific to each ligand and independent of the kinase phosphorylation state. As a consequence of this, the ability of a ligand to discriminate between active and inactive activation loop conformations was also independent of phosphorylation. Importantly, we discovered that the presence of multiple enzyme conformations can lead to a plateau in the overall ligand K d, despite increasing affinity for the chosen target conformation, and modelled the conformational discrimination necessary for a conformation-promoting ligand.

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.

Mitochondrial transplantation prolongs cold ischemia time in murine heart transplantation.

BACKGROUND: Cold ischemia time (CIT) causes ischemia‒reperfusion injury to the mitochondria and detrimentally effects myocardial function and tissue viability. Mitochondrial transplantation replaces damaged mitochondria and enhances myocardial function and tissue viability. Herein we investigated the efficacy of mitochondrial transplantation in enhancing graft function and viability after prolonged CIT. METHODS: Heterotopic heart transplantation was performed in C57BL/6J mice. Upon heart harvesting from C57BL/6J donors, 0.5 ml of either mitochondria (1 × 108 in respiration buffer; mitochondria group) or respiration buffer (vehicle group) was delivered antegrade to the coronary arteries via injection to the coronary ostium. The hearts were excised and preserved for 29 ± 0.3 hours in cold saline (4°C). The hearts were then heterotopically transplanted. A second injection of either mitochondria (1 × 108) or respiration buffer (vehicle) was delivered antegrade to the coronary arteries 5 minutes after transplantation. Grafts were analyzed for 24 hours. Beating score, graft function, and tissue injury were measured. RESULTS: Beating score, calculated ejection fraction, and shortening fraction were significantly enhanced (p < 0.05), whereas necrosis and neutrophil infiltration were significantly decreased (p < 0.05) in the mitochondria group as compared with the vehicle group at 24 hours of reperfusion. Transmission electron microscopy showed the presence of contraction bands in vehicle but not in mitochondria grafts. CONCLUSIONS: Mitochondrial transplantation prolongs CIT to 29 hours in the murine heart transplantation model, significantly enhances graft function, and decreases graft tissue injury. Mitochondrial transplantation may provide a means to reduce graft failure and improve transplantation outcomes after prolonged CIT.

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.

Efficacy, safety and pharmacokinetics of a new high-purity factor X concentrate in women and girls with hereditary factor X deficiency.

Essentials Plasma-derived factor X concentrate (pdFX) is used to treat hereditary factor X deficiency. pdFX pharmacokinetics, safety and efficacy were assessed in factor X-deficient women/girls. Treatment success rate was 98%; only 6 adverse events in 2 subjects were possibly pdFX related. On-demand pdFX 25 IU kg-1 was effective and safe in women/girls with factor X deficiency. SUMMARY: Background A high-purity, plasma-derived factor X concentrate (pdFX) has been approved for the treatment of hereditary FX deficiency, an autosomal recessive disorder. Objective To perform post hoc assessments of pdFX pharmacokinetics, safety and efficacy in women and girls with hereditary FX deficiency. Patients/Methods Subjects aged ≥ 12 years with moderate/severe FX deficiency (plasma FX activity of < 5 IU dL-1 ) received on-demand or preventive pdFX (25 IU kg-1 ) for ≤ 2 years. Results Of 16 enrolled subjects, 10 women and girls (aged 14-58 years [median, 25.5 years]) received 267 pdFX infusions. Mean monthly infusions per subject were higher among women and girls (2.48) than among men and boys (1.62). In women and girls, 132 assessable bleeding episodes (61 heavy menstrual bleeds, 47 joint bleeds, 15 muscle bleeds, and nine other bleeds) were treated with pdFX, with a 98% treatment success rate versus 100% in men and boys. Mean pdFX incremental recovery was similar in the two groups (2.05 IU dL-1 versus 1.91 IU dL-1 per IU kg-1 ), as was the mean half-life (29.3 h versus 29.5 h). Of 142 adverse events in women and girls, headache was the most common (12 events in six subjects). Six events (two infusion-site erythema, two fatigue, one back pain, one infusion-site pain) in two subjects were considered to be possibly pdFX-related. Following the trial, pdFX was used to successfully maintain hemostasis in two subjects undergoing obstetric delivery. Conclusions pdFX was well tolerated and effective in women and girls with FX deficiency. Although women and girls had different bleeding symptoms and sites than men and boys, their pdFX pharmacokinetic profile was comparable.