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Endocrine mucin-producing sweat gland carcinoma is a low-grade eyelid tumor. Small biopsies and insensitive immunohistochemistry predispose to misdiagnosis. We aimed to identify clarifying immunohistochemical markers, molecular markers, or both. Clinicopathologic data (22 cases) were reviewed. Immunohistochemistry (insulinoma-associated protein 1, BCL-2, mucin 2 [MUC2], mucin 4, androgen receptor, β-catenin, and Merkel cell polyomavirus) and next-generation sequencing (Memorial Sloan Kettering integrated mutation profiling of actionable cancer targets, 468 genes) were performed (3 cases). Female patients (n = 15) and male patients (n = 7) (mean age 71.8 years; range 53–88 years) had eyelid or periorbital tumors (>90%) with mucin-containing solid or cystic neuroendocrine pathology. Immunohistochemistry (insulinoma-associated protein 1, BCL2, androgen receptor, retinoblastoma-associated protein 1, and β-catenin) was diffusely positive (5/5), MUC2 partial, mucin 4 focal, and Merkel cell polyomavirus negative. Memorial Sloan Kettering integrated mutation profiling of actionable cancer targets identified 12 single-nucleotide variants and 1 in-frame deletion in 3 cases, each with DNA damage response or repair (BRD4, PPP4R2, and RTEL1) and tumor-suppressor pathway (BRD4, TP53, TSC1, and LATS2) mutations. Microsatellite instability, copy number alterations, and structural alterations were absent. Insulinoma-associated protein 1 and MUC2 are positive in endocrine mucin-producing sweat gland carcinoma. MUC2 positivity suggests conjunctival origin. Multistep pathogenesis involving DNA damage repair and tumor-suppressor pathways may be implicated.

Solar balloons are a promising platform for providing multi-hour flights in and above the upper cloud layer of Venus. The temperature and pressure profiles from 55–75 km above the surface of Venus are very similar to Earth’s from sea level to approximately 30 km, the site of numerous terrestrial solar balloon missions. Additionally, Venus’s highly reflective cloud layer can provide a higher intensity of reflected solar radiation. An ideal solar balloon envelope incorporates a lightweight material that has a high absorbtivity in the visual spectrum, and a low emissivity in the infrared spectrum. The envelope then absorbs direct, reflected, and diffuse radiation, as well as infrared radiation, and heats up the internal balloon air through conduction, resulting in passive flight without the need for an external heat source or lifting gas. This work builds on previous high altitude and solar balloon models and adapts them for Venus’ atmosphere to explore their expected payload mass capacity and flight times. The possible equilibrium float altitudes for various system masses and solar zenith angles are compared between Earth and Venus. Finally, an Earth simulation is compared to terrestrial solar balloon mission data to validate the model and further demonstrate the feasibility of solar balloon flight on Venus.

Epstein-Barr virus (EBV) is associated with hematologic and solid tumors. In this study, we utilized a hybridization capture-based next generation sequencing (NGS) platform that targets 400 genes associated with hematological malignancies to detect and quantify non-targeted viral-derived EBV reads that aligned to the EBV reference contig (NC_007605). We evaluated 5,234 samples from 3,636 unique patients with hematological neoplasms and found that 100 samples (1.9%) in 93 unique patients had ≥ 6 EBV reads (range, 6-32,325; mean, 827.5; median, 54). The majority (n=73, 73%) represented known EBV-associated conditions, and the most common was post-transplant lymphoproliferative disorders (n=21, 29%). Documented EBV viremia accounted for a moderate quantity of EBV reads in 4/27 samples corresponding to conditions not known to be EBV-associated, while suspected viremia or low-level activation was likely the etiology in the remaining 23 samples. A good correlation (Spearman r=0.8, 95% CI 0.74-0.85) was found between EBV reads by NGS and systematic semi-quantitative EBER ISH assessment in 162 available samples, particularly at higher level of EBV-involvement. An optimal threshold for significant morphologic EBV-involvement was found to be ≥10 reads by the receiver operating characteristic (ROC) analysis (AUC 0.990 and 95% CI 0.9974-1.000%). Thus, in addition to mutational analysis, hybrid-capture-based NGS panels can be used to detect and quantitate off-target EBV-derived viral DNA, which correlates well with morphology.

Anthelmintic resistance (AR) has thus far only rarely been reported for intestinal helminths of dogs and cats, in contrast to parasites of livestock and horses. We highlight possible reasons for this striking and important discrepancy, including ecological, biological and genetic factors and/or intervention regimens of key intestinal helminths concerning both host groups. In view of the current knowledge related to the genetics, mechanisms and principles of AR development, we point at issues which in our view contribute to a comparatively lower risk of AR development in intestinal helminths of dogs and cats. Finally, we specify research needs and provide recommendations by which, based on the available information about AR in ruminant and equine helminths, the development of AR in dog and cat helminths may best be documented, prevented or at least postponed., Graphical abstract Image 1, Highlights • Identifying causes of differences in anthelmintic resistance between livestock and dogs & cats. • Discussion of roles of AR mechanisms, genetics and husbandry in delayed AR evolution in dogs & cats. • Research needs for improved tools to monitor AR development in dogs & cats.

The mechanism of vertical transmission of Trypanosoma cruzi is poorly understood. In this study, we evaluated the role of IgG subclasses in the congenital transmission of Chagas disease. We conducted a case-control study in a public maternity hospital in Santa Cruz, Bolivia, enrolling women at delivery. Thirty women who transmitted T. cruzi to their newborns (cases), and 51 women who did not (controls) were randomly selected from 676 total seropositive women. Trypanosoma cruzi–specific IgG1, IgG2, and IgG3 levels were measured by in-house ELISA. The IgG4 levels were unmeasurable as a result of low levels in all participants. Quantitative polymerase chain reaction results and demographic factors were also analyzed. One-unit increases in normalized absorbance ratio of IgG1 or IgG2 levels increased the odds of congenital T. cruzi transmission in Chagas-seropositive women by 2.0 (95% CI: 1.1–3.6) and 2.27 (95% CI: 0.9–5.7), adjusted for age and previous blood transfusion. Odds of congenital transmission were 7.0 times higher in parasitemic mothers (95% CI: 2.3–21.3, P < 0.01) compared with nonparasitemic mothers. We observed that all mothers with IgG1 ≥ 4 were transmitters (sensitivity = 20%, specificity = 100%). Additionally, no mothers with IgG2 < 1.13 were transmitters (sensitivity = 100%, specificity = 21.6%). We demonstrated that IgG subclasses and parasite presence in blood are associated with vertical transmission of T. cruzi and could identify women at increased risk for congenital transmission by measuring IgG subclasses. These measures have potential as objective screening tests to predict the congenital transmission of Chagas.

With the wide adoption of genomic sequencing in children having seizures, an increasing number ofSCN2Agenetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by geneSCN2A, is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrentSCN2Agenetic variant is L1342P, which was identified in multiple patients with epileptic encephalopathy and intractable seizures. However, the mechanism underlying L1342P-mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human-induced pluripotent stem cell (hiPSC) line from a male donor, in which L1342P was introduced by CRISPR/Cas9-mediated genome editing. Using patch-clamping and microelectrode array (MEA) recordings, we revealed that cortical neurons derived from hiPSCs carrying heterozygous L1342P variant have significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, suggesting hyperexcitability phenotypes. Interestingly, L1342P neuronal culture displayed a degree of resistance to the anticonvulsant medication phenytoin, which recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, can potently alleviate spontaneous and chemically-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures and demonstrate the utility of genome-edited hiPSCs as anin vitroplatform to advance personalized phenotyping and drug discovery.SIGNIFICANCE STATEMENTA mounting number ofSCN2Agenetic variants have been identified from patients with epilepsy, but howSCN2Avariants affect the function of human neurons contributing to seizures is still elusive. This study investigated the functional consequences of a recurringSCN2Avariant (L1342P) using human iPSC-derived neurons and revealed both intrinsic and network hyperexcitability of neurons carrying a mutant Nav1.2 channel. Importantly, this study recapitulated elements of clinical observations of drug-resistant features of the L1342P variant, and provided a platform forin vitrodrug testing. Our study sheds light on cellular mechanism of seizures resulting from a recurring Nav1.2 variant, and helps to advance personalized drug discovery to treat patients carrying pathogenicSCN2Avariant.

Visual pathways of the brain are organized into parallel channels that code different features of the external environment. In the current study, we investigated the anatomical organization of parallel pathways from the superior colliculus (SC) to the pulvinar nucleus in the mouse. Virus injections placed in the ipsilateral and contralateral SC to induce the expression of different fluorescent proteins defines two pulvinar zones. The lateral pulvinar (Pl) receives ipsilateral SC input and the caudal medial pulvinar (Pcm) receives bilateral SC input. To examine the ultrastructure of these projections using transmission electron microscopy, we injected the SC with viruses to induce peroxidase expression within synaptic vesicles or mitochondria. We quantitatively compared the sizes of ipsilateral and contralateral tectopulvinar terminals and their postsynaptic dendrites, as well as the sizes of the overall population of synaptic terminals and their postsynaptic dendrites in the Pl and Pcm. Our ultrastructural analysis revealed that ipsilateral tectopulvinar terminals are significantly larger than contralateral tectopulvinar terminals. In particular, the ipsilateral tectopulvinar projection includes a subset of large terminals (≥ 1 μm(2)) that envelop dendritic protrusions of postsynaptic dendrites. We also found that both ipsilateral and contralateral tectopulvinar terminals are significantly larger than the overall population of synaptic terminals in both the Pl and Pcm. Thus, the ipsilateral tectopulvinar projection is structurally distinct from the bilateral tectopulvinar pathway, but both tectopulvinar channels may be considered the primary or “driving” input to the Pl and Pcm.

Tumor microenvironment-targeted therapies are emerging as promising treatment options for different cancer types. Tumor-associated macrophages and microglia (TAMs) represent an abundant nonmalignant cell type in brain metastases and have been proposed to modulate metastatic colonization and outgrowth. Here we demonstrate that targeting TAMs at distinct stages of the metastatic cascade using an inhibitor of colony-stimulating factor 1 receptor (CSF1R), BLZ945, in murine breast-to-brain metastasis models leads to antitumor responses in prevention and intervention preclinical trials. However, in established brain metastases, compensatory CSF2Rb–STAT5-mediated pro-inflammatory TAM activation blunted the ultimate efficacy of CSF1R inhibition by inducing neuroinflammation gene signatures in association with wound repair responses that fostered tumor recurrence. Consequently, blockade of CSF1R combined with inhibition of STAT5 signaling via AC4-130 led to sustained tumor control, a normalization of microglial activation states and amelioration of neuronal damage. Klemm et al. show that resistance to CSF1R inhibition in breast cancer brain metastasis is driven by compensatory activation of the CSF2Rb–STAT5 axis in macrophages, which can be alleviated by combined targeting of CSF1R and STAT5.

Background information Phosphatidylinositol (PI) is an essential phospholipid, critical to membrane bilayers. The complete deacylation of PI by B-type phospholipases produces intracellular and extracellular glycerophosphoinositol (GPI). Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters at the yeast plasma membrane. Internalized GPI is degraded to produce inositol, phosphate and glycerol, thereby contributing to these pools. GIT1 gene expression is controlled by nutrient balance, with phosphate or inositol starvation increasing GIT1 expression to stimulate GPI uptake. However, less is known about control of Git1 protein levels or localization. Results We find that the α-arrestins, an important class of protein trafficking adaptor, regulate Git1 localization and this is dependent upon their interaction with the ubiquitin ligase Rsp5. Specifically, α-arrestin Aly2 stimulates Git1 trafficking to the vacuole under basal conditions, but in response to GPI-treatment, either Aly1 or Aly2 promote Git1 vacuole trafficking. Cell surface retention of Git1, as occurs in aly1∆ aly2∆ cells, is linked to impaired growth in the presence of exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of GPI somehow causes cellular toxicity. Regulation of α-arrestin Aly1 by the protein phosphatase calcineurin improves steady-state and substrate-induced trafficking of Git1, however, calcineurin plays a larger role in Git1 trafficking beyond regulation of α-arrestins. Interestingly, loss of Aly1 and Aly2 increased phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole, and this was further exacerbated by GPI addition, suggesting that the effect is partially linked to Git1. Loss of Aly1 and Aly2 leads to increased incorporation of inositol label from [3 H]-inositol-labelled GPI into PI, confirming that internalized GPI influences PI balance and indicating a role for the a-arrestins in this regulation. Conclusions The α-arrestins Aly1 and Aly2 are novel regulators of Git1 trafficking with previously unanticipated roles in controlling phospholipid distribution and balance. Significance To our knowledge, this is the first example of α-arrestin regulation of phosphatidyliniositol-3-phosphate levels. In future studies it will be exciting to determine if other α-arrestins similarly alter PI and PIPs to change the cellular landscape.

The type VII protein secretion system (T7SS) of Staphylococcus aureus is encoded at the ess locus. T7 substrate recognition and protein transport are mediated by EssC, a membrane-bound multidomain ATPase. Four EssC sequence variants have been identified across S. aureus strains, each accompanied by a specific suite of substrate proteins. The ess genes are upregulated during persistent infection, and the secretion system contributes to virulence in disease models. It also plays a key role in intraspecies competition, secreting nuclease and membrane-depolarizing toxins that inhibit the growth of strains lacking neutralizing immunity proteins. A genomic survey indicates that the T7SS is widely conserved across staphylococci and is encoded in clusters that contain diverse arrays of toxin and immunity genes. The presence of genomic islands encoding multiple immunity proteins in species such as Staphylococcus warneri that lack the T7SS points to a major role for the secretion system in bacterial antagonism.

The American Zika virus (ZIKV) epidemic has highlighted the need to gain a better understanding of this emerging virus. The goal of this study was to describe the clinical symptoms, laboratory findings, and risk factors for symptomatic ZIKV infection in an area with ongoing transmission of other arboviral infections. We recruited patients at least 2 years of age seeking care at public health centers in León, Nicaragua, between January 2016 and August 2017, for fever, maculopapular rash, and/or nonsuppurative conjunctivitis with a duration of less than 1 week. A laboratory diagnosis of ZIKV was established using a combination of molecular and serological tests. Clinical and laboratory findings and potential risk factors were compared between participants with and without acute ZIKV infection. Fifty-eight (26%) of the 225 participants included in the analysis were found to have acute ZIKV infection. Pregnancy and reports of previous arboviral infection were associated with a higher risk of ZIKV infection. Rash, conjunctivitis, sore throat, and lower absolute neutrophil counts were associated with acute ZIKV infection. The clinical characteristics and risk factors identified were consistent with those identified by previous studies; however, we found sore throat to be a feature of ZIKV infection. We also found that neutrophil counts were lower in ZIKV-infected subjects. These clinical symptoms and laboratory data may help clinicians suspect ZIKV infection during future outbreaks.

The cortico–basal ganglia–thalamo–cortical loop is one of the fundamental network motifs in the brain. Revealing its structural and functional organization is critical to understanding cognition, sensorimotor behaviour, and the natural history of many neurological and neuropsychiatric disorders. Classically, this network is conceptualized to contain three information channels: motor, limbic and associative1–4. Yet this three-channel view cannot explain the myriad functions of the basal ganglia. We previously subdivided the dorsal striatum into 29 functional domains on the basis of the topography of inputs from the entire cortex5. Here we map the multi-synaptic output pathways of these striatal domains through the globus pallidus external part (GPe), substantia nigra reticular part (SNr), thalamic nuclei and cortex. Accordingly, we identify 14 SNr and 36 GPe domains and a direct cortico-SNr projection. The striatonigral direct pathway displays a greater convergence of striatal inputs than the more parallel striatopallidal indirect pathway, although direct and indirect pathways originating from the same striatal domain ultimately converge onto the same postsynaptic SNr neurons. Following the SNr outputs, we delineate six domains in the parafascicular and ventromedial thalamic nuclei. Subsequently, we identify six parallel cortico–basal ganglia–thalamic subnetworks that sequentially transduce specific subsets of cortical information through every elemental node of the cortico– basal ganglia–thalamic loop. Thalamic domains relay this output back to the originating corticostriatal neurons of each subnetwork in a bona fide closed loop., Mesoscale connectomic mapping of the cortico–basal ganglia–thalamic network reveals key architectural and information processing features.

Shifting range boundaries can lead to secondary contact of closely related species, which might in turn lead to hybridization when the evolution of reproductive isolation is incomplete. We examined winter nest use of northern (Glaucomys sabrinus (Shaw, 1801)) and southern (Glaucomys volans (Linnaeus, 1758)) flying squirrels in an area of recent secondary contact and known hybridization in Ontario, Canada, to test for evidence of reinforcement due to different and diverging nesting behaviours. We radio-collared 26 flying squirrels (12 G. sabrinus and 14 G. volans) between two survey periods (winters of 2008–2009 and 2019–2020) and identified all nest trees used by individuals throughout each winter. For each nest tree, we identified the nest type and collected tree classification information to compare differences in nest use between species. We also present a novel application of habitat suitability modelling to test for evidence of divergence in nest use through time, which would suggest reinforcement. We found southern flying squirrels used a higher proportion of cavities in large, hardwood trees, whereas northern flying squirrels used more external nests and softwood trees. Conditional probabilities provided some evidence for increased differentiation in nest use by flying squirrels through time. Overall, we found relatively little overlap in winter nest use between flying squirrel species, despite evidence for hybridization at this site, and some weak evidence for increased divergence between species in nest use over 11 years.

Plant cells are surrounded by a cell wall, a rigid structure that is not only important for cell and organ shape, but crucial for intercellular communication and interactions with the environment. In the flowering plant Arabidopsis thaliana, the 17 members of the Catharanthus roseus RLK1-like (CrRLK1L) receptor kinase family are involved in a multitude of physiological and developmental processes, making it difficult to assess their primary or ancestral function. To reduce genetic complexity, we characterized the single CrRLK1L gene of Marchantia polymorpha, MpFERONIA (MpFER). Plants with reduced MpFER levels show defects in vegetative development, i.e., rhizoid formation and cell expansion, and have reduced male fertility. In contrast, Mpfer null mutants and overexpression lines severely affect cell integrity and morphogenesis of the gametophyte. Thus, the CrRLK1L gene family originated from a single gene with an ancestral function in cell expansion and the maintenance of cellular integrity. During land plant evolution, this ancestral gene diversified to fulfil a multitude of specialized physiological and developmental and roles in the formation of both gametophytic and sporophytic structures essential to the life cycle of flowering plants.Summary statementThe CrRLK1L family arose in land plants and the Marchantia polymorpha genome contains a single copy, MpFER, which is broadly expressed and regulates cell expansion and cell wall integrity.

Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later1-3. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.

Background: A rapid molecular diagnostic test (MDT) is a test used to identify several different species of gram-negative bacteria and their genetic resistance markers. However, the impact of rapid MDT has not been established when combined with pharmacist involvement. Objective: To determine the impact of pharmacy involvement on patient outcomes when using rapid MDT. The primary outcome is the time from gram stain result to the first dose of the targeted antibiotic. Methods: This is a single-center, quasi-experimental, 1-group pretest-posttest design study of patients with gram-negative bacteremia in a community hospital. Hospitalized patients 18 years or older were included if they had a gram-negative blood culture. Patients were excluded if they were discharged or expired prior to culture results. Outcomes were compared between patients prior to and after implementation of the automated MDT. This research was determined to be exempt from institutional review board oversight consistent with West Florida Healthcare and in accordance with institutional policy. Results: The use of rapid MDT combined with pharmacist intervention resulted in a statistically significant decrease in the time to targeted antibiotic therapy (pre-intervention group, n = 77, 44.8 ± 17.8 hours versus post-intervention group, n= 80, 4.4 ± 5.8 hours; P ≤.001). There was no significant difference found between secondary outcomes. Limitations included small sample size as well as inconsistent documentation. Conclusions: The use of rapid MDT combined with pharmacist intervention resulted in a statistically significant decrease in the time to targeted antibiotic therapy.

A maternal Group B Streptococcus (GBS) six-valent polysaccharide conjugate vaccine (GBS6) is being developed to protect neonates and infants up to 3 months of age through passive transfer of antibodies from the mother to the infant. Fertility and developmental toxicity studies were conducted in female Sprague Dawley rats and New Zealand White rabbits with GBS6 (20 μg capsular polysaccharide/serotype formulated with or without AlPO4 , the highest clinical dose). Females were administered the full human dose of the GBS6 formulation intramuscularly twice prior to mating and twice during gestation, to ensure that high antibody levels were maintained throughout gestation and lactation. Approximately, half of the rats and rabbits were evaluated at the end of gestation, and the remainder were evaluated at the end of lactation. Maternal blood for GBS6 serology, to measure antibody titers to the GBS6 antigens, was collected prior to the first dose, prior to mating, and at each necropsy. Blood for serology was also collected from offspring at the end of gestation and lactation. In both species, there was no evidence of vaccine-related effects on fertility, embryo-fetal development, or postnatal development of the offspring, supporting regulatory guidance that single-species evaluation would have been sufficient. Functional serum antibodies to all six serotypes in the vaccine were confirmed in maternal animals and functional serum antibodies to one or more of the six serotypes was also confirmed in some rat offspring and most of the rabbit offspring. The results of these studies supported the safety of GBS6 vaccine administration to pregnant women.

The hormone oxytocin is commonly administered during childbirth to initiate and strengthen uterine contractions and prevent postpartum hemorrhage. However, patients have wide variation in the oxytocin dose required for a clinical response. To begin to uncover the mechanisms underlying this variability, we screened the 11 most prevalent missense genetic variants in the oxytocin receptor (OXTR) gene. We found that five variants, V45L, P108A, L206V, V281M, and E339K, significantly altered oxytocin-induced Ca2+ signaling or β-arrestin recruitment and proceeded to assess the effects of these variants on OXTR trafficking to the cell membrane, desensitization, and internalization. The variants P108A and L206V increased OXTR localization to the cell membrane, whereas V281M and E339K caused OXTR to be retained inside the cell. We examined how the variants altered the balance between OXTR activation and desensitization, which is critical for appropriate oxytocin dosing. The E339K variant impaired OXTR activation, internalization, and desensitization to roughly equal extents. In contrast, V281M decreased OXTR activation but had no effect on internalization and desensitization. V45L and P108A did not alter OXTR activation but did impair β-arrestin recruitment, internalization, and desensitization. Molecular dynamics simulations predicted that V45L and P108A prevent extension of the first intracellular loop of OXTR, thus inhibiting β-arrestin binding. Overall, our data suggest mechanisms by which OXTR genetic variants could alter clinical response to oxytocin.

Human tissue samples represent an invaluable source of information for the analysis of disease-specific cellular alterations and their variation between different pathologies. In cancer research, advancing a comprehensive understanding of the unique characteristics of individual tumor types and their microenvironment is of considerable importance for clinical translation. However, investigating human brain tumor tissue is challenging due to the often-limited availability of surgical specimens. Here we describe a multimodule integrated pipeline for the processing of freshly resected human brain tumor tissue and matched blood that enables analysis of the tumor microenvironment, with a particular focus on the tumor immune microenvironment (TIME). The protocol maximizes the information yield from limited tissue and includes both the preservation of bulk tissue, which can be performed within 1 h following surgical resection, as well as tissue dissociation for an in-depth characterization of individual TIME cell populations, which typically takes several hours depending on tissue quantity and further downstream processing. We also describe integrated modules for immunofluorescent staining of sectioned tissue, bulk tissue genomic analysis and fluorescence- or magnetic-activated cell sorting of digested tissue for subsequent culture or transcriptomic analysis by RNA sequencing. Applying this pipeline, we have previously described the overall TIME landscape across different human brain malignancies, and were able to delineate disease-specific alterations of tissue-resident versus recruited macrophage populations. This protocol will enable researchers to use this pipeline to address further research questions regarding the tumor microenvironment. Bulk tumor preservation and dissociation enable multiple analyses of the brain tumor immune microenvironment via immunofluorescent staining and cell sorting followed by transcriptomics, genomics or in vitro culture of specific cell populations.

Lung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable, with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations in EGFR/ErbB1 and ERBB2. Germline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and subcellular localization were assessed, and cellular proliferation, pAkt/Akt and pERK levels determined. A novel germline variant in ERBB3 (c.1946 T > G: p.Iso649Arg), coding for receptor tyrosine-protein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80 kDa form (which enhances proliferation) compared with the full-length (180 kDa) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localization pre- and post-epidermal growth factor stimulation; however, epidermal growth factor receptor (EGFR) levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared with wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 min post-stimulation compared with wtErbB3 transfection, demonstrating altered signalling pathway activation. Cumulatively, these results support this mutation as tumorogenic. This is the first reported family with a germline ERBB3 mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.

Core histones package chromosomal DNA and regulate genomic transactions, with their import and deposition involving a dedicated repertoire of molecular chaperones. Histones H3 and H4 have been predominantly characterised as obligate heterodimers, however, recent findings have alluded to the existence of a significant pool of monomeric histone H3 in the nucleoplasm. Using a combination of in vitro and in vivo experiments, here we show that monomeric H3 and H4 use an Importin 5 (Imp5) dependent pathway for their nuclear import, distinct from Importin 4 (Imp4) previously described for H3-H4 dimers. Using mutants that disrupt the histone fold, we show monomeric H3 loses its interaction with Imp4, but retains interactions with Imp5 and the chaperone NASP. H4 monomeric mutants similarly bind Imp5 and not Imp4, however, they lose interaction with NASP, retaining their interaction with the HAT1-RBBP7 complex instead. In vitro experiments revealed that Imp5 and NASP are mutually exclusive in their binding, suggesting a facilitated hand-off mechanism. Furthermore, new H3 accumulates rapidly in a NASP-bound complex after nuclear translocation. NASP can assemble into three distinct co-chaperoning complexes, including a novel complex containing NASP, H3 and the putative ubiquitin ligase UBR7, a NASP-H3-H4-RBBP7 subcomplex and the previously characterised NASP-H3-H4-ASF1-HAT1-RBBP7 multi-chaperoning complex. Here we propose an alternative import pathway and folding mechanism for monomeric H3 and H4 that involves Imp5, rather than Imp4, and hands off to nuclear chaperones NASP, RBBP7 and HAT1. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/464968v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@462172org.highwire.dtl.DTLVardef@3df5c5org.highwire.dtl.DTLVardef@1d28f01org.highwire.dtl.DTLVardef@aa8fb0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Metal dyshomeostasis, and especially overexposure, is known to cause adverse health effects due to modulation of a variety of metabolic pathways. An increasing body of literature has demonstrated that metal exposure may affect SIRT signaling, although the existing data are insufficient. Therefore, in this review we discuss the available data (PubMed-Medline, Google Scholar) on the influence of metal overload on sirtuin (SIRT) signaling and its association with other mechanisms involved in metal-induced toxicity. The existing data demonstrate that cadmium (Cd), mercury (Hg), arsenic (As), lead (Pb), aluminium (Al), hexavalent chromium (CrVI), manganese (Mn), iron (Fe), and copper (Cu) can inhibit SIRT1 activity. In addition, an inhibitory effect of Cd, Pb, As, and Fe on SIRT3 has been demonstrated. In turn, metal-induced inhibition of SIRT was shown to affect deacetylation of target proteins including FOXO, PGC1α, p53 and NF-kB. Increased acetylation downregulates PGC1α signaling pathway, resulting in cellular altered redox status and increased susceptibility to oxidative stress, as well as decreased mitochondrial biogenesis. Lower rates of LKB1 deacetylation may be responsible for metal-induced decreases in AMPK activity and subsequent metabolic disturbances. A shift to the acetylated FOXO results in increased expression of pro-apoptotic genes which upregulates apoptosis together with increased p53 signaling. Correspondingly, decreased NF-kB deacetylation results in upregulation of target genes of proinflammatory cytokines, enzymes, and cellular adhesion molecules thus promoting inflammation. Therefore, alterations in sirtuin activity may at least partially mediate metal-induced metabolic disturbances that have been implicated in neurotoxicity, nephrotoxicity, cardiotoxicity, and other toxic effects of heavy metals.

Cheliceral chelal design in free-living astigmatid mites (Arthropoda: Acari) is reviewed within a mechanical model. Trophic access (body size and cheliceral reach) and food morsel handling (chelal gape and estimated static adductive crushing force) are morphologically investigated. Forty-seven commonly occurring astigmatid mite species from 20 genera (covering the Acaridae, Aeroglyphidae, Carpoglyphidae, Chortoglyphidae, Glycyphagidae, Lardoglyphidae, Pyroglyphidae, Suidasiidae, and Winterschmidtiidae) are categorised into functional groups using heuristics. Conclusions are confirmed with statistical tests and multivariate morphometrics. Despite these saprophagous acarines in general being simple ‘shrunken/swollen’ versions of each other, clear statistical correlations in the specifics of their mechanical design (cheliceral and chelal scale and general shape) with the type of habitat and food consumed (their ‘biome’) are found. Using multivariate analyses, macro- and microsaprophagous subtypes are delineated. Relative ratios of sizes on their own are not highly informative of adaptive syndromes. Sympatric resource competition is examined. Evidence for a maximum doubling of approximate body volume within nominal taxa is detected but larger mites are not more ‘generalist’ feeding types. Two contrasting types of basic ‘Bauplan’ are found differing in general scale: (i) a large, chunk-crunching, ‘demolition’-feeding omnivore design (comprising 10 macrosaprophagous astigmatid species), and (ii) a small selective picking, squashing/slicing or fragmentary/‘plankton’ feeding design (which may indicate obligate fungivory/microbivory) comprising 20 microsaprophagous acarid-shaped species. Seventeen other species appear to be specialists. Eleven of these are either: small (interstitial/burrowing) omnivores—or a derived form designed for processing large hard food morsels (debris durophagy, typified by the pyroglyphid Dermatophagoides farinae), or a specialist sub-type of particular surface gleaning/scraping fragmentary feeding. Six possible other minor specialist gleaning/scraping fragmentary feeders types each comprising one to two species are described. Details of these astigmatid trophic-processing functional groups need field validation and more corroborative comparative enzymology. Chelal velocity ratio in itself is not highly predictive of habitat but with cheliceral aspect ratio (or chelal adductive force) is indicative of life-style. Herbivores and pest species are typified by a predicted large chelal adductive force. Pest species may be ‘shredders’ derived from protein-seeking necrophages. Carpoglyphus lactis typifies a mite with tweezer-like chelae of very feeble adductive force. It is suggested that possible zoophagy (hypocarnivory) is associated with low chelal adductive force together with a small or large gape depending upon the size of the nematode being consumed. Kuzinia laevis typifies an oophagous durophage. Functional form is correlated with taxonomic position within the Astigmata—pyroglyphids and glycyphagids being distinct from acarids. A synthesis with mesostigmatid and oribatid feeding types is offered together with clarification of terminologies. The chelal lyrifissure in the daintiest chelicerae of these astigmatids is located similar to where the action of the chelal moveable digit folds the cheliceral shaft in uropodoids, suggesting mechanical similarities of function. Acarid astigmatids are trophically structured like microphytophagous/fragmentary feeding oribatids. Some larger astigmatids (Aleuroglyphus ovatus, Kuzinia laevis, Tyroborus lini) approximate, and Neosuidasia sp. matches, the design of macrophytophagous oribatids. Most astigmatid species reviewed appear to be positioned with other oribatid secondary decomposers. Only Dermatophagoides microceras might be a primary decomposer approximating a lichenivorous oribatid (Austrachipteria sp.) in trophic form. Astigmatid differences are consilient with the morphological trend from micro- to macrophytophagy in oribatids. The key competency in these actinotrichid mites is a type of ‘gnathosomisation’ through increased chelal and cheliceral height (i.e., a shape change that adjusts the chelal input effort arm and input adductive force) unrestricted by the dorsal constraint of a mesostigmatid-like gnathotectum. A predictive nomogram for ecologists to use on field samples is included. Future work is proposed in detail.

Case series summary Described are four cats diagnosed with ophthalmomyiasis externa caused by Cuterebra larvae. Medical records were retrospectively reviewed to identify cats with ophthalmomyiasis externa between 2005 and 2020 at Cornell University Hospital for Animals. Signalment, history, clinical and diagnostic findings, treatment and outcome were recorded. All cats were young (Relevance and novel information To the authors’ knowledge, this is the first report of feline ophthalmomyiasis externa caused by Cuterebra species. Parasite removal was successful in restoring comfort and resolving clinical signs in all cats with adequate follow-up information.

A multitude of concurrent biological and physical processes contribute to microbial community turnover, especially in highly dynamic coastal environments. Characterizing what factors contribute most to shifts in microbial community structure and the specific organisms that correlate with changes in the products of photosynthesis improves our understanding of nearshore microbial ecosystem functions. We conducted high frequency sampling in nearshore Southern California in order to capture sub-weekly microbial community dynamics. Microbial communities were characterized by flow cytometry and 16S rRNA gene sequencing, and placed in the context of physicochemical parameters. Within our time-series, season and nutrient availability corresponded to changes in dominant microbial community members. Concurrent aseasonal drivers with overlapping scales of variability were also apparent when we used network analysis to assess the microbial community as subsets of the whole. Our analyses revealed the microbial community as a mosaic, with overlapping groups of taxa that varied on different timescales and correlated with unique abiotic and biotic factors. Specifically, a subnetwork associated with chlorophyll a exhibited rapid turnover, indicating that ecologically important subsets of the microbial community can change on timescales different than and in response to factors other than those that govern turnover of most members of the assemblage.

Athrotaxis cupressoides is an iconic Tasmanian palaeoendemic conifer that is vulnerable to fire. A survey of three populations burnt by severe fire in 2016, conducted 1 year post-fire, found 33% of stems were still alive, with many surviving stems suffering some canopy scorch. We re-surveyed these populations to quantify delayed mortality, resprouting, and presence of juveniles, and to determine whether fire impacts can be reliably assessed after 1 year. We applied three measures of fire severity: canopy scorched, canopy consumed, and the minimum burnt twig diameter of neighbouring shrubs. We found overall stem survival in 2020 was 31%, and that 97% of stems that were dead 4 years post-fire had died within the first year. Our best predictor of stem mortality was percentage canopy scorched. Overall, 1.8% of burnt stems resprouted, but severely burnt stems did not resprout. Juveniles were present ~9.9% of burnt trees in 2017, and only 1.8% in 2020. We conclude that A. cupressoides stems are not unusually fire sensitive, but rather, that the species’ vulnerability to severe fire results from its lack of reliable recovery mechanisms. This study shows that fire-caused mortality can be reliably assessed 1 year post-fire, and possibly earlier. Interventions such as sowing seed or transplanting seedlings could be necessary to re-establish fire-killed populations.

Background The hookworm, Ancylostoma caninum, is a common and important zoonotic intestinal nematode parasite that infects dogs globally. Both the immature and adult stages of A. caninum ingest large volumes of blood during the feeding process and can cause severe anemia and death in young dogs, even before patent infections can be diagnosed using routine faecal examination methods. Thus, effective treatment of any pre-patent stages of immature hookworms can reduce or eliminate the risk of clinical disease in infected dogs and additionally reduce environmental contamination of eggs and infective larvae. Two randomized, blinded, GCP-compliant, pivotal laboratory dose confirmation studies were conducted to evaluate the effectiveness and safety of a new novel combination of lotilaner and milbemycin oxime tablets (Credelio Plus®) administered orally to dogs experimentally infected with immature (L4 and immature adult [L5]) stages of A. caninum. Methods Treatments using the intended global commercial tablet formulation of Credelio Plus were administered in a time frame relative to inoculation with infective larvae so that effectiveness could be assessed against each specific immature stage of A. caninum. In each study, dogs were randomized to one of six (study 1) or four (study 2) treatment groups. Each treatment group contained 8 (study 1) or 10 (study 2) dogs that had been experimentally inoculated with infective A. caninum larvae on day 0 and were dosed once on day 7 or day 11. Enrolled subjects were administered placebo tablets, Credelio Plus tablets, or lotilaner mono tablets to provide minimum dosages of 0.75 mg/kg of milbemycin oxime and 20 mg/kg of lotilaner. All dogs were necropsied 5 days after their respective treatment. All nematodes recovered from the gastrointestinal tract at necropsy were counted by species and stage. Results For both dose confirmation studies and based on geometric mean worm counts, efficacy of Credelio Plus was ≥ 97.3% against L4 larval stage of A. caninum and ≥ 98.7% against immature adult (L5) A. caninum. Conclusions These studies demonstrated that the orally administered Credelio Plus combination tablet was highly efficacious in treating immature (L4 and immature adult [L5]) stages of A. caninum in experimentally infected dogs. Graphical Abstract

Background The ascarid, Toxocara canis, is a common and important zoonotic intestinal nematode parasite that infects dogs globally. An effective treatment that kills any pre-patent stages of immature T. canis could additionally reduce or eliminate the development of patent infections that can result in clinical disease in infected dogs and would further reduce environmental contamination of eggs. Two randomized, blinded, GCP-compliant, pivotal laboratory dose confirmation studies were conducted to assess the effectiveness and safety of a new novel combination of lotilaner and milbemycin oxime tablets (Credelio Plus) administered orally to dogs that were experimentally infected with immature (L4 or immature adult [L5]) stages of T. canis. Methods The commercial tablet formulation of Credelio Plus® was administered in a time frame relative to inoculation with infective eggs. This allowed for effectiveness to be assessed against each specific immature stage of T. canis. In each study, dogs were randomized and allocated to one of four treatment groups. Each treatment group contained ten dogs that had been experimentally inoculated on Day 0 with infective T. canis eggs and then were dosed once on Day 14 or Day 24 using either placebo tablets or Credelio Plus tablets (IP) to provide minimum dosages of 0.75 mg/kg of milbemycin oxime and 20 mg/kg of lotilaner. All dogs were necropsied 5 or 6 days after their respective treatment. At necropsy, all nematodes recovered from the gastrointestinal tract were counted by species and stage. Results In both dose confirmation studies using geometric mean worm counts, effectiveness of Credelio Plus was ≥ 98.6% and ≥ 96.8% against L4 larval stage T. canis and immature adult [L5] T. canis in both studies, respectively. Conclusions These studies demonstrated that the Credelio Plus combination tablet administered orally to dogs was highly efficacious against experimental infections with L4 and immature adult [L5] stages of T. canis. Graphical Abstract

Background Vertical transmission of Trypanosoma cruzi infection from mother to infant accounts for a growing proportion of new cases of Chagas disease. However, no systematic reviews of risk factors for T. cruzi vertical transmission have been performed. Methods We performed a systematic review of the literature in PubMed, LILACS, and Embase databases, following PRISMA guidelines. Studies were not excluded based on language, country of origin, or date of publication. Results Our literature review yielded 27 relevant studies examining a wide variety of risk factors, including maternal age, parasitic load, immunologic factors, vector exposure, and more. Several studies suggested that mothers with higher parasitic loads may have a greater risk of vertical transmission, and a meta-analysis of two studies found a significantly higher parasitic load among transmitting than non-transmitting mothers with T. cruzi infection. A second meta-analysis of ten studies demonstrated that maternal age was not significantly associated with vertical transmission risk. Conclusions The current literature suggests that high maternal parasitic load may be a risk factor for congenital Chagas disease among infants of T. cruzi seropositive mothers. Given the considerable heterogeneity and risk of bias among current literature, additional studies are warranted to assess potential risk factors for vertical transmission of T. cruzi infection.

Municipal wastewater treatment plants (WWTPs) are a significant source of anthropogenic pollutants and are a serious environmental stressor in Laurentian Great Lakes ecosystems. In this study, we examined whether three freshwater fish species (bluegill sunfish Lepomis macrochirus, green sunfish Lepomis cyanellus, and round goby Neogobius melanostomus) collected near two wastewater effluent outflows in Lake Ontario showed altered measures of somatic investment and thermal tolerance. Fish of all three species collected near the WWTPs were larger with 50–60% heavier body masses compared to those collected at reference sites. Green sunfish had higher body condition and increased haematocrit at wastewater-contaminated sites, and both round goby and bluegill sunfish had larger livers (controlling for body mass) at wastewater-contaminated sites. Thermal tolerance (critical thermal maximum, CTmax) differed between species (green sunfish > bluegill sunfish > round goby), but was similar in fish collected at wastewater-contaminated sites compared to cleaner reference sites. Wastewater-contaminated sites had poorer water quality, higher nutrient loadings, and higher concentrations of anthropogenic contaminants (measured via polar organic chemical integrative samplers, POCIS) than reference sites. Our results suggest that fish in the wild may have some capacity to cope with WWTP effluent and avoid any potential impairments in thermal tolerance. Our findings also suggest that treated wastewater is changing water quality locally in Great Lakes watersheds, and that many fish species may be able to access extra nutrients provided by such effluent outflows. However, if outflow areas become preferred foraging areas this will inadvertently increase exposure to anthropogenic stressors and pollutants.

European guidelines for testing attractant and repellent efficacy (i.e., Product type 19 [PT19]) have been in revision since 2017. A key topic of discussion is the current approach to evaluating topical repellents. The European Chemical Agency has stated field testing should be avoided because of mosquito-borne disease risks. However, the most common laboratory method, the arm-in-cage (AIC) test, may limit the reliable extrapolation of lab results to field conditions. This study’s main goal was to assess alternative laboratory methods for evaluating topical mosquito repellents that use mosquito landing rates more representative of those in the field. The study took place at three European testing labs using 30 study participants per test and the mosquito, Aedes albopictus (Skuse, 1894, Diptera: Culicidae). In phase 1, a conventional AIC test and a sleeved AIC test were performed. Respectively, the arm area exposed was 600 and 100 cm2, and cage volume was 0.040 and 0.064 m3. Mosquito density was the same for both: 1 female/840 cm3. In phase 2, room-based testing (40 ± 5 mosquitoes in 25–30 m3) was used as a proxy for field testing. The mosquito repellent employed was 15% N,N-diethyl-m-toluamide in ethanol at two doses: 1 and 0.5 g/600 cm2. The protection times measured at each laboratory were analyzed both separately and together using nonparametric (Kruskal–Wallis) test. The two alternatives methods showed to be potential alternatives to the current AIC method recreated field mosquito landing rates and achieved reproducible protection times across laboratories., Graphical Abstract Graphical Abstract

Despite signs of infection—including taste loss, dry mouth and mucosal lesions such as ulcerations, enanthema and macules— the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly understood. To address this, we generated and analyzed two single-cell RNA sequencing datasets of the human minor salivary glands and gingiva (9 samples, 13,824 cells), identifying 50 cell clusters. Using integrated cell normalization and annotation, we classified 34 unique cell subpopulations between glands and gingiva. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry factors such as ACE2 and TMPRSS members were broadly enriched in epithelial cells of the glands and oral mucosae. Using orthogonal RNA and protein expression assessments, we confirmed SARS-CoV-2 infection in the glands and mucosae. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting ACE2 and TMPRSS expression and sustained SARS-CoV-2 infection. Acellular and cellular salivary fractions from asymptomatic individuals were found to transmit SARS-CoV-2 ex vivo. Matched nasopharyngeal and saliva samples displayed distinct viral shedding dynamics, and salivary viral burden correlated with COVID-19 symptoms, including taste loss. Upon recovery, this asymptomatic cohort exhibited sustained salivary IgG antibodies against SARS-CoV-2. Collectively, these data show that the oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.

Liver resident CD8 T cells have an essential role in immunopathology in a mouse model of nonalcoholic steatohepatitis, by becoming auto-aggressive following sequential transcriptional and metabolic activation steps . Nonalcoholic steatohepatitis (NASH) is a manifestation of systemic metabolic disease related to obesity, and causes liver disease and cancer(1,2). The accumulation of metabolites leads to cell stress and inflammation in the liver(3), but mechanistic understandings of liver damage in NASH are incomplete. Here, using a preclinical mouse model that displays key features of human NASH (hereafter, NASH mice), we found an indispensable role for T cells in liver immunopathology. We detected the hepatic accumulation of CD8 T cells with phenotypes that combined tissue residency (CXCR6) with effector (granzyme) and exhaustion (PD1) characteristics. Liver CXCR6(+) CD8 T cells were characterized by low activity of the FOXO1 transcription factor, and were abundant in NASH mice and in patients with NASH. Mechanistically, IL-15 induced FOXO1 downregulation and CXCR6 upregulation, which together rendered liver-resident CXCR6(+) CD8 T cells susceptible to metabolic stimuli (including acetate and extracellular ATP) and collectively triggered auto-aggression. CXCR6(+) CD8 T cells from the livers of NASH mice or of patients with NASH had similar transcriptional signatures, and showed auto-aggressive killing of cells in an MHC-class-I-independent fashion after signalling through P2X7 purinergic receptors. This killing by auto-aggressive CD8 T cells fundamentally differed from that by antigen-specific cells, which mechanistically distinguishes auto-aggressive and protective T cell immunity.

Desmoplastic small round cell tumor (DSRCT) is characterized by the EWSR1–WT1 t(11;22) (p13:q12) translocation. Few additional putative drivers have been identified, and research has suffered from a lack of model systems. Next-generation sequencing (NGS) data from 68 matched tumor-normal samples, whole-genome sequencing data from 10 samples, transcriptomic and affymetrix array data, and a bank of DSRCT patient-derived xenograft (PDX) are presented. EWSR1–WT1 fusions were noted to be simple, balanced events. Recurrent mutations were uncommon, but were noted in TERT (3%), ARID1A (6%), HRAS (5%), and TP53 (3%), and recurrent loss of heterozygosity (LOH) at 11p, 11q, and 16q was identified in 18%, 22%, and 34% of samples, respectively. Comparison of tumor-normal matched versus unmatched analysis suggests overcalling of somatic mutations in prior publications of DSRCT NGS data. Alterations in fibroblast growth factor receptor 4 (FGFR4) were identified in 5 of 68 (7%) of tumor samples, whereas differential overexpression of FGFR4 was confirmed orthogonally using 2 platforms. PDX models harbored the pathognomic EWSR1–WT1 fusion and were highly representative of corresponding tumors. Our analyses confirm DSRCT as a genomically quiet cancer defined by the balanced translocation, t(11;22)(p13:q12), characterized by a paucity of secondary mutations but a significant number of copy number alterations. Against this genomically quiet background, recurrent activating alterations of FGFR4 stood out, and suggest that this receptor tyrosine kinase, also noted to be highly expressed in DSRCT, should be further investigated. Future studies of DSRCT biology and preclinical therapeutic strategies should benefit from the PDX models characterized in this study.Implications:These data describe the general quiescence of the desmoplastic small round cell tumor (DSRCT) genome, present the first available bank of DSRCT model systems, and nominate FGFR4 as a key receptor tyrosine kinase in DSRCT, based on high expression, recurrent amplification, and recurrent activating mutations.

Plasmacytoid dendritic cells (pDCs) are the principal natural type I interferon–producing dendritic cells. Neoplastic expansion of pDCs and pDC precursors leads to blastic plasmacytoid dendritic cell neoplasm (BPDCN), and clonal expansion of mature pDCs has been described in chronic myelomonocytic leukemia. The role of pDC expansion in acute myeloid leukemia (AML) is poorly studied. Here, we characterize patients with AML with pDC expansion (pDC-AML), which we observe in ∼5% of AML cases. pDC-AMLs often possess cross-lineage antigen expression and have adverse risk stratification with poor outcome. RUNX1 mutations are the most common somatic alterations in pDC-AML (>70%) and are much more common than in AML without pDC expansion and BPDCN. We demonstrate that pDCs are clonally related to, as well as originate from, leukemic blasts in pDC-AML. We further demonstrate that leukemic blasts from RUNX1-mutated AML upregulate a pDC transcriptional program, poising the cells toward pDC differentiation and expansion. Finally, tagraxofusp, a targeted therapy directed to CD123, reduces leukemic burden and eliminates pDCs in a patient-derived xenograft model. In conclusion, pDC-AML is characterized by a high frequency of RUNX1 mutations and increased expression of a pDC transcriptional program. CD123 targeting represents a potential treatment approach for pDC-AML.