Droplet digital PCR for oncogenic KMT2A fusion detection

Acute myeloid leukemia (AML) is an aggressive blood cancer diagnosed in approximately 120,000 individuals worldwide each year. During treatment for AML, detecting residual disease is essential for prognostication and treatment decision-making. Currently, methods for detecting residual AML are limited to identifying approximately 1:100 to 1:1000 leukemic cells (morphology and DNA sequencing) or are difficult to implement (flow cytometry). AML arising after chemotherapy or radiation exposure is termed therapy-related AML (t-AML) and is exceptionally aggressive and treatment resistant. t-AML is often driven by oncogenic fusions that result from prior treatments that introduce double-strand DNA breaks. The most common t-AML-associated translocations affect KMT2A. There are at least 80 known KMT2A fusion partners, but approximately 80% of fusions involve only five partners-AF9, AF6, AF4, ELL, and ENL. We present a novel droplet digital PCR assay targeting the most common KMT2A-rearrangements to enable detection of rare AML cells harboring these fusions. This assay was benchmarked in cell lines and patient samples harboring oncogenic KMT2A fusions and demonstrated a limit of detection of approximately 1:1,000,000 cells. Future application of this assay could improve disease detection and treatment decision-making for patients with t-AML with KMT2A fusions and premalignant oncogenic fusion detection in at-risk individuals after chemotherapy exposure

Influence of salinity on SAV distribution in a series of intermittently connected coastal lakes

Intermittently closed and open lakes and lagoons (ICOLLs) are coastal lakes that intermittently exchange water with the sea and experience saline intrusions. Understanding effects of seawater exchange on local biota is important to preserve ecosystem functioning and ecological integrity. Coastal dune lakes of northwest Florida are an understudied group of ICOLLs in close geographic proximity and with entrance regimes operating along a frequency continuum. We exploited this natural continuum and corresponding water chemistry gradient to determine effects of water chemistry on resident submersed aquatic vegetation (SAV) distributions in these ecosystems. SAV distribution decreased with increases in salinity, but was unaffected by variation in nitrogen, phosphorous, and turbidity. Salinity perturbations corresponding with water exchange with the Gulf of Mexico were associated with reductions in SAV in coastal dune lakes. Potential impacts associated with changes in global climate may increase the frequency of seawater exchange across all coastal dune lakes and potentially reduce the distribution of oligohaline macrophytes among these ecosystems

Spatiotemporal Modeling of Nursery Habitat Using Bayesian Inference: Environmental Drivers of Juvenile Blue Crab Abundance

Nursery grounds provide conditions favorable for growth and survival of juvenile fish and crustaceans through abundant food resources and refugia, and enhance secondary production of populations. While small-scale studies remain important tools to assess nursery value of structured habitats and environmental factors, targeted applications that unify survey data over large spatial and temporal scales are vital to generalize inference of nursery function, identify highly productive regions, and inform management strategies. Using 21 years of spatio-temporally indexed survey data (i.e., water chemistry, turbidity, blue crab, and predator abundance) and GIS information on potential nursery habitats (i.e., seagrass, salt marsh, and unvegetated shallow bottom), we constructed five Bayesian hierarchical models with varying spatial and temporal dependence structures to infer variation in nursery habitat value for young juveniles (20–40 mm carapace width) of the blue crab Callinectes sapidus within three tributaries (James, York and Rappahannock Rivers) in lower Chesapeake Bay. Out-of-sample predictions of juvenile blue crab counts from a model considering fully nonseparable spatiotemporal dependence outperformed predictions from simpler models. Salt marsh surface area and turbidity were the strongest determinants of crab abundance (positive association in both cases). Highest crab abundances occurred near the turbidity maximum where relative salt marsh area was greatest. Relative seagrass area, which has been emphasized as the most valuable nursery in studies conducted at small spatial scales, was not associated with high crab abundance within the three tributaries. Hence, salt marshes should be considered a key nursery habitat for the blue crab, even where extensive seagrass beds occur. The patterns between juvenile blue crab abundance and environmental variables also indicated that identification of nurseries should be based on investigations at broad spatial and temporal scales incorporating multiple potential nursery habitats, and based on statistical analyses that address spatial and temporal statistical dependence

Variation in Seagrass-Associated Macroinvertebrate Communities Along the Gulf Coast of Peninsular Florida: An Exploration of Patterns and Ecological Consequences

Seagrasses form vast meadows of structurally complex habitat that support faunal communities with greater numbers of species and individuals than nearby unstructured habitats. The Gulf coast of peninsular Florida represents a natural laboratory ideally suited to the study of processes that shape seagrass-associated invertebrate and fish communities within meadows of a single species of seagrass, Thalassia testudinum. This suitability arises from a pronounced structural and chemical gradient that exists over ecologically relevant spatial and temporal scales, as revealed by extensive monitoring of water quality and seagrass. We hypothesized that seagrass-associated invertebrate communities would vary across five estuarine systems spread along a spatial gradient in phosphorus concentration, an important driver of seagrass and phytoplankton growth in this region. The quantitative results based on data acquired at 25 stations (75 samples, 52,086 specimens, and 161 taxa) indicated that each of the five estuarine systems were distinct with regard to species composition and differences among systems were driven by abundant or relatively common species. In addition, we found evidence to indicate food webs in seagrass meadows along this gradient may differ, especially in the relative dominance of algal grazers and predatory invertebrates. These changes in species composition and trophic roles could be driven by phosphorus directly, through increases in rates of primary production with higher concentrations of phosphorus, or indirectly, through nutrient-mediated changes in the physical structure of the seagrass canopy. Our results suggest that differences in the habitat created by T. testudinum under differing phosphorus supplies lead to ecologically significant shifts in macroinvertebrate communities

Divergent effects of DNMT3A and TET2 mutations on hematopoietic progenitor cell fitness

The DNA methylation regulators DNMT3A and TET2 are recurrently mutated in hematological disorders. Despite possessing antagonistic biochemical activities, loss-of-function murine models show overlapping phenotypes in terms of increased hematopoietic stem cell (HSC) fitness. Here, we directly compared the effects of these mutations on hematopoietic progenitor function and disease initiation. In contrast to Dnmt3a-null HSCs, which possess limitless self-renewal in vivo, Tet2-null HSCs unexpectedly exhaust at the same rate as control HSCs in serial transplantation assays despite an initial increase in self-renewal. Moreover, loss of Tet2 more acutely sensitizes hematopoietic cells to the addition of a common co-operating mutation (Flt

Spatial mapping of hematopoietic clones in human bone marrow

UNLABELLED: Clonal hematopoiesis (CH) is the expansion of somatically mutated cells in the hematopoietic compartment of individuals without hematopoietic dysfunction. Large CH clones (i.e., \u3e2% variant allele fraction) predispose to hematologic malignancy, but CH is detected at lower levels in nearly all middle-aged individuals. Prior work has extensively characterized CH in peripheral blood, but the spatial distribution of hematopoietic clones in human bone marrow is largely undescribed. To understand CH at this level, we developed a method for spatially aware somatic mutation profiling and characterized the bone marrow of a patient with polycythemia vera. We identified the complex clonal distribution of somatic mutations in the hematopoietic compartment, the restriction of somatic mutations to specific subpopulations of hematopoietic cells, and spatial constraints of these clones in the bone marrow. This proof of principle paves the way to answering fundamental questions regarding CH spatial organization and factors driving CH expansion and malignant transformation in the bone marrow. SIGNIFICANCE: CH occurs commonly in humans and can predispose to hematologic malignancy. Although well characterized in blood, it is poorly understood how clones are spatially distributed in the bone marrow. To answer this, we developed methods for spatially aware somatic mutation profiling to describe clonal heterogeneity in human bone marrow. See related commentary by Austin and Aifantis, p. 139

Molecular toolkit development for gene expression and gene silencing technologies in the homobasidiomycete Fungi Agaricus bisporus and Coprinus cinereus

Resumen de la conferencia presentada al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.We have developed a “Molecular Toolkit” comprising interchangeable promoters and marker genes to facilitate transformation of homobasidiomycete mushrooms and subsequent analysis of gene expression. We will describe the testing of a wide range of promoters in both Agaricus bisporus and Coprinus cinereus when linked to a range of selectable and visual marker genes, along with the parameters required to successfully achieve foreign gene expression within these organisms. It has been previously demonstrated that a prerequisite for GFP expression in A. bisporus and C. cinereus is an intron. We describe the construction of an expression vector containing a multiple cloning site linked to an intron thus allowing different genes to be easily expressed in A. bisporus and C. cinereus. We report on the development of gene silencing technologies within A. bisporus and C. cinereus. In particular the serine protease has been targeted for gene silencing in A. bisporus. Serine protease has been implicated in post-harvest and age-related senescence of sporophores. On harvesting, mushrooms degenerate rapidly to give browned caps and loss of texture in the fruit body, and such problems can dramatically reduce sale ability of the mushrooms. Suppression of genes involved in these pathways could increase mushroom shelf-life and profitability for mushroom growers, or help to further elucidate the complex biochemical pathways involved in post-harvest degradation. Progress will also be reported on gene silencing in C. cinereus

Pevonedistat targets malignant cells in myeloproliferative neoplasms in vitro and in vivo via NFκB pathway inhibition

Targeted inhibitors of JAK2 (eg ruxolitinib) often provide symptomatic relief for myeloproliferative neoplasm (MPN) patients, but the malignant clone persists and remains susceptible to disease transformation. These observations suggest that targeting alternative dysregulated signaling pathways may provide therapeutic benefit. Previous studies identified NFκB pathway hyperactivation in myelofibrosis (MF) and secondary acute myeloid leukemia (sAML) that was insensitive to JAK2 inhibition. Here, we provide evidence that NFκB pathway inhibition via pevonedistat targets malignant cells in MPN patient samples as well as in MPN and patient-derived xenograft mouse models that are nonredundant with ruxolitinib. Colony forming assays revealed preferential inhibition of MF colony growth compared with normal colony formation. In mass cytometry studies, pevonedistat blunted canonical TNFα responses in MF and sAML patient CD34+ cells. Pevonedistat also inhibited hyperproduction of inflammatory cytokines more effectively than ruxolitinib. Upon pevonedistat treatment alone or in combination with ruxolitinib, MPN mouse models exhibited reduced disease burden and improved survival. These studies demonstrating efficacy of pevonedistat in MPN cells in vitro as well as in vivo provide a rationale for therapeutic inhibition of NFκB signaling for MF treatment. Based on these findings, a Phase 1 clinical trial combining pevonedistat with ruxolitinib has been initiated

Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the "button mushroom" forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost andduringmushroomformation.The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation aremore highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics

Combined multiplex panel test results are a poor estimate of disease prevalence without adjustment for test error

This is the uncorrected proof.Data Availability: All data and code used for running experiments, model fitting, and plotting is available on a GitHub repository at https://bristol-vaccine-centre.github.io/testerror/. This is in the form of an R package providing methods to support the estimation of epidemiological parameters based on the results of multiplex panel tests and it is deployed on the Bristol Vaccine Centre r-universe (https://bristol-vaccine-centre.r-universe.dev/testerror). We have also used Zenodo to assign a DOI to the repository: doi:10.5281/zenodo.7691196.Multiplex panel tests identify many individual pathogens at once, using a set of component tests. In some panels the number of components can be large. If the panel is detecting causative pathogens for a single syndrome or disease then we might estimate the burden of that disease by combining the results of the panel, for example determining the prevalence of pneumococcal pneumonia as caused by many individual pneumococcal serotypes. When we are dealing with multiplex test panels with many components, test error in the individual components of a panel, even when present at very low levels, can cause significant overall error. Uncertainty in the sensitivity and specificity of the individual tests, and statistical fluctuations in the numbers of false positives and false negatives, will cause large uncertainty in the combined estimates of disease prevalence. In many cases this can be a source of significant bias. In this paper we develop a mathematical framework to characterise this issue, we determine expressions for the sensitivity and specificity of panel tests. In this we identify a counter-intuitive relationship between panel test sensitivity and disease prevalence that means panel tests become more sensitive as prevalence increases. We present novel statistical methods that adjust for bias and quantify uncertainty in prevalence estimates from panel tests, and use simulations to test these methods. As multiplex testing becomes more commonly used for screening in routine clinical practice, accumulation of test error due to the combination of large numbers of test results needs to be identified and corrected for.Engineering and Physical Sciences Research Council (EPSRC)Medical Research Council (MRC)National Institute for Health and Care Research (NIHR