Infection of zebrafish embryos with intracellular bacterial pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions (1). The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage (2, 3). The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle (4-6). In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection (7). A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization (8). Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system

Phagocytosis of mycobacteria by zebrafish macrophages is dependent on the scavenger receptor Marco, a key control factor of pro-inflammatory signalling

Animal science

An annotation database for chemicals of emerging concern in exposome research

International audienceBackground: Chemicals of Emerging Concern (CECs) include a very wide group of chemicals that are suspected to be responsible for adverse effects on health, but for which very limited information is available. Chromatographic techniques coupled with high-resolution mass spectrometry (HRMS) can be used for non-targeted screening and detection of CECs, by using comprehensive annotation databases. Establishing a database focused on the annotation of CECs in human samples will provide new insight into the distribution and extent of exposures to a wide range of CECs in humans.Objectives: This study describes an approach for the aggregation and curation of an annotation database (CECscreen) for the identification of CECs in human biological samples.Methods: The approach consists of three main parts. First, CECs compound lists from various sources were aggregated and duplications and inorganic compounds were removed. Subsequently, the list was curated by standardization of structures to create "MS-ready" and "QSAR-ready" SMILES, as well as calculation of exact masses (monoisotopic and adducts) and molecular formulas. The second step included the simulation of Phase I metabolites. The third and final step included the calculation of QSAR predictions related to physicochemical properties, environmental fate, toxicity and Absorption, Distribution, Metabolism, Excretion (ADME) processes and the retrieval of information from the US EPA CompTox Chemicals Dashboard.Results: All CECscreen database and property files are publicly available (DOI: https://doi.org/10.5281/zenodo.3956586). In total, 145,284 entries were aggregated from various CECs data sources. After elimination of duplicates and curation, the pipeline produced 70,397 unique "MS-ready" structures and 66,071 unique QSAR-ready structures, corresponding with 69,526 CAS numbers. Simulation of Phase I metabolites resulted in 306,279 unique metabolites. QSAR predictions could be performed for 64,684 of the QSAR-ready structures, whereas information was retrieved from the CompTox Chemicals Dashboard for 59,739 CAS numbers out of 69,526 inquiries. CECscreen is incorporated in the in silico fragmentation approach MetFrag.Discussion: The CECscreen database can be used to prioritize annotation of CECs measured in non-targeted HRMS, facilitating the large-scale detection of CECs in human samples for exposome research. Large-scale detection of CECs can be further improved by integrating the present database with resources that contain CECs (metabolites) and meta-data measurements, further expansion towards in silico and experimental (e.g., MassBank) generation of MS/MS spectra, and development of bioinformatics approaches capable of using correlation patterns in the measured chemical features

An efficient strategy for evaluating new non-invasive screening tests for colorectal cancer: the guiding principles.

New screening tests for colorectal cancer (CRC) are rapidly emerging. Conducting trials with mortality reduction as the end point supporting their adoption is challenging. We re-examined the principles underlying evaluation of new non-invasive tests in view of technological developments and identification of new biomarkers. A formal consensus approach involving a multidisciplinary expert panel revised eight previously established principles. Twelve newly stated principles emerged. Effectiveness of a new test can be evaluated by comparison with a proven comparator non-invasive test. The faecal immunochemical test is now considered the appropriate comparator, while colonoscopy remains the diagnostic standard. For a new test to be able to meet differing screening goals and regulatory requirements, flexibility to adjust its positivity threshold is desirable. A rigorous and efficient four-phased approach is proposed, commencing with small studies assessing the test's ability to discriminate between CRC and non-cancer states (phase I), followed by prospective estimation of accuracy across the continuum of neoplastic lesions in neoplasia-enriched populations (phase II). If these show promise, a provisional test positivity threshold is set before evaluation in typical screening populations. Phase III prospective studies determine single round intention-to-screen programme outcomes and confirm the test positivity threshold. Phase IV studies involve evaluation over repeated screening rounds with monitoring for missed lesions. Phases III and IV findings will provide the real-world data required to model test impact on CRC mortality and incidence. New non-invasive tests can be efficiently evaluated by a rigorous phased comparative approach, generating data from unbiased populations that inform predictions of their health impact

Epidemiology and Treatment Guidelines of Negative Symptoms in Schizo-phrenia in Central and Eastern Europe: A Literature Review

AIM: To gather and review data describing the epidemiology of schizophrenia and clinical guidelines for schizophrenia therapy in seven Central and Eastern European countries, with a focus on negative symptoms. Methods : A literature search was conducted which included publications from 1995 to 2012 that were indexed in key databases. Results : Reports of mean annual incidence of schizophrenia varied greatly, from 0.04 to 0.58 per 1,000 population. Lifetime prevalence varied from 0.4% to 1.4%. One study reported that at least one negative symptom was present in 57.6% of patients with schizophrenia and in 50-90% of individuals experiencing their first episode of schizophrenia. Primary negative symptoms were observed in 10-30% of patients. Mortality in patients with schizophrenia was greater than in the general population, with a standardized mortality ratio of 2.58-4.30. Reasons for higher risk of mortality in the schizophrenia population included increased suicide risk, effect of schizophrenia on lifestyle and environment, and presence of comorbidities. Clinical guidelines overall supported the use of second-generation antipsychotics in managing negative symptoms of schizophrenia, although improved therapeutic approaches are needed. Conclusion : Schizophrenia is one of the most common mental illnesses and poses a considerable burden on patients and healthcare resources alike. Negative symptoms are present in many patients and there is an unmet need to improve treatment offerings for negative symptoms beyond the use of second-generation antipsychotics and overall patient outcomes

Search for Gravitational Waves Associated with Gamma-Ray Bursts Detected by Fermi and Swift during the LIGO-Virgo Run O3b

We search for gravitational-wave signals associated with gamma-ray bursts (GRBs) detected by the Fermi and Swift satellites during the second half of the third observing run of Advanced LIGO and Advanced Virgo (2019 November 1 15:00 UTC-2020 March 27 17:00 UTC). We conduct two independent searches: A generic gravitational-wave transients search to analyze 86 GRBs and an analysis to target binary mergers with at least one neutron star as short GRB progenitors for 17 events. We find no significant evidence for gravitational-wave signals associated with any of these GRBs. A weighted binomial test of the combined results finds no evidence for subthreshold gravitational-wave signals associated with this GRB ensemble either. We use several source types and signal morphologies during the searches, resulting in lower bounds on the estimated distance to each GRB. Finally, we constrain the population of low-luminosity short GRBs using results from the first to the third observing runs of Advanced LIGO and Advanced Virgo. The resulting population is in accordance with the local binary neutron star merger rate. © 2022. The Author(s). Published by the American Astronomical Society

Narrowband Searches for Continuous and Long-duration Transient Gravitational Waves from Known Pulsars in the LIGO-Virgo Third Observing Run

Isolated neutron stars that are asymmetric with respect to their spin axis are possible sources of detectable continuous gravitational waves. This paper presents a fully coherent search for such signals from eighteen pulsars in data from LIGO and Virgo's third observing run (O3). For known pulsars, efficient and sensitive matched-filter searches can be carried out if one assumes the gravitational radiation is phase-locked to the electromagnetic emission. In the search presented here, we relax this assumption and allow both the frequency and the time derivative of the frequency of the gravitational waves to vary in a small range around those inferred from electromagnetic observations. We find no evidence for continuous gravitational waves, and set upper limits on the strain amplitude for each target. These limits are more constraining for seven of the targets than the spin-down limit defined by ascribing all rotational energy loss to gravitational radiation. In an additional search, we look in O3 data for long-duration (hours-months) transient gravitational waves in the aftermath of pulsar glitches for six targets with a total of nine glitches. We report two marginal outliers from this search, but find no clear evidence for such emission either. The resulting duration-dependent strain upper limits do not surpass indirect energy constraints for any of these targets. © 2022. The Author(s). Published by the American Astronomical Society