Translocation events are frequent in cancer and may create chimeric fusions or 'regulatory rearrangements' that drive oncogene overexpression. Here we identify super-enhancer translocations that ...drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Remarkably, MYB protein binds to the translocated enhancers, creating a positive feedback loop that sustains its expression. MYB also binds enhancers that drive different regulatory programs in alternate cell lineages in ACC, cooperating with TP63 in myoepithelial cells and a Notch program in luminal epithelial cells. Bromodomain inhibitors slow tumor growth in ACC primagraft models in vivo. Thus, our study identifies super-enhancer translocations that drive MYB expression and provides insight into downstream MYB functions in alternate ACC lineages.
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IJS, NUK, SBMB, UL, UM, UPUK
Influence of Triplet Instabilities in TDDFT Peach, Michael J. G; Williamson, Matthew J; Tozer, David J
Journal of chemical theory and computation,
11/2011, Volume:
7, Issue:
11
Journal Article
Peer reviewed
Open access
Singlet and triplet vertical excitation energies from time-dependent density functional theory (TDDFT) can be affected in different ways by the inclusion of exact exchange in hybrid or ...Coulomb-attenuated/range-separated exchange–correlation functionals; in particular, triplet excitation energies can become significantly too low. To investigate these issues, the explicit dependence of excitation energies on exact exchange is quantified for four representative molecules, paying attention to the effect of constant, short-range, and long-range contributions. A stability analysis is used to verify that the problematic TDDFT triplet excitations can be understood in terms of the ground state triplet instability problem, and it is proposed that a Hartree–Fock stability analysis should be used to identify triplet excitations for which the presence of exact exchange in the TDDFT functional is undesirable. The use of the Tamm–Dancoff approximation (TDA) significantly improves the problematic triplet excitation energies, recovering the correct state ordering in benzoquinone; it also affects the corresponding singlet states, recovering the correct state ordering in naphthalene. The impressive performance of the TDA is maintained for a wide range of molecules across representative functionals.
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IJS, KILJ, NUK, PNG, UL, UM
Networks of no-take marine reserves (NTMRs) are widely advocated for preserving exploited fish stocks and for conserving biodiversity. We used underwater visual surveys of coral reef fish and benthic ...communities to quantify the short- to medium-term (5 to 30 years) ecological effects of the establishment of NTMRs within the Great Barrier Reef Marine Park (GBRMP). The density, mean length, and biomass of principal fishery species, coral trout (Plectropomus spp., Variola spp.), were consistently greater in NTMRs than on fished reefs over both the short and medium term. However, there were no clear or consistent differences in the structure of fish or benthic assemblages, non-target fish density, fish species richness, or coral cover between NTMR and fished reefs. There was no indication that the displacement and concentration of fishing effort reduced coral trout populations on fished reefs. A severe tropical cyclone impacted many survey reefs during the study, causing similar declines in coral cover and fish density on both NTMR and fished reefs. However, coral trout biomass declined only on fished reefs after the cyclone. The GBRMP is performing as expected in terms of the protection of fished stocks and biodiversity for a developed country in which fishing is not excessive and targets a narrow range of species. NTMRs cannot protect coral reefs directly from acute regional-scale disturbance but, after a strong tropical cyclone, impacted NTMR reefs supported higher biomass of key fishery-targeted species and so should provide valuable sources of larvae to enhance population recovery and long-term persistence.
•Expansion of a GBR reserve network led to substantial increases in coral trout stocks•A large tropical cyclone impacted the reserve network, causing widespread damage•Impacts on coral trout biomass were smaller inside NTMRs compared to fished reefs•NTMRs may provide greater potential for replenishment after disturbances
No-take marine reserves (NTMRs) are widely advocated for conserving exploited fish stocks and biodiversity. Emslie et al. show that expanding NTMR networks had clear benefits for fishery target, but not non-target, species. A cyclone caused widespread degradation, but target species biomass was retained within NTMRs, with greater recovery potential.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
ABSTRACT
The gut microbiome supplies essential metabolites such as short‐chain fatty acids to skeletal muscle mitochondria, and the composition and activity of the microbiota is in turn affected by ...muscle fitness. To further our understanding of the complex interactions between the gut microbiome and muscle, we examined the effect of microbiota‐derived phenolic metabolites on the ability of human muscle cells to take up and metabolize glucose. As a model, we used the differentiated human skeletal muscle myoblast line, LHCN‐M2, which expresses typical muscle phenotypic markers. We initially tested a selected panel of parent phenolic compounds and microbial metabolites, and their respective phenolic conjugates, as found in blood. Several of the tested compounds increased glucose uptake and metabolism, notably in high glucose—and insulin‐treated myotubes. One of the most effective was isovanillic acid 3‐O‐sulfate (IVAS), a metabolite from the microbiome found in the blood, primarily derived from consumed cyanidin 3‐O‐glucoside, a major compound in berry fruits. IVAS stimulated a dose‐dependent increase in glucose transport through glucose transporter GLUT4‐ and PI3K‐dependent mechanisms. IVAS also up‐regulated GLUT1, GLUT4, and PI3K p85α protein, and increased phosphorylation of Akt. The stimulation of glucose uptake and metabolism by a unique microbiome metabolite provides a novel link among diet, gut microbiota, and skeletal muscle energy source utilization.—Houghton, M. J., Kerimi, A., Mouly, V., Tumova, S., Williamson, G. Gut microbiome catabolites as novel modulators of muscle cell glucose metabolism. FASEB J. 33, 1887–1898 (2019). www.fasebj.org
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Nitrogen (N) and phosphorus (P) inputs influence algal community structure and function. The rates and ratios of N and P supply, and different N forms (e.g., NO₃ and NH₄), from external loading and ...internal cycling can be highly seasonal. However, the interaction between seasonality in nutrient supply and algal nutrient limitation remains poorly understood. We examined seasonal variation in nutrient limitation and response to N form in a hypereutrophic reservoir that experiences elevated, but seasonal, nutrient inputs and ratios. External N and P loading is high in spring and declines in summer, when internal loading because more important, reducing loading N:P ratios. Watershed NO₃ dominates spring N supply, but internal NH₄ supply becomes important during summer.We quantified how phytoplankton groups (diatoms, chlorophytes, and cyanobacteria) are limited by N or P, and their N form preference (NH₄ vs. NO₃), with weekly experiments (May–October). Phytoplankton were P-limited in spring, transitioned to N limitation or colimitation (primary N) in summer, and returned to P limitation following fall turnover. UnderNlimitation (or colimitation), chlorophytes and cyanobacteriaweremore strongly stimulated by NH₄ whereas diatoms were often equally, or more strongly, stimulated by NO₃ addition. Cyanobacteria heterocyte development followed the onset of N-limiting conditions,with a severalweek lag time, but heterocyte production did not fully alleviate N-limitation. We show that phytoplankton groups vary seasonally in limiting nutrient and N formpreference, suggesting that dual nutrient management strategies incorporating both N and P, and N formare needed to manage eutrophication.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
•α-Amylase inhibition was studied using a chromatographic HPAE-PAD method.•Maltoheptaoside (Mal-7) was used as the substrate.•Phenolics inhibit α-amylase through direct inhibition and by complexing ...with starch.•Conventional α-amylase methods detect both direct and indirect inhibition.•Short maltooligosaccharides can be used to assess direct α-amylase inhibition.
The aim was to determine inhibition of human α-amylase activity by (poly)phenols using maltoheptaoside as substrate with direct chromatographic product quantification, compared to hydrolysis of amylose and amylopectin estimated using 3,5-dinitrosalicylic acid. Acarbose exhibited similar IC50 values (50% inhibition) with maltoheptaoside, amylopectin or amylose as substrates (2.37 ± 0.11, 3.71 ± 0.12 and 2.08 ± 0.01 µM respectively). Epigallocatechin gallate, quercetagetin and punicalagin were weaker inhibitors of hydrolysis of maltoheptaoside (<50% inhibition) than amylose (IC50: epigallocatechin gallate = 20.41 ± 0.25 µM, quercetagetin = 30.15 ± 2.05 µM) or amylopectin. Interference using 3,5-dinitrosalicylic acid was in the order punicalagin > epigallocatechin gallate > quercetagetin, with minimal interference using maltoheptaoside as substrate. The main inhibition mechanism of epigallocatechin gallate and punicalagin was through complexation with starch, especially amylose, whereas only quercetagetin additionally binds to the α-amylase active site. Interference is minimised using maltoheptaoside as substrate with product detection by chromatography, potentially allowing assessment of direct enzyme inhibition by almost any compound.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of ...Pseudomonas aeruginosa growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms. Selective green fluorescent protein (GFP) labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNA levels for genes regulated by the hypoxia-induced regulator Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-term anoxia. Transcripts for ribosomal proteins were associated primarily with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (the rmf and PA4463 genes) at the bottom of the biofilms. The dormant biofilm cells of a P. aeruginosa Δrmf strain had decreased membrane integrity, as shown by propidium iodide staining. Using selective GFP labeling and cell sorting, we show that the dividing cells are more susceptible to killing by tobramycin and ciprofloxacin. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state.
The bacterial heat-shock response is regulated by the alternative sigma factor, σ
(RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoters for genes required ...for protein refolding or degradation. In
, RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for
. We placed the
gene under control of the arabinose-inducible P
promoter, then deleted the chromosomal
allele. This allowed transcriptomic analysis of the RpoH (σ
) regulon following a short up-shift in the cellular concentration of RpoH by arabinose addition, in the absence of a sudden change in temperature. The
∆
(P
-
) strain grew in the absence of arabinose, indicating that some
expression occurred without arabinose induction. When arabinose was added, the
mRNA abundance of
∆
(P
-
) measured by RT-qPCR increased five-fold within 15 min of arabinose addition. Transcriptome results showed that
genes required for protein repair or degradation are induced by increased RpoH levels, and that many genes essential for
growth are induced by RpoH. Other stress response genes induced by RpoH are involved in damaged nucleic acid repair and in amino acid metabolism. Annotation of the hypothetical proteins under RpoH control included proteins that may play a role in antibiotic resistances and in non-ribosomal peptide synthesis. Phenotypic analysis of
∆
(P
-
) showed that it is impaired in its ability to survive during starvation compared to the wild-type strain.
∆
(P
-
) also had increased sensitivity to aminoglycoside antibiotics, but not to other classes of antibiotics, whether cultured planktonically or in biofilms. The enhanced aminoglycoside sensitivity of the mutant strain may be due to indirect effects, such as the build-up of toxic misfolded proteins, or to the direct effect of genes, such as aminoglycoside acetyl transferases, that are regulated by RpoH. Overall, the results demonstrate that RpoH regulates genes that are essential for viability of
, that it protects
from damage from aminoglycoside antibiotics, and that it is required for survival during nutrient-limiting conditions.
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Stem cells reside in 'niches', where support cells provide critical signalling for tissue renewal. Culture methods mimic niche conditions and support the growth of stem cells in vitro. However, ...current functional assays preclude statistically meaningful studies of clonal stem cells, stem cell-niche interactions, and genetic analysis of single cells and their organoid progeny. Here, we describe a 'microraft array' (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for quantitative PCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell-niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
C. neoformans is a leading cause of fatal mycosis linked to CNS dissemination. Laccase, encoded by the LAC1 gene, is an important virulence factor implicated in brain dissemination yet little is ...known about the mechanism(s) accounting for this observation. Here, we investigated whether the presence or absence of laccase altered the local immune response in the lungs by comparing infections with the highly virulent strain, H99 (which expresses laccase) and mutant strain of H99 deficient in laccase (lac1Δ) in a mouse model of pulmonary infection. We found that LAC1 gene deletion decreased the pulmonary fungal burden and abolished CNS dissemination at weeks 2 and 3. Furthermore, LAC1 deletion lead to: 1) diminished pulmonary eosinophilia; 2) increased accumulation of CD4+ and CD8+ T cells; 3) increased Th1 and Th17 cytokines yet decreased Th2 cytokines; and 4) lung macrophage shifting of the lung macrophage phenotype from M2- towards M1-type activation. Next, we used adoptively transferred CD4+ T cells isolated from pulmonary lymph nodes of mice infected with either lac1Δ or H99 to evaluate the role of laccase-induced immunomodulation on CNS dissemination. We found that in comparison to PBS treated mice, adoptively transferred CD4+ T cells isolated from lac1Δ-infected mice decreased CNS dissemination, while those isolated from H99-infected mice increased CNS dissemination. Collectively, our findings reveal that immune modulation away from Th1/Th17 responses and towards Th2 responses represents a novel mechanism through which laccase can contribute to cryptococcal virulence. Furthermore, our data support the hypothesis that laccase-induced changes in polarization of CD4+ T cells contribute to CNS dissemination.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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