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Acknowledgements: This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and National Health Service (NHS) England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support. GS was performed by Illumina at Illumina Laboratory Services and was overseen by Genomics England. We thank all NHS clinicians who have contributed clinical phenotype data to the 100,000 Genomes Rare Diseases Programme and all staff at Genomics England who have contributed to the sequencing, maintenance of the research environment and assembly of the standard bioinformatic files that were required for our analyses. We thank the participants of the rare diseases program who made this research possible. We are grateful to V. Keeley for providing access to paternal DNA (ERG), F. Elmslie for inviting a patient to the clinic (ERG) and T. Jaworek for technical assistance (GPR156). D.G. was supported by the Cambridge British Heart Foundation (BHF) Centre of Research Excellence (RE/18/1/34212) and the Wellcome Collaborative (219506/Z/19/Z). V.H. was supported by an Medical Research Council (MRC)/National Institute for Health and Care Research Clinical Academic Research Partnership (MR/V037617/1). G.M.B. and K. Frudd were funded by BHF (PG/17/33/32990). G.M.B. and D.P. were funded by BHF (PG/20/16/35047). E.S. was supported by the Swiss Federal National Fund for Scientific Research (CRSII5_177191/1). S.M. and P.O. were supported by the MRC (MR/P011543/1) and BHF (RG/17/7/33217). K. Freson was supported by Katholieke Universiteit (KU) Leuven Special Research Fund (BOF) (C14/19/096) and Research Foundation – Flanders (G072921N). Work at the University of Maryland, Baltimore was supported by the National Institute on Deafness and Other Communication Disorders/National Institutes of Health (R01DC016295 to Z.M.A.). M.A.-O., F.I. and K.R. were supported by the King Salman Center for Disability Research (85722). E.T. was supported by the Mindich Child Health and Development Institute, the Charles Bronfman Institute for Personalized Medicine and the Lowy Foundation USA., Funder: Cambridge BHF Centre of Research Excellence [RE/18/1/34212] and Wellcome Collaborative Award 219506/Z/19/Z, Funder: BHF Project grant PG/17/33/32990, Funder: Swiss National Science Foundation grant CRSII5_177191, Funder: King Salman Center for Disability Research # 85722, Funder: MRC/NIHR Clinical Academic Research Partnership MR/V037617/1, Funder: Medical Research Council grant MR/P011543/1 and British Heart Foundation grant RG/17/7/33217, Funder: NIDCD/NIH R01DC016295, Funder: BHF Project grants PG/20/16/35047 & PG/17/33/32990, Funder: KULeuven BOF grant C14/19/096, FWO grant G072921N, The genetic etiologies of more than half of rare diseases remain unknown. Standardized genome sequencing and phenotyping of large patient cohorts provide an opportunity for discovering the unknown etiologies, but this depends on efficient and powerful analytical methods. We built a compact database, the ‘Rareservoir’, containing the rare variant genotypes and phenotypes of 77,539 participants sequenced by the 100,000 Genomes Project. We then used the Bayesian genetic association method BeviMed to infer associations between genes and each of 269 rare disease classes assigned by clinicians to the participants. We identified 241 known and 19 previously unidentified associations. We validated associations with ERG, PMEPA1 and GPR156 by searching for pedigrees in other cohorts and using bioinformatic and experimental approaches. We provide evidence that (1) loss-of-function variants in the Erythroblast Transformation Specific (ETS)-family transcription factor encoding gene ERG lead to primary lymphoedema, (2) truncating variants in the last exon of transforming growth factor-β regulator PMEPA1 result in Loeys–Dietz syndrome and (3) loss-of-function variants in GPR156 give rise to recessive congenital hearing impairment. The Rareservoir provides a lightweight, flexible and portable system for synthesizing the genetic and phenotypic data required to study rare disease cohorts with tens of thousands of participants.

Merkel cell carcinoma (MCC) is a rare, but aggressive skin cancer the incidence of which has increased significantly in recent years. The majority of MCCs have incorporated Merkel cell polyomavirus (VP-MCC) while the remainder are virus-negative (VN-MCC). Although a variety of therapeutic options have shown promise in treating MCC, there remains a need for additional therapeutics as well as probes for better understanding MCC. A high-throughput screening campaign was used to assess the ability of > 25,000 synthetic and natural product compounds as well as > 20,000 natural product extracts to affect growth and survival of VN-MCC and VP-MCC cell lines. Sixteen active compounds were identified that have mechanisms of action reported in the literature along with a number of compounds with unknown mechanisms. Screening results with pure compounds suggest a range of potential targets for MCC including DNA damage, inhibition of DNA or protein synthesis, reactive oxygen species, and proteasome inhibition as well as NFκB inhibition while also suggesting the importance of zinc and/or copper binding. Many of the active compounds, particularly some of the natural products, have multiple reported targets suggesting that this strategy might be a particularly fruitful approach. Processing of several active natural product extracts resulted in the identification of additional MCC-active compounds. Based on these results, further investigations focused on natural products sources, particularly of fungal origin, are expected to yield further potentially useful modulators of MCC.

KCNJ16 encodes Kir5.1 and acts in combination with Kir4.1, encoded by KCNJ10, to form an inwardly rectifying K+ channel expressed at the basolateral membrane of epithelial cells in the distal nephron. This Kir4.1/Kir5.1 channel is critical for controlling basolateral membrane potential and K+ recycling, the latter coupled to Na-K-ATPase activity, which determines renal Na+ handling. Previous work has shown that Kcnj16−/− mice and SSKcnj16−/− rats demonstrate hypokalemic, hyperchloremic metabolic acidosis. Here, we present the first report of a patient identified to have biallelic loss-of-function variants in KCNJ16 by whole exome sequencing who presented with chronic metabolic acidosis with exacerbations triggered by minor infections.

Lysosomal acid lipase (LAL) deficiency, or cholesterol ester storage disease, is a disorder affecting the breakdown of cholesterol esters and triglycerides within lysosomes. Clinical findings include hepatomegaly, hepatic dysfunction, and dyslipidemia, with a wide range of phenotypic variability and age of onset (1-3). The available clinical and molecular information of the patient presented herein was consistent with a diagnosis of LAL deficiency, but her LAL activity assay repeatedly showed normal or borderline low results. Her response to enzyme replacement therapy (4,5) and demonstrable deficiency on a newer specific enzymatic assay (6) ultimately confirmed her diagnosis of LAL deficiency.

Secreted phospholipase A(2) (sPLA(2)) enzymes release free fatty acids, including arachidonic acid and generate lysophosphospholipds from phospholipids, including membrane phospholipids from cells and bacteria and surfactant phospholipids. We have shown that an endogenous enzyme sPLA(2) group X (sPLA(2)-X) is elevated in the airways of asthmatics and that mice lacking the sPLA(2)-X gene (Pla2g10) display attenuated airway hyperresponsiveness (AHR), innate and adaptive immune responses, and type-2 cytokine production in a model of airway sensitization and challenge using a complete allergen that induces endogenous adjuvant activity. This complete allergen also induces the expression of sPLA(2)-X/Pla2g10. In the periphery, a sPLA(2) found in bee venom (bee venom PLA(2), bvPLA(2)) administered with the incomplete antigen ovalbumin (OVA) leads to an antigen specific immune response. In this study, we demonstrate that both bvPLA(2) and murine sPLA(2)-X have adjuvant activity, leading to a type-2 immune response in the lung with features of airway hyperresponsiveness (AHR) and antigen-specific type-2 airway inflammation following peripheral sensitization and subsequent airway challenge with OVA. Further, the adjuvant effects of sPLA(2)-X that result in the type-2 biased OVA-specific adaptive immune response in the lung was dependent upon the catalytic activity of the enzyme, as a catalytically inactive mutant form of sPLA(2)-X does not elicit the adaptive component of the immune response, although other components of the immune response were induced by the inactive enzyme suggesting receptor mediated effects. Our results demonstrate that exogenous and endogenous sPLA(2)s play an important role in peripheral sensitization resulting in airway responses to inhaled antigens.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spot (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed clear deficit in the enzymatic activity, and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patient and for monitoring the efficacy of therapeutic treatments. On the other hand, ARSA activity was measured in DBS for the first time without an antibody. Currently, this new ARSA DBS assay serves as a second-tier test following the sulfatide measurement in DBS in our large-scale de-identified pilot study for the screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay. We are also evaluating its potential utility as the first-tier test for the newborn screening of MLD.

Niemann-Pick disease type C1 (NPC1) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, there is an urgency to improve diagnostics and monitor therapeutic efficacy with biomarkers. In this study, we sought to define the structure of an unknown lipid biomarker for NPC1 with [M + H](+) ion at m/z 509.3351, previously designated as lysoSM-509. The structure of N-palmitoyl-O-phosphocholineserine (PPCS) was proposed for the lipid biomarker based on the results from mass spectrometric analyses and chemical derivatizations. As no commercial standard is available, authentic PPCS was chemically synthesized, and the structure was confirmed by comparison of endogenous and synthetic compounds as well as their derivatives using liquid chromatography-tandem mass spectrometry (LC-MS/MS). PPCS is the most abundant species among N-acyl-O-phosphocholineserines (APCS), a class of lipids that have not been previously detected in biological samples. Further analysis demonstrated that all APCS species with acyl groups ranging from C14 to C24 were elevated in NPC1 plasma. PPCS is also elevated in both central and peripheral tissues of the NPC1 cat model. Identification of APCS structures provide an opportunity for broader exploration of the roles of these novel lipids in NPC1 disease pathology and diagnosis.

Elevated secreted phospholipase A2 (sPLA2) activity in the airways has been implicated in the pathogenesis of asthma and allergic disease for some time. The identity and function of these enzymes in asthma is becoming clear from work in our lab and others. We focused on sPLA2 group X (sPLA2-X) after identifying increased levels of this enzyme in asthma, and that it is responsible for a large portion of sPLA2 activity in the airways and that the levels are strongly associated with features of airway hyperresponsiveness (AHR). In this review, we discuss studies that implicated sPLA2-X in human asthma, and murine models that demonstrate a critical role of this enzyme as a regulator of type-2 inflammation, AHR and production of eicosanoids. We discuss the mechanism by which sPLA2-X acts to regulate eicosanoids in leukocytes, as well as effects that are mediated through the generation of lysophospholipids and through receptor-mediated functions. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.

The mechanisms responsible for driving endogenous airway hyperresponsiveness (AHR) in the form of exercise-induced bronchoconstriction (EIB) are not fully understood. We examined alterations in airway phospholipid hydrolysis, surfactant degradation, and lipid mediator release in relation to AHR severity and changes induced by exercise challenge. Paired induced sputum ( n = 18) and bronchoalveolar lavage (BAL) fluid ( n = 11) were obtained before and after exercise challenge in asthmatic subjects. Samples were analyzed for phospholipid structure, surfactant function, and levels of eicosanoids and secreted phospholipase A2group 10 (sPLA2-X). A primary epithelial cell culture model was used to model effects of osmotic stress on sPLA2-X. Exercise challenge resulted in increased surfactant degradation, phospholipase activity, and eicosanoid production in sputum samples of all patients. Subjects with EIB had higher levels of surfactant degradation and phospholipase activity in BAL fluid. Higher basal sputum levels of cysteinyl leukotrienes (CysLTs) and prostaglandin D2(PGD2) were associated with direct AHR, and both the postexercise and absolute change in CysLTs and PGD2levels were associated with EIB severity. Surfactant function either was abnormal at baseline or became abnormal after exercise challenge. Baseline levels of sPLA2-X in sputum and the absolute change in amount of sPLA2-X with exercise were positively correlated with EIB severity. Osmotic stress ex vivo resulted in movement of water and release of sPLA2-X to the apical surface. In summary, exercise challenge promotes changes in phospholipid structure and eicosanoid release in asthma, providing two mechanisms that promote bronchoconstriction, particularly in individuals with EIB who have higher basal levels of phospholipid turnover.

The mucopolysaccharidoses (MPSs) are a class of inborn errors of metabolism caused by deficiency of each of the enzymes involved in the lysosomal degradation of mucopolysaccharides. Newborn screening panels worldwide have been recently expanded to include one or more MPS disorders, as treatments are available and are most efficacious if initiated early in life. Here we report the first multiplex assay of 10 enzymatic activities in dried blood spots and fibroblast lysates that allow newborn screening and diagnosis of all MPS disorders except the ultrarare MPS-IX. The assay consists of incubation of enzyme-specific substrates with dried blood spot punches or fibroblast lysate followed by quantification of enzymatic products using liquid chromatography-tandem mass spectrometry (LC-MS/MS) together with internal standards. Assay of all MPS enzymes using fluorimetric or other methods has not been possible. The steps of the LC-MS/MS assay are sufficiently simple and rapid to be used in newborn screening and diagnostic laboratories. Assays showed acceptable precision, and enzymatic activities measured in confirmed MPS samples are well below the reference range.

All newborn screening (NBS) for mucopolysaccharidosis-I (MPS-I) is carried out by the measurement of &alpha, iduronidase (IDUA) enzymatic activity in dried blood spots (DBS). The majority of low enzyme results are due to pseudodeficiencies, and studies from the Mayo Clinic have shown that the false positive rate can be greatly reduced by including a second-tier analysis of glycosaminoglycans (GAGs) in DBS as part of NBS. In the present study, we obtained newborn DBS from 13 patients with severe MPS-I and 2 with attenuated phenotypes. These samples were submitted to four different GAG mass spectrometry analyses in a comparative study: (1) internal disaccharide, (2) endogenous disaccharide, (3) Sensi-Pro, (4) Sensi-Pro Lite (a variation of Sensi-Pro with a simplified workflow). Patients with attenuated MPS-I show less GAG elevation than those with severe disease, and all MPS-I patients were separated from the reference range using all four methods. The minimal differential factor (lowest GAG marker level in MPS-I samples divided by highest level in the reference range of 30 random newborns) was about two for internal disaccharide, Sensi-Pro, and Sensi-Pro Lite methods. The endogenous disaccharide was clearly the best method with a minimal differential of 16-fold. This study supports use of second-tier GAG analysis of newborn DBS, especially the endogenous disaccharide method, as part of NBS to reduce the false positive rate.

Background The necessity of early treatment for lysosomal storage diseases (LSDs) has triggered the development of newborn screening for LSDs in recent years. Here we report the first 70,000 newborns screened for Mucopolysaccharidosis (MPS) type 4A (Morquio syndrome) and other LSDs by an 8-plex assay including the original 4-plex LSD screening tandem mass spectrometry (MS/MS) assay for Pompe disease, Fabry disease, Gaucher disease, and MPS I disease. Methods The additional reaction for MPS II, MPS 3B, MPS 4A, and MPS 6 enzymes was performed separately from the 4-plex reaction. The two reactions were quenched and extracted, then combined before carrying out a single 2-min UPLC-MS/MS analysis. Results From Mar. 2018 to Apr. 2019, 73,743 newborns were screened with the 8-plex LSD screening assay. The 8-plex assay revealed a better analytical precision than the previous 4-plex assay possibly because the 8-plex was carried out using UPLC-MS/MS. Six newborns were found to have low MPS-4A enzyme (N-acetylgalactosamine-6-sulfatase) activity and biallelic GALNS pathogenic mutations in trans; these patients are presumably affected with MPS4A, making an incidence of one in 12,291 (95% confident interval (CI): 5633-26,817). One mutation, c.857C > T (p.T286 M) of the GALNS gene, accounted 5 of the 12 mutated alleles. These newborns had immature vertebral bodies at 1 month of age, and one case was treated with elosulfase alfa 2 mg/kg/week starting from 4 months of age. Among other MPSs screened, one case of MPS I, 3 cases of MPS II, and 3 cases of MPS 3B were detected. One case of mucolipidosis type III was also diagnosed. In conjunction with another 9 patients of Pompe disease, Gaucher disease, and classical Fabry disease, making an incidence of LSDs as one in 3206 newborns (95% CI: 2137 - 4811). The one with infantile-onset Pompe disease and the one with Gaucher disease were treated since the age of 8 days and 41 days respectively. Conclusions Routine newborn screening of MPS 4A and other LSDs were made possible by the 8-plex LSD screening assay. However, detailed phenotype prediction and the time to start treatment will need further elucidation.

Lipoprotein(a), Lp(a), is a modified low-density lipoprotein particle that contains apolipoprotein(a), encoded by LPA, and is a highly heritable, causal risk factor for cardiovascular diseases that varies in concentrations across ancestries. Here, we use deep-coverage whole genome sequencing in 8392 individuals of European and African ancestry to discover and interpret both single-nucleotide variants and copy number (CN) variation associated with Lp(a). We observe that genetic determinants between Europeans and Africans have several unique determinants. The common variant rs12740374 associated with Lp(a) cholesterol is an eQTL for SORT1 and independent of LDL cholesterol. Observed associations of aggregates of rare non-coding variants are largely explained by LPA structural variation, namely the LPA kringle IV 2 (KIV2)-CN. Finally, we find that LPA risk genotypes confer greater relative risk for incident atherosclerotic cardiovascular diseases compared to directly measured Lp(a), and are significantly associated with measures of subclinical atherosclerosis in African Americans., Circulating lipoprotein(a) is an important risk factor for cardiovascular disease and shows variability between different ethnic groups. Here, Zekavat et al. perform whole-genome sequencing in individuals of European and African ancestries and find ancestry-specific genetic determinants for lipoprotein(a) levels.

Protic ionic liquids (PILs) form through proton transfer from a Brønsted acid to a Brønsted base. In this work we use molecular dynamics simulation to study how PIL properties vary with χ, the extent of the proton transfer reaction. Three PILs are considered: N-propylammonium acetate, [N3][Ac], N-butylammonium acetate, [N4][Ac], and N, N-dimethylbutylammonium acetate, [N114][Ac]. In all cases density and viscosity increase with increasing χ, while diffusivities of all species decrease with increasing χ. In each PIL the ionic conductivity exhibits a maximum at intermediate χ due to competition between increasing ion concentration and decreasing ion mobility. Ionicity analysis suggests that strongly correlated behavior is present at all χ. Finally, we determine the χ for which the properties of each simulated PIL best agree with experimental data; these are χ = 0.86, 0.80, and 0.18 for [N3][Ac], [N4][Ac], and [N114][Ac], respectively. These results suggest that proton transfer is nearly complete in the primary ammonium PILs but not in the tertiary ammonium PIL, consistent with recent experimental observations. We propose that this difference is due to cooperative production of hydrogen bonds with increasing χ in the primary ammonium PILs, which does not occur in the tertiary ammonium PIL.

The time of origin of Earth’s dynamo is unknown. Detrital zircon crystals containing ferromagnetic inclusions from the Jack Hills of Western Australia have the potential to contain the oldest records of the geodynamo. It has recently been argued that magnetization in these zircons indicates that an active dynamo existed as far back as 4.2 Ga. However, the ages of ferromagnetic inclusions in the zircons are unknown. Here we present the first detailed characterization of the mineralogy and spatial distribution of ferromagnetic minerals in Jack Hills detrital zircons. We demonstrate that ferromagnetic minerals in most Jack Hills zircons are commonly located in cracks and on the zircons’ exteriors. Hematite is observed to dominate the magnetization of many zircons, while other zircons also contain significant quantities of magnetite and goethite. This indicates that the magnetization of most zircons is likely to be dominantly carried by secondary minerals that could be hundreds of millions to billions of years younger than the zircons’ crystallization ages. We conclude that the existence of the geodynamo prior to 3.5 Ga has yet to be established.

BACKGROUND Deficiency of the lysosomal enzyme galactosylcerebrosidase (GALC) causes Krabbe disease. Newborn screening for Krabbe disease is ongoing, but improved methods for follow-up analysis of screen-positive babies are needed to better advise families and to optimize treatment. We report a new assay for the enzymatic activity of GALC in lymphocytes. METHODS T lymphocytes were isolated from venous blood by magnetic bead technology. The assay used a close structural analog of the natural substrate and LC-MS/MS to quantify the amount of product with the aid of a chemically identical internal standard. RESULTS The analytical range of the assay (ratio of assay response for the QC high standard to that from all non–enzymatic-dependent processes) was 20-fold greater than that for the conventional radiometric GALC assay. The LC-MS/MS could distinguish cells that were null in GALC from those that contained traces of active enzyme (down to 0.3% of normal). There was a good correlation between the level of residual GALC activity in lymphocytes and the severity of Krabbe disease. CONCLUSIONS The new assay can measure small amounts of residual GALC activity in leukocytes with high accuracy compared to previous assays and can contribute, along with genotyping, biomarker analysis, and neurological imaging, a better plan for post–newborn screening follow-up for Krabbe disease.

A high throughput screening and subsequent hit validation identified compound 1 as an inhibitor of Trypanosoma brucei parasite growth. Extensive structure–activity relationship optimization based on antiparasitic activity led to the highly potent compounds, 1-(4-fluorobenzyl)-3-(4-dimethylamino-3-chlorophenyl)-2-thiohydantoin (68) and 1-(2-chloro-4-fluorobenzyl)-3-(4-dimethylamino-3-methoxyphenyl)-2-thiohydantoin (76), with a T. brucei EC50 of 3 and 2 nM, respectively. This represents >100-fold improvement in potency compared to compound 1. In vivo efficacy experiments of 68 and 76 in an acute mouse model of Human African Trypanosomiasis showed a 100% cure rate after 4 days of oral treatment at 50 mg/kg twice per day.

BACKGROUND We expanded the use of tandem mass spectrometry combined with liquid chromatography (LC-MS/MS) for multiplex newborn screening of seven lysosomal enzymes in dried blood spots (DBS). The new assays are for enzymes responsible for the mucopolysaccharidoses (MPS-I, -II, -IIIB, -IVA, -VI, and -VII) and type 2 neuronal ceroid lipofuscinosis (LINCL). METHODS New substrates were prepared and characterized for tripeptidyl peptidase 1 (TPP1), α-N-acetylglucosaminidase (NAGLU), and lysosomal β-glucuronidase (GUSB). These assays were combined with previously developed assays to provide a multiplex LC-MS/MS assay of 7 lysosomal storage diseases. Multiple reaction monitoring of ion dissociations for enzyme products and deuterium-labeled internal standards was used to quantify the enzyme activities. RESULTS Deidentified DBS samples from 62 nonaffected newborns were analyzed to simultaneously determine (run time 2 min per DBS) the activities of TPP1, NAGLU, and GUSB, along with those for α-iduronidase (IDUA), iduronate-2-sulfatase (I2S), N-acetylgalactosamine-6-sulfatase (GALNS), and N-acetylgalactosamine-4-sulfatase (ARSB). The activities measured in the 7-plex format showed assay response-to-blank-activity ratios (analytical ranges) of 102–909 that clearly separated healthy infants from affected children. CONCLUSIONS The new multiplex assay provides a robust comprehensive newborn screening assay for the mucopolysaccharidoses. The method has been expanded to include additional lysosomal storage diseases.

Objective Expression of Spam1/PH20 and its modulation of high/low molecular weight hyaluronan substrate have been proposed to play an important role in murine oligodendrocyte precursor cell (OPC) maturation in vitro and in normal and demyelinated central nervous system (CNS). We reexamined this using highly purified PH20. Methods Steady‐state expression of mRNA in OPCs was evaluated by quantitative polymerase chain reaction; the role of PH20 in bovine testicular hyaluronidase (BTH) inhibition of OPC differentiation was explored by comparing BTH to a purified recombinant human PH20 (rHuPH20). Contaminants in commercial BTH were identified and their impact on OPC differentiation characterized. Spam1/PH20 expression in normal and demyelinated mouse CNS tissue was investigated using deep RNA sequencing and immunohistological methods with two antibodies directed against recombinant murine PH20. Results BTH, but not rHuPH20, inhibited OPC differentiation in vitro. Basic fibroblast growth factor (bFGF) was identified as a significant contaminant in BTH, and bFGF immunodepletion reversed the inhibitory effects of BTH on OPC differentiation. Spam1 mRNA was undetected in OPCs in vitro and in vivo; PH20 immunolabeling was undetected in normal and demyelinated CNS. Interpretation We were unable to detect Spam1/PH20 expression in OPCs or in normal or demyelinated CNS using the most sensitive methods currently available. Further, “BTH” effects on OPC differentiation are not due to PH20, but may be attributable to contaminating bFGF. Our data suggest that caution be exercised when using some commercially available hyaluronidases, and reports of Spam1/PH20 morphogenic activity in the CNS may be due to contaminants in reagents.

© 2020 American Chemical Society. Microbially-mediated methylation of arsenic (As) plays an important role in the As biogeochemical cycle, particularly in rice paddy soils where methylated As, generated microbially, is translocated into rice grains. The presence of the arsenite (As(III)) methyltransferase gene (arsM) in soil microbes has been used as an indication of their capacity for As methylation. Here, we evaluate the ability of seven microorganisms encoding active ArsM enzymes to methylate As. Amongst those, only the aerobic species were efficient methylators. The anaerobic microorganisms presented high resistance to As exposure, presumably through their efficient As(III) efflux, but methylated As poorly. The only exception were methanogens, for which efficient As methylation was seemingly an artifact of membrane disruption. Deletion of an efflux pump gene (acr3) in one of the anaerobes, Clostridium pasteurianum, rendered the strain sensitive to As and capable of more efficiently methylating As. Our results led to the following conclusions: (i) encoding a functional ArsM enzyme does not guarantee that a microorganism will actively drive As methylation in the presence of the metalloid and (ii) there is an inverse relationship between efficient microbial As efflux and its methylation, because the former prevents the intracellular accumulation of As.

Ion mobility spectrometry (IMS) is of significant interest as a platform for glycoanalysis. While much attention has been focused on the resolution of isomeric carbohydrates and glycoconjugates, another appealing aspect of IMS is the ability to sort different classes of biomolecules into distinct regions of mass vs. mobility space. This capability has potential to greatly simplify glycoproteomic analyses, as glycosylated and non-glycosylated peptides can be rapidly partitioned in the gas phase. Nevertheless, the physical and chemical characteristics of glycopeptides that dictate their mass vs. mobility loci have yet to be systematically investigated. This report presents an IMS study of model protonated glycopeptide ions with systematically varied oligosaccharide topologies, polypeptide sequences, and charge states. In all, over 110 ion-neutral collision cross sections (CCSs) were measured and analyzed in the context of the physicochemical characteristics of the analytes. Glycan size and composition emerged as a decisive factor in dictating the CCS space occupied by the glycopeptides and exerted this influence in a charge state dependent fashion. Furthermore, elongation of the glycan group was found to either increase or decrease glycopeptide CCSs depending on the ion charge state and the size of the glycan. Molecular dynamics (MD) simulations of the gas phase structures and CCSs of selected glycopeptides revealed that the experimental observations were consistent with a glycan size and charge state dependent interplay between destabilizing coulombic repulsion effects (tending to result in more extended structures) and stabilizing charge solvation effects in which the glycan plays a major role (tending to result in more compact structures). Taken together, these IMS and MD findings suggest the possibility of predicting and delineating glycopeptide-enriched regions of mass vs. mobility space for applications in glycoproteomics.

The protozoan parasite Entamoeba histolytica can induce amebic colitis and amebic liver abscess. First-line drugs for the treatment of amebiasis are nitroimidazoles, particularly metronidazole. Metronidazole has side effects and potential drug resistance is a concern. Schistosomiasis, a chronic and painful infection, is caused by various species of the Schistosoma flatworm. There is only one partially effective drug, praziquantel, a worrisome situation should drug resistance emerge. As many essential metabolic pathways and enzymes are shared between eukaryotic organisms, it is possible to conceive of small molecule interventions that target more than one organism or target, particularly when chemical matter is already available. Farnesyltransferase (FT), the last common enzyme for products derived from the mevalonate pathway, is vital for diverse functions, including cell differentiation and growth. Both E. histolytica and Schistosoma mansoni genomes encode FT genes. In this study, we phenotypically screened E. histolytica and S. mansoni in vitro with the established FT inhibitors, lonafarnib and tipifarnib, and with 125 tipifarnib analogs previously screened against both the whole organism and/or the FT of Trypanosoma brucei and Trypanosoma cruzi. For E. histolytica, we also explored whether synergy arises by combining lonafarnib and metronidazole or lonafarnib with statins that modulate protein prenylation. We demonstrate the anti-amebic and anti-schistosomal activities of lonafarnib and tipifarnib, and identify 17 tipifarnib analogs with more than 75% growth inhibition at 50 μM against E. histolytica. Apart from five analogs of tipifarnib exhibiting activity against both E. histolytica and S. mansoni, 10 additional analogs demonstrated anti-schistosomal activity (severe degenerative changes at 10 μM after 24 h). Analysis of the structure-activity relationship available for the T. brucei FT suggests that FT may not be the relevant target in E. histolytica and S. mansoni. For E. histolytica, combination of metronidazole and lonafarnib resulted in synergism for growth inhibition. Also, of a number of statins tested, simvastatin exhibited moderate anti-amebic activity which, when combined with lonafarnib, resulted in slight synergism. Even in the absence of a definitive molecular target, identification of potent anti-parasitic tipifarnib analogs encourages further exploration while the synergistic combination of metronidazole and lonafarnib offers a promising treatment strategy for amebiasis.

Introduction: BT8009 consists of a bicyclic peptide targeting the tumor antigen Nectin-4, linked to the cytotoxin monomethyl auristatin E (MMAE) via a molecular spacer and cleavable linker. BT8009 is currently being investigated in a Phase 1/2 clinical trial (BT8009-100, NCT04561362) in relapsed and/or refractory solid tumor patients. Nectin-4 positive tumor tissue will be a requirement for enrolment eligibility (with the exception of bladder cancer) starting in dose escalation level 3. Nectin-4 positivity will be determined by IHC on tumor tissue, requiring either tumor membrane or tumor cytoplasmic H-score ≥ 100. Methods: Nectin-4 is not included on commercial NGS panels or routinely screened by IHC. Therefore, the work reported here aimed to identify a routinely measured molecular surrogate of tumor Nectin-4 protein expression (i.e. somatic mutation or gene amplification) that could potentially increase patient screening frequency, yield, and likelihood of response to BT8009. Here we demonstrate that Nectin-4 copy number is associated with Nectin-4 mRNA expression in 9 cancer indications in TCGA including breast, bladder, and lung cancer. In addition, across >30 cancer indications in TCGA, copy number of Nectin-4 is highly correlated with SDHC copy number (these two genes are ~225 kb apart on 1q23), which is included on multiple commercially available NGS panels. Taken together, these results suggest that the existence of an SDHC amplification could be used as an enrichment tool to identify patients with Nectin-4 positive tumors. To test this hypothesis, we obtained 100 TNBC human tumor samples and generated data to determine Nectin-4 and SDHC copy number as well as Nectin-4 protein expression status by IHC. Results: SDHC and Nectin-4 copy number were observed to be highly positively correlated (Spearman's Rho=0.93). Furthermore, all (N = 22) TNBC tumors with a Nectin-4 copy number ≥ 3 were identified as positive for Nectin-4 protein expression and were at or above the H-score threshold required for enrolment to BT8009-100. In contrast, within the subset of tumors that were diploid for Nectin-4/SDHC (n=30), there were both Nectin-4 positive and negative tumors. This suggests that the presence of SDHC/Nectin-4 amplification can be used to increase the probability of identifying Nectin-4 expressing tumors. Conclusions: We demonstrate the potential utility of using pre-existing patient NGS data with SDHC amplifications to increase the frequency of identifying patients with high Nectin-4 expressing tumors via IHC. The use of this strategy is ongoing at all current sites enrolling patients to BT8009-100. Citation Format: Tara Gelb, Sean Santos, Amy Dickson, Carly Campbell, Mike Rigby, Andrew J. Mckenzie, Dan Rozelle, Stephen J. Blakemore. Molecular-based enrichment strategy for Nectin-4 targeted Bicycle toxin conjugate BT8009 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 391.

Introduction: Toxin-conjugated agents targeting cell-surface antigens (e.g. antibody-drug conjugates) show promising clinical activity for some solid tumors, but none yet for OS. There is an urgent need to identify high-confidence OS cell-surface antigens and evaluate the antitumor activity of toxin conjugates that can deliver cytotoxic payloads to OS tumors expressing these targets. Methods: To identify cell-surface proteins that are highly expressed in OS but not in normal tissues, we prepared surface protein extracts from 4 standard, 4 patient-derived OS cell lines, and 10 OS PDX tissues. Quantitative mass spectrometry (MS) was used to profile the surface proteins. We included the proteomics data of OS and 39 types of normal human tissues via a public MS database (ProteomicsDB). We compared RNA-sequencing data from 101 OS patient samples via the NCI TARGET project with the RNA-sequencing data of 54 types of normal human tissue (GTEx, 17382 samples). We integrated the MS and RNA sequencing datasets to prioritize candidate surface targets by differential and absolute expression at both protein and mRNA levels. The cell-surface expression of the targets was further validated by IHC and flow cytometry with OS cell lines and tumor tissue microarray. BT1769, which consists of a bicyclic peptide targeting the tumor antigen MT1-MMP, linked to the cytotoxin monomethyl auristatin E (MMAE) via a molecular spacer and cleavable linker was tested in 6 OS PDX models using standard PPTC methods at 3 mg/kg given IV weekly x 4. Ewing sarcoma (EWS) models lacking MT1-MMP expression were also tested. Results: MS identified 1156 surface proteins as highly expressed in OS cell lines/PDX tissues. 129 proteins overexpressed in OS were identified from the public MS database. RNA sequencing data identified 195 overexpressed plasma membrane-associated genes in OS compared to normal tissues. The targets identified by both proteomics and transcriptomic data analysis were cross-referenced with existing toxin-conjugated agents. Three potential targets, MT1-MMP, CD276, and MRC2 were validated and MT1-MMP was prioritized because it had the highest absolute expression level in OS. BT1769 significantly inhibited tumor growth in 6/6 OS PDX models when compared to vehicle, with complete response maintained at 6 weeks in 3/6 models. Minimal activity was observed in 2/2 EWS models. Correlation analysis between the MT1-MMP gene expression level, IHC staining intensity and drug response are underway and will be presented. Conclusion: Integrated proteomic and transcriptomic OS surfaceome profiling identified high-confidence OS cell-surface antigens as therapeutic targets. MT1-MMP was highly expressed in most OS patient samples and PDX models, and BT1769 was highly active against select OS PDX models. Citation Format: Yifei Wang, Wendong Zhang, Zhongting Zhang, Xiangjun Tian, Rossana N. Lazcano Segura, Pooja Hingorani, Michael Roth, Jonathan Gill, Douglas Harrison, Zhaohui Xu, Jing Wang, Eric J. Earley, Stephen W. Erickson, Edward A. Kolb, Malcolm A. Smith, Peter Houghton, Raushan Kurmasheva, Tara Gelb, Philip Huxley, Johanna Lahdenranta, Gemma Mudd, Richard Gorlick. Integrative surfaceome profiling identifies immunotherapeutic targets in osteosarcomas (OS); Preclinical testing of BT1769, an MT1-MMP-targeted Bicycle Toxin Conjugate, in OS by the Pediatric Preclinical Testing Consortium (PPTC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB008.

Introduction: Bicycles® are a novel class of fully synthetic, short, constrained peptides. BT8009 consists of a bicyclic peptide targeting the tumor antigen Nectin-4, linked to the cytotoxin monomethyl auristatin E (MMAE) via a molecular spacer and cleavable linker. Nectin-4 is a validated tumor target and is highly expressed in a wide range of tumors. A Phase I/II dose escalation/expansion study, BT8009-100 (NCT04561362), enrolling patients with advanced solid tumors associated with Nectin-4 expression is ongoing in the US. Here we report the analysis of tumor tissue microarrays (TMAs) stained with a Nectin-4 immunohistochemistry (IHC) assay to support patient selection for the BT8009-100 study. Methods: We developed a clinical grade Nectin-4 IHC assay on the Leica platform using a proprietary rabbit monoclonal α-Nectin-4 primary antibody (Abcam, Burlingame, CA) and the BOND Polymer Refine detection kit. TMAs of cancer types reported to have high Nectin-4 expression, including esophageal, pancreatic, bladder, head & neck, stomach, non-small cell lung, breast, and ovarian cancer, were stained and manually scored for Nectin-4 levels, with the tumor microenvironment evaluated in a subset of samples. Nectin-4 H-scores (stain intensity on a scale of 0-3 x percent positive tumor cells) were generated by a pathologist independently for tumor cell membrane and tumor cytoplasm. H-scores ≥ 100 in the tumor membrane or cytoplasm were considered positive. Results: In all indications tested, a greater degree of Nectin-4 positivity was observed in the tumor cytoplasm as compared to tumor membrane. Breast and bladder cancer had the greatest frequency of Nectin-4 positivity. Subtype analysis in breast cancer identified enrichment of Nectin-4 expression in hormone receptor negative and human epidermal growth factor receptor 2 positive tumors. Conclusion: The frequency of Nectin-4 expression as measured by IHC across multiple tumor types may guide clinical strategy for the BT8009 program. TableIndicationTotal cores (n)Percent Tumor Membrane PositivePercent Tumor Cytoplasm PositivePercent Tumor Membrane or Cytoplasm PositiveBreast225164957Bladder142274256Esophagus14073032Head & Neck69102532Lung15731717Ovarian8921315Pancreas96123Stomach131022 Citation Format: Carly T. Campbell, Robert Smale, Michael Rigby, Shawn Watson, Amy Dickson, Tara Gelb, Heather Cohen, Pierre Lee, Josette William Ragheb, Stephen J. Blakemore. A multi tumor survey of Nectin-4 expression to guide BT8009 indication selection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1197.

Instability to storage and shipping conditions and injection administration remain major challenges in treating chronic conditions with biopharmaceuticals. Herein, formulations of poly(trehalose methacrylate) (pTrMA) were successfully optimized to stabilize insulin without appreciably increasing viscosity. Polymers were synthesized (2,400 – 29,200 Da), and added to insulin at different concentrations. pTrMA maintained >95% intact insulin against 250 rpm at 37 °C for 3 hours with at least 10 mol. eq. of 5.0 kDa, 7.5 mol. eq. of 9.4 kDa, 5 mol. eq. of 12.8 kDa, 1 mol. eq. of 19.8 kDa, and 0.5 mol. eq. of 29.2 kDa polymers, compared to 13.1% of insulin alone. The lowest pTrMA concentration formulations were more viscous than insulin alone, but the highest viscosity, U-600 with 10 mol. eq. of 5 kDa pTrMA, was only 1.43 cP at 25 °C. This data demonstrates that pTrMA is a promising low viscosity additive to stabilize the diabetes therapeutic insulin.