CAR-engineered lymphocyte persistence is governed by a FAS ligand/FAS auto-regulatory circuit.

Chimeric antigen receptor (CAR)-engineered T and NK cells can cause durable remission of B-cell malignancies; however, limited persistence restrains the full potential of these therapies in many patients. The FAS ligand (FAS-L)/FAS pathway governs naturally-occurring lymphocyte homeostasis, yet knowledge of which cells express FAS-L in patients and whether these sources compromise CAR persistence remains incomplete. Here, we constructed a single-cell atlas of diverse cancer types to identify cellular subsets expressing FASLG, the gene encoding FAS-L. We discovered that FASLG is limited primarily to endogenous T cells, NK cells, and CAR-T cells while tumor and stromal cells express minimal FASLG. To establish whether CAR-T/NK cell survival is regulated through FAS-L, we performed competitive fitness assays using lymphocytes modified with or without a FAS dominant negative receptor (ΔFAS). Following adoptive transfer, ΔFAS-expressing CAR-T and CAR-NK cells became enriched across multiple tissues, a phenomenon that mechanistically was reverted through FASLG knockout. By contrast, FASLG was dispensable for CAR-mediated tumor killing. In multiple models, ΔFAS co-expression by CAR-T and CAR-NK enhanced antitumor efficacy compared with CAR cells alone. Together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS-L/FAS auto-regulatory circuit.

Early but not late convalescent plasma is associated with better survival in moderate-to-severe COVID-19.

BACKGROUND: Limited therapeutic options exist for coronavirus disease 2019 (COVID-19). COVID-19 convalescent plasma (CCP) is a potential therapeutic, but there is limited data for patients with moderate-to-severe disease. RESEARCH QUESTION: What are outcomes associated with administration of CCP in patients with moderate-to-severe COVID-19 infection? STUDY DESIGN AND METHODS: We conducted a propensity score-matched analysis of patients with moderate-to-severe COVID-19. The primary endpoints were in-hospital mortality. Secondary endpoints were number of days alive and ventilator-free at 30 days; length of hospital stay; and change in WHO scores from CCP administration (or index date) to discharge. Of 151 patients who received CCP, 132 had complete follow-up data. Patients were transfused after a median of 6 hospital days; thus, we investigated the effect of convalescent plasma before and after this timepoint with 77 early (within 6 days) and 55 late (after 6 days) recipients. Among 3,217 inpatients who did not receive CCP, 2,551 were available for matching. RESULTS: Early CCP recipients, of whom 31 (40%) were on mechanical ventilation, had lower 14-day (15% vs 23%) and 30-day (38% vs 49%) mortality compared to a matched unexposed cohort, with nearly 50% lower likelihood of in-hospital mortality (HR 0.52, [95% CI 0.28-0.96]; P = 0.036). Early plasma recipients had more days alive and ventilator-free at 30 days (+3.3 days, [95% CI 0.2 to 6.3 days]; P = 0.04) and improved WHO scores at 7 days (-0.8, [95% CI: -1.2 to -0.4]; P = 0.0003) and hospital discharge (-0.9, [95% CI: -1.5 to -0.3]; P = 0.004) compared to the matched unexposed cohort. No clinical differences were observed in late plasma recipients. INTERPRETATION: Early administration of CCP improves outcomes in patients with moderate-to-severe COVID-19, while improvement was not observed with late CCP administration. The importance of timing of administration should be addressed in specifically designed trials.

Assessment of semi-natural habitats and landscape features on Irish farmland: New insights to inform EU Common Agricultural Policy implementation.

Concerns over the loss of biodiversity and ecosystem services in farmland have prompted the development of agri-environment policy measures aimed at reducing farming pressure and maintaining semi-natural habitats in farmed landscapes. However, further knowledge is needed to guarantee successful agri-environment measures implementation. The current study assessed the quantity and the quality of semi-natural habitats in farms across a gradient of farming intensities in two contrasting regions in Ireland. Policy protection seemed fundamental for semi-natural habitats preservation. Habitats not protected by agricultural policy relied on extensive farming and are in danger of disappearing if they are intensified or abandoned. Due to the lack of policy incentives for habitat quality, no correlations were found between farming intensity and share of semi-natural habitats with habitat quality. Therefore, extensive farming and retention of habitats alone may not reverse the decline of farmland quality and biodiverisity and, thus, measures incentivising the environmental quality may be more successful.

Catching Flies With Honey(dew): Adult Marsh Flies (Diptera: Sciomyzidae) Utilize Sugary Secretions for High-Carbohydrate Diets.

Marsh flies are a diverse family that provide valuable ecosystem services, including the biological control of mollusks that are agricultural pests and vectors of animal and human parasitic diseases. In addition, some species may serve as important ecological bioindicators. Despite the extensive research on this family, most have centered on larval diet and behavior, as this is the life stage primarily used for biological control; virtually nothing is known about the natural dietary components of adult marsh flies. Our study aimed to close this knowledge gap by examining the dietary range and preference of adult marsh fly species. Individual flies were provided with five food choices in cafeteria-style food choice trials, consisting of crushed snail, freshly killed slug, glucose solution, honey-yeast mixture (the standard laboratory rearing diet), or water. Sciomyzidae at family level displayed significant differences in food selection (P = 0.0212), with carbohydrates (honey-yeast and glucose solution) significantly preferred over protein options (mollusk tissue) or the water control (P < 0.001). This suggests that marsh flies may naturally maintain a carbohydrate-rich diet. Because many plants typical at field sites produce little or no nectar, a second experiment aimed to determine the source of these carbohydrates in nature. When presented with honeydew harvested from aphids (Hemiptera: Auchenorrhycha), Tetanocera elata Fabricius (Diptera: Sciomyzidae) individuals were observed to feed on dry honeydew and honeydew solution significantly more frequently than the water control (P < 0.001 and P = 0.01969, respectively), suggesting that honeydew may play an important role in adult marsh fly diet.

Matrix Screen Identifies Synergistic Combination of PARP Inhibitors and Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors in Ewing Sarcoma.

Purpose: Although many cancers are showing remarkable responses to targeted therapies, pediatric sarcomas, including Ewing sarcoma, remain recalcitrant. To broaden the therapeutic landscape, we explored the in vitro response of Ewing sarcoma cell lines against a large collection of investigational and approved drugs to identify candidate combinations.Experimental Design: Drugs displaying activity as single agents were evaluated in combinatorial (matrix) format to identify highly active, synergistic drug combinations, and combinations were subsequently validated in multiple cell lines using various agents from each class. Comprehensive metabolomic and proteomic profiling was performed to better understand the mechanism underlying the synergy. Xenograft experiments were performed to determine efficacy and in vivo mechanism.Results: Several promising candidates emerged, including the combination of small-molecule PARP and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, a rational combination as NAMPTis block the rate-limiting enzyme in the production of nicotinamide adenine dinucleotide (NAD+), a necessary substrate of PARP. Mechanistic drivers of the synergistic cell killing phenotype of these combined drugs included depletion of NMN and NAD+, diminished PAR activity, increased DNA damage, and apoptosis. Combination PARPis and NAMPTis in vivo resulted in tumor regression, delayed disease progression, and increased survival.Conclusions: These studies highlight the potential of these drugs as a possible therapeutic option in treating patients with Ewing sarcoma. Clin Cancer Res; 23(23); 7301-11. ©2017 AACR.

Quantifying phenotype-environment matching in the protected Kerry spotted slug (Mollusca: Gastropoda) using digital photography: exposure to UV radiation determines cryptic colour morphs.

BACKGROUND: Animal colours and patterns commonly play a role in reducing detection by predators, social signalling or increasing survival in response to some other environmental pressure. Different colour morphs can evolve within populations exposed to different levels of predation or environmental stress and in some cases can arise within the lifetime of an individual as the result of phenotypic plasticity. Skin pigmentation is variable for many terrestrial slugs (Mollusca: Gastropoda), both between and within species. The Kerry spotted slug Geomalacus maculosus Allman, an EU protected species, exhibits two distinct phenotypes: brown individuals occur in forested habitats whereas black animals live in open habitats such as blanket bog. Both colour forms are spotted and each type strongly resembles the substrate of their habitat, suggesting that G. maculosus possesses camouflage. RESULTS: Analysis of digital images of wild slugs demonstrated that each colour morph is strongly and positively correlated with the colour properties of the background in each habitat but not with the substrate of the alternative habitats, suggesting habitat-specific crypsis. Experiments were undertaken on laboratory-reared juvenile slugs to investigate whether ultraviolet (UV) radiation or diet could induce colour change. Exposure to UV radiation induced the black (bog) phenotype whereas slugs reared in darkness did not change colour. Diet had no effect on juvenile colouration. Examination of skin tissue from specimens exposed to either UV or dark treatments demonstrated that UV-exposed slugs had significantly higher concentrations of black pigment in their epithelium. CONCLUSIONS: These results suggest that colour dimorphism in G. maculosus is an example of phenotypic plasticity which is explained by differential exposure to UV radiation. Each resulting colour morph provides incidental camouflage against the different coloured substrate of each habitat. This, to our knowledge, is the first documented example of colour change in response to UV radiation in a terrestrial mollusc. Pigmentation appears to be correlated with a number of behavioural traits in G. maculosus, and we suggest that understanding melanisation in other terrestrial molluscs may be useful in the study of pestiferous and invasive species. The implications of colour change for G. maculosus conservation are also discussed.

Selective inhibitors of trypanosomal uridylyl transferase RET1 establish druggability of RNA post-transcriptional modifications.

Non-coding RNAs are crucial regulators for a vast array of cellular processes and have been implicated in human disease. These biological processes represent a hitherto untapped resource in our fight against disease. In this work we identify small molecule inhibitors of a non-coding RNA uridylylation pathway. The TUTase family of enzymes is important for modulating non-coding RNA pathways in both human cancer and pathogen systems. We demonstrate that this new class of drug target can be accessed with traditional drug discovery techniques. Using the Trypanosoma brucei TUTase, RET1, we identify TUTase inhibitors and lay the groundwork for the use of this new target class as a therapeutic opportunity for the under-served disease area of African Trypanosomiasis. In a broader sense this work demonstrates the therapeutic potential for targeting RNA post-transcriptional modifications with small molecules in human disease.

Small molecule inhibition of FOXM1: How to bring a novel compound into genomic context.

Deregulation of transcription factor (TF) networks is emerging as a major pathogenic event in many human cancers (Darnell, 2002 [1]; Libermann and Zerbini, 2006 [2]; Laoukili et al., 2007 [3]). Small molecule intervention is an attractive avenue to understand TF regulatory mechanisms in healthy and disease state, as well as for exploiting these targets therapeutically (Koehler et al., 2003 [4]; Berg, 2008 [5]; Koehler, 2010 [6]). However, because of their physico-chemical properties, TF targeting has been proven to be difficult (Verdine and Walensky, 2007 [7]). The TF FOXM1 is an important mitotic player (Wonsey and Follettie, 2005 [8]; Laoukili et al., 2005 [9]; McDonald, 2005 [10]) also implicated in cancer progression (Laoukili et al., 2007 [3]; Teh, 2011 [11]; Koo, 2012 [12]) and drug resistance development (Kwok et al., 2010 [13]; Carr et al., [14]). Therefore, its inhibition is an attractive goal for cancer therapy. Here, we describe a computational biology approach, by giving detailed insights into methodologies and technical results, which was used to analyze the transcriptional RNA-Seq data presented in our previous work (Gormally et al., 2014 [20]). Our Bioinformatics analysis shed light on the cellular effect of a novel FOXM1 inhibitor (FDI-6) newly identified through a biophysical screen. The data for this report is available at the public GEO repository (accession number http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58626).

Registered report: Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukemia.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from 'Inhibition of bromodomain and extra terminal (BET) recruitment to chromatin as an effective treatment for mixed-lineage leukemia (MLL)-fusion leukemia' by Dawson and colleagues, published in Nature in 2011 (Dawson et al., 2011). The experiments to be replicated are those reported in Figures 2A, 3D, 4B, 4D and Supplementary Figures 11A-B and 16A. In this study, BET proteins were demonstrated as potential therapeutic targets for modulating aberrant gene expression programs associated with MLL-fusion leukemia. In Figure 2A, the BET bromodomain inhibitor I-BET151 was reported to suppress growth of cells harboring MLL-fusions compared to those with alternate oncogenic drivers. In Figure 3D, treatment of MLL-fusion leukemia cells with I-BET151 resulted in transcriptional suppression of the anti-apoptotic gene BCL2. Figures 4B and 4D tested the therapeutic efficacy of I-BET151 in vivo using mice injected with human MLL-fusion leukemia cells and evaluated disease progression following I-BET151 treatment. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

FOXM1 binds directly to non-consensus sequences in the human genome.

BACKGROUND: The Forkhead (FKH) transcription factor FOXM1 is a key regulator of the cell cycle and is overexpressed in most types of cancer. FOXM1, similar to other FKH factors, binds to a canonical FKH motif in vitro. However, genome-wide mapping studies in different cell lines have shown a lack of enrichment of the FKH motif, suggesting an alternative mode of chromatin recruitment. We have investigated the role of direct versus indirect DNA binding in FOXM1 recruitment by performing ChIP-seq with wild-type and DNA binding deficient FOXM1. RESULTS: An in vitro fluorescence polarization assay identified point mutations in the DNA binding domain of FOXM1 that inhibit binding to a FKH consensus sequence. Cell lines expressing either wild-type or DNA binding deficient GFP-tagged FOXM1 were used for genome-wide mapping studies comparing the distribution of the DNA binding deficient protein to the wild-type. This shows that interaction of the FOXM1 DNA binding domain with target DNA is essential for recruitment. Moreover, analysis of the protein interactome of wild-type versus DNA binding deficient FOXM1 shows that the reduced recruitment is not due to inhibition of protein-protein interactions. CONCLUSIONS: A functional DNA binding domain is essential for FOXM1 chromatin recruitment. Even in FOXM1 mutants with almost complete loss of binding, the protein-protein interactions and pattern of phosphorylation are largely unaffected. These results strongly support a model whereby FOXM1 is specifically recruited to chromatin through co-factor interactions by binding directly to non-canonical DNA sequences.

Child car seats - a habitat for house dust mites and reservoir for harmful allergens.

INTRODUCTION AND OBJECTIVE: House dust mites produce allergens which can cause or aggravate diseases such as asthma, eczema and rhinitis. The objectives of this study are to quantify typical house dust mite and Der p 1 allergen levels in child car seats, and to determine external variables that may influence mite populations in cars. MATERIALS AND METHODS: Dust samples were collected from the child car seats and driver seats of 106 cars using a portable vacuum sampling pump over a two minute sampling period. Mites were counted and identified and results were expressed as mites per gram (mites/g) of dust, while Der p 1 content of samples were measured by enzyme-linked immunosorbent assay (ELISA). Questionnaires were completed by participants to identify environmental and behavioural effects on mite populations. Results were analysed using General Linear Model (GLM) procedures. RESULTS: Twelve species of mites, of which nine are known to produce harmful allergens, were recorded from 212 dust samples. Over 80% of drivers' seats and over 77% of child car seats harboured dust mites with a significant correlation (p = 0.001) between the mites/g of dust and Der p 1 content recovered from each seat. A mean of 53 mites/g of dust per seat was recovered, with a mean Der p 1 level of 1.1µg/g. Over 12% of driver seats and 15% of child car seats contained house dust mite levels sufficient to be risk factors for sensitisation and allergic reactions. CONCLUSIONS: Child car seats and driver seats are habitats to a range of mite species which can be present in sufficient concentrations to cause or aggravate allergen related illnesses in individuals who are genetically predisposed.

Dynamics of house dust mite transfer in modern clothing fabrics.

BACKGROUND: Clothing is largely presumed as being the mechanism by which house dust mites are distributed among locations in homes, yet little research to date has investigated the capacity with which various clothing fabric types serve as vectors for their accumulation and dispersal. Although previous research has indicated that car seats provide a habitat for mite populations, dynamics involved in the transfer of mites to clothing via car seat material is still unknown. OBJECTIVE: To investigate the dynamics involved in the transfer of house dust mites from car seat material to modern clothing fabrics. METHODS: A total of 480 samples of car seat material were seeded with mites and subjected to contact with plain woven cotton, denim, and fleece. Contact forces equivalent to the mass of a typical adult and child were administered for different durations of contact. RESULTS: Mean transfer efficiencies of mites from car seat material to receiving clothing fabrics ranged from 7.2% to 19.1%. Fabric type, mite condition (live or dead), and the force applied all revealed a significant effect (P < .001 for each variable) on the transfer efficiency of house dust mites from seeded material to receiving fabrics, whereas duration of contact revealed no effect (P = .20). In particular, mean numbers of mites transferred to fleece (compared with denim and plain woven cotton) were greater for each treatment. CONCLUSION: These findings indicate that clothing type can have important implications for the colonization of other biotopes by house dust mites, with potential for affecting an individuals' personal exposure to dust mite allergens.

Suppression of the FOXM1 transcriptional programme via novel small molecule inhibition.

The transcription factor FOXM1 binds to sequence-specific motifs on DNA (C/TAAACA) through its DNA-binding domain (DBD) and activates proliferation- and differentiation-associated genes. Aberrant overexpression of FOXM1 is a key feature in oncogenesis and progression of many human cancers. Here--from a high-throughput screen applied to a library of 54,211 small molecules--we identify novel small molecule inhibitors of FOXM1 that block DNA binding. One of the identified compounds, FDI-6 (NCGC00099374), is characterized in depth and is shown to bind directly to FOXM1 protein, to displace FOXM1 from genomic targets in MCF-7 breast cancer cells, and induce concomitant transcriptional downregulation. Global transcript profiling of MCF-7 cells by RNA-seq shows that FDI-6 specifically downregulates FOXM1-activated genes with FOXM1 occupancy confirmed by ChIP-PCR. This small molecule-mediated effect is selective for FOXM1-controlled genes with no effect on genes regulated by homologous forkhead family factors.

Assessing metal contamination from construction and demolition (C&D) waste used to infill wetlands: using Deroceras reticulatum (Mollusca: Gastropoda).

Large quantities of construction and demolition waste (C&D) are produced globally every year, with little known about potential environmental impacts. In the present study, the slug, Deroceras reticulatum (Mollusca: Gastropoda) was used as the first biomonitor of metals (Ag, As, Ba, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, Sb, Se, Ti, Tl, V and Zn) on wetlands post infilling with construction and demolition (C&D) waste. The bioaccumulation of As, Ba, Cd, Co, Sb, Se and Tl were found to be significantly elevated in slugs collected on C&D waste when compared to unimproved pastures (control sites), while Mo, Se and Sr had significantly higher concentrations in slugs collected on C&D waste when compared to known contaminated sites (mining locations), indicating the potential hazardous nature of C&D waste to biota. Identifying exact sources for these metals within the waste can be problematic, due to its heterogenic nature. Biomonitors are a useful tool for future monitoring and impact studies, facilitating policy makers and regulations in other countries regarding C&D waste infill. In addition, improving separation of C&D waste to allow increased reuse and recycling is likely to be effective in reducing the volume of waste being used as infill, subsequently decreasing potential metal contamination.

Light-mediated in cell downregulation of G-quadruplex-containing genes using a photo-caged ligand.

The use of a caged G-quadruplex ligand allows for transcriptional control of quadruplex-containing genes using UV light as an external trigger. An important oncogene, SRC, involved in the initiation and proliferation of epithelial tumours is shown to be significantly downregulated in cells treated by the caged ligand in synergy with UV light treatment.

Enhanced iron availability by protein glycation may explain higher infection rates in diabetics.

Serum proteins exist in a state of higher glycation among individuals with poor glycemic control, notably diabetics. These non-enzymatic modifications via the Maillard reaction have far reaching effects on metabolism and regulation, and may be responsible for increased infection rates within this population. Here we explore the effects of glycation on iron metabolism and innate immunity by investigating the interaction between siderophores and bovine serum albumin (BSA). Using a quartz crystal microbalance with dissipation monitoring to quantify association rates, glycated BSA exhibited a significantly reduced affinity for apo and holo enterobactin compared to a non-glycated BSA standard. Bacterial growth assays in the presence of BSA and under iron-limited conditions indicated the growth rate of enterobactin-producing E. coli increased significantly when the BSA was in a glycated form. The results, in addition to data in the literature, support the hypothesis that glycation of serum proteins may effectively increase the available free iron pool for bacteria in blood serum and weaken our innate immunity. This phenomenon may be partially responsible for higher infection rates in some diabetics, especially those with poor glycemic control.

Controlling tyrosinase activity on charged polyelectrolyte surfaces: a QCM-D analysis.

The quartz crystal microbalance (QCM) was used to monitor the immobilization of tyrosinase on polycationic and polyanionic precursor assemblies in situ and in real-time. The resulting enzymatic surfaces were then exposed to various flavonoids, and the degree of binding was measured using QCM. We show that enzyme activity is retained when immobilized on polycationic films (flavonoid binding observed), while the active site is blocked when assembled on a polyanionic film (no flavonoid binding to the enzyme). We rationalize these observations by considering a combination of interlayer interpenetration and strong electrostatic interactions between the polyelectrolyte and tyrosinase's dicopper 2(+) center. Ion-pair formation between anionic moieties of the polyanion and the metal-coordinated active site is suggested as the dominant mechanism leading to the deactivation of tyrosinase. We are currently working to expand this research to achieve a more general theory of how various metal-coordinated enzymes react with polyelectrolyte surfaces of varying structural morphology, charge density, and chemical composition.

Dual-beam polarization interferometry resolves mechanistic aspects of polyelectrolyte adsorption.

The electrostatically driven binding dynamics of a polyelectrolyte multilayer (PEMU) film was investigated in real-time using dual-beam polarization interferometry (DPI) and independently supported by quartz crystal microbalance with dissipation monitoring (QCM-D) studies. Multilayer assemblies of the polyanions poly[1-[4[(3-carboxy-4-hydroxyphenylazo)benzenesulfonamido]-1,2-ethanediyl sodium salt] (PAZO) and poly(styrene sulfonate) (PSS) were respectively constructed with the polycation poly(ethylenimine) (PEI) on anionic functionalized substrates using the layer-by-layer electrostatic self-assembly method. DPI measurements indicate that polyelectrolyte adsorption occurs in three distinct stages. In the first stage, for approximately 5 s, coil-like segments of polyanion partially tether to the surface of the oppositely charged PEI. In the second stage, these coils unfurl over a period of approximately 10 s to cover the surface resulting in an increase in average density of the film. During the final adsorption step, the surface-bound polyelectrolyte diffuses into the multilayer assembly, exposing the surface to further deposition. This last step occurs over a much longer time period and results in a highly interpenetrated film containing a charge-overcompensated region at the film surface.