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Biology subjects

Brown, L. E.

Publications and source records attributed to Brown, L. E..

13 recordsLinked to original sources

Diatom biodiversity response to shrinking glaciers in the Peruvian Andes

Alpine river biodiversity is under threat as climate change is driving extensive glacier retreat worldwide, altering aquatic habitats and restructuring biotic communities. However, with the exception of macroinvertebrates, the responses of aquatic groups to deglaciation have received little attention, particularly in tropical ecosystems where species may respond differently owing to additional pressures such as distinct wet and dry seasons, limited snowmelt and extreme high altitude and thus lower oxygen availability. Utilising a continuum of glacier cover across 23 rivers in the Peruvian Andes (Cordilleras Blanca and Vilcanota), we demonstrate that diatoms, abundant primary producers in glacier-fed rivers, show regionally consistent increased -diversity and density responses with deglaciation. However, {beta}-diversity was found to decline, potentially reflecting the reduction of habitat heterogeneity and niche space both within and between alpine rivers as glaciers are lost. High regional biodiversity, with just 23% of taxa found in both regions, was closely linked to the distinct underlying geologies of the Andes which influence water chemistry. Thirty-eight taxa were found exclusively at sites with glacier cover [≥] 25%, which is considerably higher than numbers recorded in European alpine rivers. However, 31 taxa were identified only at non-glacial sites suggesting deglaciation could open up more habitat for these species. Peruvian rivers overall showed similar responses to glacier retreat as temperate alpine systems, but the large number of taxa found only at mid-high glacial influence sites suggests tropical diatom assemblages may be especially vulnerable to climate change.

ecology↗

Structural Basis for the Improved RNA-Clamping of Amidino-Rocaglates to eIF4A1

Eukaryotic Initiation Factor 4A-1 (eIF4A1) is an ATP-dependent RNA helicase that unwinds 5-UTR mRNA secondary structures to facilitate cap-dependent translation initiation. Rocaglates, a class of natural products typified by rocaglamide A (RocA), possess anti-neoplastic and anti-infectious activity mediated by their interaction with eIF4A1. Rocaglates inhibit cap-dependent translation initiation by "clamping" eIF4A1 onto polypurine RNA, impeding ribosome scanning. A novel class of rocaglate derivatives, amidino-rocaglates (ADRs) which feature an amidine ring fused to the rocaglate core, are particularly effective at promoting eIF4A1-RNA-clamping compared to other rocaglate congeners. Herein, we present the X-ray crystal structure of an ADR in complex with eIF4A1, the nonhydrolyzable ATP ground-state mimic adenylyl-imidodiphosphate (AMPPNP), and poly r(AG)5 RNA refined to 1.69 [A] resolution. The binding pose and interactions of the ADR with eIF4A1 do not differ substantially from those of RocA, prompting investigation of the basis for enhanced target engagement. Computational modeling suggests that the rigidified ADR scaffold is inherently preorganized in an eIF4A1-RNA-binding-competent conformation, thereby avoiding entropic penalties associated with RocA binding. This study illustrates how conformational rigidification of the rocaglate scaffold can be leveraged to improve potency for the development of rocaglates as potential anticancer and anti-infectious agents. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/616674v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@3c6dcorg.highwire.dtl.DTLVardef@1f4ae24org.highwire.dtl.DTLVardef@d4e264org.highwire.dtl.DTLVardef@5b1bde_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

In vitro discovery of a therapeutic lead for HFMD from a library screen of rocaglates/aglains

The lack of an effective antiviral treatment for enteroviruses, including the human enterovirus A71 (EV-A71), has resulted in an immense global healthcare burden associated with hand-foot-and-mouth disease (HFMD). Rocaglates and aglains belong to a family of compounds produced by Aglaia genus plants. Since the initial discovery of rocaglates in 1982, various rocaglates and aglains have been synthesized and extensively studied as anticancer and antiviral agents. Here, we report our studies towards the discovery of a novel aglain derivative as an EV-A71 inhibitor and work to decipher its antiviral effect. From an immunofluorescence-based phenotypic screen of a library of 296 rocaglate and aglain derivatives, we identified a lead aglain derivative which effectively suppressed EV-A71 replication by 2.3 log fold at a non-cytotoxic concentration. Further validation revealed inhibition of EV-A71 across multiple cell types and a pan-enterovirus inhibitory spectrum against other enteroviruses. Subsequent mechanistic investigation revealed interference with EV-A71 intracellular post-entry events including viral RNA transcription and translation. Findings from this study have established a strong foundation for development of aglain scaffolds as much needed antiviral agents for HFMD, paving the way for future medicinal chemistry optimization and in vivo studies.

microbiology↗

Single Cell Profiling Distinguishes Leukemia-Selective Chemotypes

A central challenge in chemical biology is to distinguish molecular families in which small structural changes trigger large changes in cell biology. Such families might be ideal scaffolds for developing cell-selective chemical effectors - for example, molecules that activate DNA damage responses in malignant cells while sparing healthy cells. Across closely related structural variants, subtle structural changes have the potential to result in contrasting bioactivity patterns across different cell types. Here, we tested a 600-compound Diversity Set of screening molecules from the Boston University Center for Molecular Discovery (BU-CMD) in a novel phospho-flow assay that tracked fundamental cell biological processes, including DNA damage response, apoptosis, M-phase cell cycle, and protein synthesis in MV411 leukemia cells. Among the chemotypes screened, synthetic congeners of the rocaglate family were especially bioactive. In follow-up studies, 37 rocaglates were selected and deeply characterized using 12 million additional cellular measurements across MV411 leukemia cells and healthy peripheral blood mononuclear cells. Of the selected rocaglates, 92% displayed significant bioactivity in human cells, and 65% selectively induced DNA damage responses in leukemia and not healthy human blood cells. Furthermore, the signaling and cell-type selectivity were connected to structural features of rocaglate subfamilies. In particular, three rocaglates from the rocaglate pyrimidinone (RP) structural subclass were the only molecules that activated exceptional DNA damage responses in leukemia cells without activating a detectable DNA damage response in healthy cells. These results indicate that the RP subset should be extensively characterized for anticancer therapeutic potential as it relates to the DNA damage response. This single cell profiling approach advances a chemical biology platform to dissect how systematic variations in chemical structure can profoundly and differentially impact basic functions of healthy and diseased cells.

systems biology↗

Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR)

Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge and serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies of substrate-dependent target engagement remain sparse. Herein, we describe a strategy for the discovery of substrate-dependent ligand binding. Using proteome integral solubility alteration (PISA) assays, we show that simple biochemical additives can enable detection of RNA-protein-small molecule complexes in native cell lysates. We apply our approach to rocaglates, molecules that specifically clamp RNA to eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), and potentially other members of the DEAD-box (DDX) helicase family. To identify unexpected interactions, we used a target class-specific thermal window and compared ATP analog and RNA base dependencies for key rocaglate-DDX interactions. We report and validate novel DDX targets of high-profile rocaglates - including the clinical candidate Zotatifin - using limited proteolysis-mass spectrometry and fluorescence polarization (FP) experiments. We also provide structural insight into divergent DDX3X affinities between synthetic rocaglates. Taken together, our study provides a model for screening uncompetitive inhibitors using a chemical proteomics approach and uncovers actionable DDX clamping targets, clearing a path towards characterization of novel molecular clamps and associated RNA helicases.

biochemistry↗

The MYCN 5' UTR as a therapeutic target in neuroblastoma

Tumor cell amplification of the MYCN transcription factor is seen in half of patients with high-risk neuroblastoma, where it functions as an oncogenic driver associated with metastatic disease and poor survival. Yet, direct targeting of MYCN has been met with little success, prompting efforts to inhibit its expression at multiple levels. MYCN-amplified neuroblastoma cells have an increased requirement for protein synthesis to meet the overwhelming transcriptional burden imposed by oncogenic MYCN. Here, we take advantage of this vulnerability to interrogate the therapeutic potential of inhibiting the activity of the eukaryotic translation initiation factor 4A1 (eIF4A1), an RNA-helicase responsible for resolving structural barriers such as polypurine preponderance within 5' untranslated regions (UTRs). We observed that eIF4A1 is a key regulator of transcript-specific mRNA recruitment in MYCN-overexpressing neuroblastomas and MYCN-associated transcripts rank highly in polypurine-rich 5' UTR sequences, the majority of which have critical roles in cell proliferation. Using CMLD012824, a novel synthetic amidino-rocaglate (ADR) derivative, we demonstrate selectively increased eIF4A1 affinity for polypurine-rich 5' UTRs, including the MYCN mRNA, leading to translation inhibition and cytotoxicity in human neuroblastoma cell lines and animal models. Through ribosome profiling and PAR-CLIP analysis, we show that ADR-mediated clamping of eIF4A1 onto mRNA spans the full lengths of target transcripts, whereas translational inhibition is mediated selectively through 5' UTR binding. Both cap-dependent and cap-independent translation of MYCN are disrupted, pointing to the ability of CMLD012824 to disrupt non-canonical translation initiation. Our studies provide insights into the functional role of eIF4A1 in meeting the increased protein synthesis demands of MYCN-amplified neuroblastoma and suggest that its disruption may be therapeutically beneficial in this disease.

cancer biology↗

A new concept in antiviral drug design

Contemporary antiviral development, whether by rational drug design or forward pharmacology, primarily strives to produce lock and key inhibitors. While the technology to identify druggable targets and create compounds to bind them has improved dramatically over the last century it has always been constrained by the finite availability of suitable binding sites that antiviral compounds can occupy. Here we present a new approach to drug design that utilizes compounds devised to alter the microenvironment of the virion surface making it incompatible with virus entry and illustrate this strategy with inhibitors of influenza virus. We show that compounds that produce a proton-rich mantle above the virion surface induce a conformational change in the viral hemagglutinin (HA) rendering the virus unable to interact with cellular receptors and gain entry to the cell. The compounds show exceptional antiviral activity both in vitro and in vivo and protect against influenza illness in mice and ferrets after a single dose, either therapeutically or prophylactically. The work presented here lays the foundation for a brand-new category of inhibitors that could be engineered to counter many different viruses and potentially other pathogens.

microbiology↗

Self-organized anteroposterior regionalization of early midbrain and hindbrain using micropatterned human embryonic stem cells

Anterior-posterior (AP) spatial regionalization is crucial for central nervous system development. Previous studies suggest that morphogen gradients can induce AP patterning in animal and organoid models. While self-organization in early embryogenesis and neurogenesis has been found using geometrically confined microtissues, spontaneously induced AP patterning has not been reported. Here, we show that circularly micropatterned human pluripotent stem cells self-organize into spatially distinct FOXG1-FOXA1+OTX2+ midbrain-like and HOXB4+ hindbrain/spinal cord-like regions. Notably, the tissue then folds inwardly to form a 3D annular structure, maintaining a distinct boundary between OTX2+ and HOXB4+ zones. The reaction-diffusion of BMP/Noggin plays a key role in the mechanism of AP patterning. Our model is validated by its capability to distinguish the teratogenic effects of valproic acid and isotretinoin. Our work suggests a novel regulatory mechanism for AP patterning and provides a tool for fast screening of teratogens.

bioengineering↗

BRCA mutational status shapes the stromal microenvironment of pancreatic cancer linking CLU+ CAF expression with HSF1 signaling

Cancer-associated fibroblasts (CAFs) give rise to desmoplastic stroma, which supports tumor progression and metastasis, and comprises up to 90% of the tumor mass in pancreatic cancer. Recent work by us and others has shown that CAFs are transcriptionally rewired by adjacent cancer cells to form heterogeneous subtypes. Whether this rewiring is differentially affected by different driver mutations in cancer cells is largely unknown. Here we address this question by dissecting and comparing the stromal landscape of BRCA-mutated and BRCA Wild-type (WT) pancreatic ductal adenocarcinoma (PDAC). We comprehensively analyze PDAC samples from a cohort of 42 patients by laser-capture microdissection, RNA-sequencing and multiplexed immunofluorescence, revealing different CAF subtype compositions in germline BRCA-mutated vs. BRCA-WT tumors. In particular, we detect an increase in a subset of Clusterin (CLU)-positive CAFs in BRCA-mutated tumors. We further unravel a network of stress responses upregulated in BRCA-mutated tumors. Using cancer organoids and cell co-cultures, we show that the transcriptional shift of pancreatic stellate cells into CLU+ CAFs is mediated through activation of heat-shock factor 1 (HSF1), the transcriptional regulator of Clu. Our findings unravel a new dimension of stromal heterogeneity, influenced by germline mutations in cancer cells, with direct translational implications for clinical research. SignificanceBRCA1/2 mutations initiate some of the deadliest cancers, yet the fibroblastic microenvironment of BRCA-mutated cancers remains uncharted. Our work addresses a major unsolved question - to what extent is the tumor microenvironment determined by cancer mutations? We find that BRCA mutations in the cancer cells affect the composition of CAFs in PDAC. These findings have direct implications for diagnosis and for efforts to exploit CAFs for therapy.

cancer biology↗

The role of gene segment interactions in driving the emergence of dominant gene constellations during influenza virus reassortment

A segmented genome enables influenza virus to undergo reassortment when two viruses infect the same cell. Resulting reassorted progeny have a spectrum of gene constellations and potentially different phenotypes. Although reassortment is involved in the creation of pandemic influenza strains and is routinely used to produce influenza vaccines, our understanding of the factors that drive the emergence of dominant gene constellations during this process is incomplete. Using an influenza vaccine seed production model, reassortant genotypes were tracked through the reassortment process under antibody selective pressure. We discovered that certain gene constellations conferring low replicative fitness were selected at the expense of more fit progeny. Nevertheless, relatively unfit reassortants likely provide high hemagglutinin antigen yields through co-production of non-infectious particles and/or by more hemagglutinin molecules per virion. Our data illustrate the dynamics and complexity of reassortment and highlight how gene segment interactions formed during packaging, in addition to antibody pressure, restrict the final viruses that dominate.

microbiology↗

Condensation tendency of connected contractile tissue with planar isotropic actin network

ABSTRACTIt has been found that many types of cells form nematic symmetry on confined planar substrates. Such observation has been satisfactorily explained by modeling cells as crowded self-propelled rods. In this work, we report that rat embryonic fibroblast (REF) cells when confined in circular mesoscale patterns, form a new type of symmetry where cells align radially at the boundary. Unlike NIH-3T3 and MDCK monolayers, the REF monolayer presents a supracellular actin gradient with isotropic meshwork. In addition, the contractile REF cells present strong adhesive interactions with neighboring cells, which confers the monolayer with significant condensation tendency. We found the loss of condensation tendency by inhibiting the cell contractility or disrupting cell-cell adhesion led to the disappearance of the radial alignment. In theory, we found the prestretch due to condensation tendency with differential cell stiffness is sufficient to explain the new symmetry within a confined tissue continuum.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗

Contextualising UK moorland burning studies: geographical versus potential sponsorship-bias effects on research conclusions

O_LIIt has recently been claimed that geographical variability resulted in false conclusions from some studies examining the impacts of prescribed moorland burning, including the Effects of Moorland Burning on the Ecohydrology of River basins (EMBER) project. We provide multiple lines of evidence to contradict these claims and show that the EMBER results are reliable.\nC_LIO_LIA systematic review of the literature also confirms that EMBER conclusions were not out of line with the majority of other published UK studies on responses to prescribed burning of Sphagnum growth/abundance, soil properties, hydrological change, or peat exposure and erosion.\nC_LIO_LIWe suggest that sponsorship-bias is associated with some recent research conclusions related to moorland burning. Thus, it is of grave concern when sponsorship or other potential conflicts of interest are not declared on publications related to moorland burning.\nC_LIO_LIWe show that sponsorship and other conflicts of interest were not declared on a recent publication that criticised the EMBER project, thereby entirely undermining that critical assessment.\nC_LIO_LIPolicy implications: The EMBER findings are robust. Our study suggests that publications on moorland burning that have been funded by pro-burning groups should be treated with extreme caution by the policy community. Publications that have been shown to have failed to declare conflicts of interest from the outset, when first submitted to a journal, should be disregarded by the policy community because peer reviewers and editors may have been unable to evaluate those pieces of work properly.\nC_LI

ecology↗

Channeling macrophage polarization by using a selective translation inhibitor

Macrophages contribute to host immunity and tissue homeostasis via alternative activation programs. M1-like macrophages control intracellular bacterial pathogens and tumor progression. In contrast, M2-like macrophages shape reparative microenvironments that can be conducive for pathogen survival or tumor growth. An imbalance of these macrophages phenotypes may perpetuate sites of chronic unresolved inflammation, such as infectious granulomas and solid tumors. We have found that plant-derived and synthetic rocaglates sensitize macrophages to low concentrations of the M1-inducing cytokine IFN-gamma and inhibit their responsiveness to IL-4, a prototypical activator of the M2-like phenotype. Treatement of primary macrophages with rocaglates increased their resilience to oxidative stress, stimulated autophagy and killing of intracellular mycobacteria. Thus, rocaglates represent a novel class of immunomodulators that can direct macrophage polarization towards the M1-like phenotype in complex microenvironments associated with hypofunction of type 1 and/or hyperactivation of type 2 immunity, e.g. chronic bacterial infections, allergies and, possibly, certain tumors.

immunology↗