Diseases of failed inflammation resolution are common and largely incurable. Therapeutic induction of inflammation resolution is an attractive strategy to bring about healing without increasing ...susceptibility to infection. However, therapeutic targeting of inflammation resolution has been hampered by a lack of understanding of the underlying molecular controls. To address this drug development challenge, we developed an in vivo screen for proresolution therapeutics in a transgenic zebrafish model. Inflammation induced by sterile tissue injury was assessed for accelerated resolution in the presence of a library of known compounds. Of the molecules with proresolution activity, tanshinone IIA, derived from a Chinese medicinal herb, potently induced inflammation resolution in vivo both by induction of neutrophil apoptosis and by promoting reverse migration of neutrophils. Tanshinone IIA blocked proinflammatory signals in vivo, and its effects are conserved in human neutrophils, supporting a potential role in treating human inflammation and providing compelling evidence of the translational potential of this screening strategy.
The zebrafish is an outstanding model for intravital imaging of inflammation due to its optical clarity and the ability to express fluorescently labelled specific cell types by transgenesis. However, ...although several transgenic labelling myeloid cells exist, none allow distinction of macrophages from neutrophils. This prevents simultaneous imaging and examination of the individual contributions of these important leukocyte subtypes during inflammation. We therefore used Bacterial Artificial Chromosome (BAC) recombineering to generate a transgenic Tg(fms:GAL4.VP16)i186 , in which expression of the hybrid transcription factor Gal4-VP16 is driven by the fms (CSF1R) promoter. This was then crossed to a second transgenic expressing a mCherry-nitroreductase fusion protein under the control of the Gal4 binding site (the UAS promoter), allowing intravital imaging of mCherry-labelled macrophages. Further crossing this compound transgenic with the neutrophil transgenic Tg(mpx:GFP)i114 allowed clear distinction between macrophages and neutrophils and simultaneous imaging of their recruitment and behaviour during inflammation. Compared with neutrophils, macrophages migrate significantly more slowly to an inflammatory stimulus. Neutrophil number at a site of tissue injury peaked around 6 hours post injury before resolving, while macrophage recruitment increased until at least 48 hours. We show that macrophages were effectively ablated by addition of the prodrug metronidazole, with no effect on neutrophil number. Crossing with Tg(Fli1:GFP)y1 transgenic fish enabled intravital imaging of macrophage interaction with endothelium for the first time, revealing that endothelial contact is associated with faster macrophage migration. Tg(fms:GAL4.VP16)i186 thus provides a powerful tool for intravital imaging and functional manipulation of macrophage behaviour during inflammation.
Abstract
The next core-collapse supernova in the Milky Way or its satellites will represent a once-in-a-generation opportunity to obtain detailed information about the explosion of a star and provide ...significant scientific insight for a variety of fields because of the extreme conditions found within. Supernovae in our galaxy are not only rare on a human timescale but also happen at unscheduled times, so it is crucial to be ready and use all available instruments to capture all possible information from the event. The first indication of a potential stellar explosion will be the arrival of a bright burst of neutrinos. Its observation by multiple detectors worldwide can provide an early warning for the subsequent electromagnetic fireworks, as well as signal to other detectors with significant backgrounds so they can store their recent data. The supernova early warning system (SNEWS) has been operating as a simple coincidence between neutrino experiments in automated mode since 2005. In the current era of multi-messenger astronomy there are new opportunities for SNEWS to optimize sensitivity to science from the next galactic supernova beyond the simple early alert. This document is the product of a workshop in June 2019 towards design of SNEWS 2.0, an upgraded SNEWS with enhanced capabilities exploiting the unique advantages of prompt neutrino detection to maximize the science gained from such a valuable event.
During inflammation, dysregulated neutrophil behaviour can play a major role in a range of chronic inflammatory diseases, for many of which current treatments are generally ineffective. Recently, ...specific naturally occurring tanshinones have shown promising anti-inflammatory effects by targeting neutrophils in vivo, yet such tanshinones, and moreover, their isomeric isotanshinone counterparts, are still a largely underexplored class of compounds, both in terms of synthesis and biological effects. To explore the anti-inflammatory effects of isotanshinones, and the tanshinones more generally, a series of substituted tanshinone and isotanshinone analogues was synthesised, alongside other structurally similar molecules. Evaluation of these using a transgenic zebrafish model of neutrophilic inflammation revealed differential anti-inflammatory profiles in vivo, with a number of compounds exhibiting promising effects. Several compounds reduce initial neutrophil recruitment and/or promote resolution of neutrophilic inflammation, of which two also result in increased apoptosis of human neutrophils. In particular, the methoxy-substituted tanshinone 39 specifically accelerates resolution of inflammation without affecting the recruitment of neutrophils to inflammatory sites, making this a particularly attractive candidate for potential pro-resolution therapeutics, as well as a possible lead for future development of functionalised tanshinones as molecular tools and/or chemical probes. The structurally related β-lapachones promote neutrophil recruitment but do not affect resolution. We also observed notable differences in toxicity profiles between compound classes. Overall, we provide new insights into the in vivo anti-inflammatory activities of several novel tanshinones, isotanshinones, and structurally related compounds.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
TNFα overexpression has been associated with several chronic inflammatory diseases, including psoriasis, lichen planus, rheumatoid arthritis, and inflammatory bowel disease. Paradoxically, numerous ...studies have reported new-onset psoriasis and lichen planus following TNFα antagonist therapy. Here, we show that genetic inhibition of Tnfa and Tnfr2 in zebrafish results in the mobilization of neutrophils to the skin. Using combinations of fluorescent reporter transgenes, fluorescence microscopy, and flow cytometry, we identified the local production of dual oxidase 1 (Duox1)-derived H₂O₂ by Tnfa- and Tnfr2-deficient keratinocytes as a trigger for the activation of the master inflammation transcription factor NF-κB, which then promotes the induction of genes encoding pro-inflammatory molecules. In addition, pharmacological inhibition of Duox1 completely abrogated skin inflammation, placing Duox1-derived H₂O₂ upstream of this positive feedback inflammatory loop. Strikingly, DUOX1 was drastically induced in the skin lesions of psoriasis and lichen planus patients. These results reveal a crucial role for TNFα/TNFR2 axis in the protection of the skin against DUOX1-mediated oxidative stress and could establish new therapeutic targets for skin inflammatory disorders.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Species richness is a metric of biodiversity that represents the number of species present in a community. Traditional fisheries assessments that rely on capture of organisms often underestimate true ...species richness. Environmental DNA (eDNA) metabarcoding is an alternative tool that infers species richness by collecting and sequencing DNA present in the ecosystem. Our objective was to determine how spatial distribution of samples and “bioinformatic stringency” affected eDNA-metabarcoding estimates of species richness compared with capture-based estimates in a 2.2 ha reservoir. When bioinformatic criteria required species to be detected only in a single sample, eDNA metabarcoding detected all species captured with traditional methods plus an additional 11 noncaptured species. However, when we required species to be detected with multiple markers and in multiple samples, eDNA metabarcoding detected only seven of the captured species. Our analysis of the spatial patterns of species detection indicated that eDNA was distributed relatively homogeneously throughout the reservoir, except near the inflowing stream. We suggest that interpretation of eDNA metabarcoding data must consider the potential effects of water body type, spatial resolution, and bioinformatic stringency.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We compared the adequacy and accuracy of fineneedle aspiration (FNA) with core needle aspiration in a total of 377 patients who underwent both tests. The adequacy rate for core needle biopsy (82.2%) ...was significantly higher than that of FNA (70.3%; P < .001), but the combined adequacy was significantly higher than that for either test alone (88.9%; P < .001). Overall concordance between the tests was 67.9%. In 70 cases, the core was adequate and negative (55 cases) or atypical (15 cases) and the aspirate was nondiagnostic; in 25 cases, the aspirate was adequate and negative (15 cases) or atypical (10 cases) and the core was nondiagnostic. In 21 cases, the FNA diagnosis was atypical and the core was negative; histologic follow-up supported the FNA diagnosis in all 14 cases with resection, of which 9 were malignant, and 8 of the 9 were papillary carcinoma. On review, it seemed that the core biopsy missed the lesion. Core needle biopsy has a higher adequacy rate than FNA but seems less sensitive, especially for papillary carcinoma. The combination of FNA with core needle biopsy seems to have the highest adequacy rate and sensitivity.
To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human ...pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population "bottleneck" whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Bacteria belonging to the "Burkholderia cepacia complex" (Bcc) often cause fatal pulmonary infections in cystic fibrosis patients, yet little is know about the underlying molecular mechanisms. These ...Gram-negative bacteria can adopt an intracellular lifestyle, although their ability to replicate intracellularly has been difficult to demonstrate. Here we show that Bcc bacteria survive and multiply in macrophages of zebrafish embryos. Local dissemination by nonlytic release from infected cells was followed by bacteremia and extracellular replication. Burkholderia cenocepacia isolates belonging to the epidemic electrophoretic type 12 (ET12) lineage were highly virulent for the embryos; intravenous injection of <10 bacteria of strain K56-2 killed embryos within 3 days. However, small but significant differences between the clonal ET12 isolates K56-2, J2315, and BC7 were evident. In addition, the innate immune response in young embryos was sufficiently developed to control infection with other less virulent Bcc strains, such as Burkholderia vietnamiensis FC441 and Burkholderia stabilis LMG14294. A K56-2 cepR quorum-sensing regulator mutant was highly attenuated, and its ability to replicate and spread to neighboring cells was greatly reduced. Our data indicate that the zebrafish embryo is an excellent vertebrate model to dissect the molecular basis of intracellular replication and the early innate immune responses in this intricate host-pathogen interaction.
Prompt neutrophil arrival is critical for host defense immediately after injury 1–3. Following wounding, a hydrogen peroxide (H2O2) burst generated in injured tissues is the earliest known leukocyte ...chemoattractant 4. Generating this tissue-scale H2O2 gradient uses dual oxidase 4 and neutrophils sense H2O2 by a mechanism involving the LYN Src-family kinase 5, but the molecular mechanisms responsible for H2O2 clearance are unknown 6. Neutrophils carry abundant amounts of myeloperoxidase, an enzyme catalyzing an H2O2-consuming reaction 7, 8. We hypothesized that this neutrophil-delivered myeloperoxidase downregulates the high tissue H2O2 concentrations that follow wounding. This was tested in zebrafish using simultaneous fluorophore-based imaging of H2O2 concentrations and leukocytes 4, 9–11 and a new neutrophil-replete but myeloperoxidase-deficient mutant (durif). Leukocyte-depleted zebrafish had an abnormally sustained wound H2O2 burst, indicating that leukocytes themselves were required for H2O2 downregulation. Myeloperoxidase-deficient zebrafish also had abnormally sustained high wound H2O2 concentrations despite similar numbers of arriving neutrophils. A local H2O2/myeloperoxidase interaction within wound-recruited neutrophils was demonstrated. These data demonstrate that leukocyte-delivered myeloperoxidase cell-autonomously downregulates tissue-generated wound H2O2 gradients in vivo, defining a new requirement for myeloperoxidase during inflammation. Durif provides a new animal model of myeloperoxidase deficiency closely phenocopying the prevalent human disorder 7, 12, 13, offering unique possibilities for investigating its clinical consequences.
► Leukocytes themselves downregulate the H2O2 burst after wounding ► They do so through neutrophil delivery of myeloperoxidase ► At the wound, myeloperoxidase and H2O2 interact directly within neutrophils ► A zebrafish myeloperoxidase-deficient mutant phenocopies the common human disorder
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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