Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets.

Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations.

Invertebrate immunity is associated with natural mechanisms that include cellular and humoral elements, similar to those that play a role in vertebrate innate immune responses Formation of extracellular traps (ETs) is a newly discovered mechanism to combat pathogens, operating not only in vertebrate leucocytes but also in invertebrate immune cells The ET components include extracellular DNA (exDNA), antimicrobial proteins and histones Formation of mammalian ETs depends on enzymes such as neutrophil elastase, myeloperoxidase, the citrullination of histones and protease activity It was confirmed that coelomocytes—immunocompetent cells of the earthworm Eisenia andrei—are also able to release ETs in a protease-dependent manner, dependent or independent of the formation of reactive oxygen species and rearrangement of the cell cytoskeleton Similar to vertebrate leukocytes (eg, neutrophil), coelomocytes are responsible for many immune functions like phagocytosis, cytotoxicity and secretion of humoral factors ETs formed by coelomocyte analogues to neutrophil ETs consist of exDNA, histone H3 and attached to these structures proteins, eg, heat shock proteins HSP27 The latter fact confirms that mechanisms of ET release are conserved in evolution The study on Annelida adds this animal group to the list of invertebrates capable of ET release, but most importantly provides insides into innate mechanisms of ET formation in lower animal taxa

/pdf/earthworm-coelomocyte-extracellular-traps-structural-and-336t7v52ww.pdf

Earthworm coelomocyte extracellular traps: structural and functional similarities with neutrophil NETs.

The tube-within-tube body plan of earthworms is appropriate for studying the interactions of microorganisms with the immune system of body cavities such as the digestive tract and coelom. This study aims to describe the immune response on the molecular and cellular level in the coelomic cavity and the gut of the earthworm Eisenia andrei after experimental microbial challenge by administering two bacterial strains (Escherichia coli and Bacillus subtilis) or yeast Saccharomyces cerevisiae to the environment. The changes in mRNA levels of defense molecules (pattern recognition receptor CCF, lysozyme, fetidin/lysenins) in the coelomocytes and gut tissue were determined by quantitative PCR. The immune response at a cellular level was captured in histological sections, and the expression of CCF was localized using in situ hybridization. Coelomocytes respond to the presence of bacteria in the coelomic cavity by increasing the mRNA levels of defense molecules, especially CCF. The immune response in gut tissue is less affected by microbial stimulation because the epithelial cells of gut exhibit basically strong mRNA synthesis of ccf as a defense against the continuous microbial load in the gut lumen. The cellular immune response is mediated by coelomocytes released from the mesenchymal lining of the coelomic cavity. These combined immune mechanisms are necessary for the survival of earthworms in the microbially rich environment of soil.

Sensing microorganisms in the gut triggers the immune response in Eisenia andrei earthworms.

Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates.

/pdf/conservative-mechanisms-of-extracellular-trap-formation-by-15zacy825r.pdf

Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement.

Nanomaterials (NMs) can interact with the innate immunity of organisms. It remains, however, unclear whether these interactions can compromise the immune functioning of the host when faced with a disease threat. Co-exposure with pathogens is thus a powerful approach to assess the immuno-safety of NMs. In this paper, we studied the impacts of in vivo exposure to a biocidal NM on the gut microbiome, host immune responses, and susceptibility of the host to a bacterial challenge in an earthworm. Eisenia fetida were exposed to CuO-nanoparticles in soil for 28 days, after which the earthworms were challenged with the soil bacterium Bacillus subtilis. Immune responses were monitored by measuring mRNA levels of known earthworm immune genes. Effects of treatments on the gut microbiome were also assessed to link microbiome changes to immune responses. Treatments caused a shift in the earthworm gut microbiome. Despite these effects, no impacts of treatment on the expression of earthworm immune markers were recorded. The methodological approach applied in this paper provides a useful framework for improved assessment of immuno-safety of NMs. In addition, we highlight the need to investigate time as a factor in earthworm immune responses to NM exposure.

https://www.mdpi.com/2079-4991/10/7/1337/pdf

The Effects of In Vivo Exposure to Copper Oxide Nanoparticles on the Gut Microbiome, Host Immunity, and Susceptibility to a Bacterial Infection in Earthworms

Due to the specific habitat conditions in which they live, earthworms are constantly exposed to pathogens. Consequently, they have evolved various immuno-defense mechanisms, including cellular (coelomocytes) and humoral responses, which may help to eliminate deleterious micro-organisms but also repair and/or protect host cells and tissues. Similar to mammalian phagocytes, coelomocytes can kill ingested pathogens with reactive oxygen species (ROS) and nitric oxide. In the present work, we studied the effects of the dermal exposure of Eisenia andrei earthworms to different immuno-stimulants: phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS) or concanavalin A (ConA). After 3 days of treatment with all immuno-stimulants, decreased numbers and changed composition of the coelomocytes were observed. The immuno-stimulants also induced numerous changes in bactericidal activity, including ROS production. Furthermore, all stimulants increased cell proliferation while only LPS-treatment significantly elevated apoptosis of coelomocytes. These results demonstrate that in vivo treatment of earthworms with immuno-stimulants induces various changes in their coelomocyte response.

https://link.springer.com/content/pdf/10.1007%2Fs00360-012-0710-7.pdf

Dermal exposure to immunostimulants induces changes in activity and proliferation of coelomocytes of Eisenia andrei

although a relatively short text, the abstract of a paper summarizes the most important issues raised in the main text. The abstract is, at least initially, the key text on which journal editors, reviewers and eventually readers form their initial judgement on the overall quality of the full manuscript. Therefore, it is essential to execute this step of the writing process well. In this article, we discuss the purpose of an abstract, why it is important, and how to write a good abstract. Increasingly, journal abstracts are structured to follow the IMRaD format (Introduction, Methods, Results, and Discussion). We provide examples of well written and badly written abstracts, with explanatory notes, to help readers understand the key points that need to be addressed and mistakes that should be avoided. Since international abstracts are generally written in english, preparing an abstract can be especially challenging for researchers who are not native speakers of english. We close this article with general linguistic advice, paying particular attention to key terms and word choice than can meaningfully express an author’s intention in a concise way. The points raised in this article will help authors improve their scientific writing and enable their findings to be expressed with clarity. KEyWORDS: scientific writing, writing advice, abstract, IMRaD HoW To WRITe a gooD abSTRaCT foR a bIoMeDICal PaPeR Mark Jeremy Hunt1 e,f • ORCID: 0000-0001-7741-2191 Klaudia Raflik1,2 e,f • ORCID: 0000-0002-4453-259X Dawid Wesołowski1,3 e,f • ORCID: 0000-0003-1306-1139 agata Sibilska-Woźniacka1 e,f • ORCID: 0000-0002-7397-2826 Dorota eliza Sakowska-Hunt1 e,f • ORCID: 0000-0002-1325-5681 1 eCoRReCToR ltd, newton, Cambridge, uK 2 adam Mickiewicz university in Poznan, Poznan, Poland 3 Jagiellonian university in Cracow, Cracow, Poland A – study design, B – data collection, C – statistical analysis, D – interpretation of data, E – manuscript preparation, F – literature review, G – sourcing of funding What is an abstract? all researchers will have written an abstract for journal submissions or conference proceedings. The abstract is essentially a mini version of the full paper. The slightly unusual origin of the word, abstract, actually sums up its purpose very nicely. abstract, in the context of academic articles, comes from the latin abstractus – meaning to draw away. In relation to writing, this means that the central points of the paper are extracted into the form of a very short version of the full manuscript. This text, the abstract, is then a self-contained text that can be understood on its own, including all of the key aspects of the study that is being described. The abstract is drawn away from the main text and is presented at the beginning of the paper. Its purpose is to help readers decide whether or not the study is of sufficient interest for them to read the full manuscript. Why is an abstract important? You may think your abstract is of the same importance as the text in the main body of the paper. In fact, as I will show, the abstract and title are the most important part of your written work. This is best understood in terms of who the readers of your work are. as a general rule, a good abstract indicates a good paper, whereas a bad abstract – a weak one. Potential Readers. The most obvious targets are other scientists. Many scientists may read the abstract but far fewer read the paper. There needs to be enough information so that readers can decide whether or not to read the full paper. Reviewers. before your paper is even considered for publication it needs to be reviewed. Typically, 2-3 reviewers will read and evaluate your manuscript for its strengths and weaknesses. based on this, they will accept or reject the publication or recommend minor/ 58 Mark Jeremy Hunt, Klaudia Raflik, Dawid Wesołowski, agata Sibilska-Woźniacka, et al. www.medicalsciencepulse.com major revisions. When reviewers are invited to review a text the only information they have to go on is the title and abstract (the full text is usually only accessible if the reviewer agrees). Reviewers are often busy and do not have to review a paper. Indeed, reviewers may be close to making a final decision on the whole paper after having read the abstract alone. Therefore, it is fundamental that your title, and abstract, are well written to even be considered for publication. Search Engines. gone are the days when students and researchers searched through library journals to find papers, photocopying, and then continuing their search for more related articles. as a student, it always felt very rewarding to find a long sought-after paper. nowadays, once a manuscript has been accepted by a journal it is uploaded to various electronic libraries, and all searching can be done with the click of a button. However, in most cases only the title, and abstract, are visible to internet search engines. The main text often remains inaccessible behind paywalls. as such, your published abstract will be readily available on the internet, and the choice of keywords you use influences how easily your abstract can be found. The structure of an abstract early journals often published reports as chapters or letters where the writing style was usually descriptive and often chronological, e.g., “first I did [...] then I did [...]”. Key points of a paper, such as motivations, assumptions, results, and methodologies, were often buried in the text. as techniques became more advanced and the need for research reproducibility grew, the importance of clear scientific writing became a prominent issue. In the 1950s, Sir austin bradford Hill, a british epidemiologist and statistician who was increasingly frustrated with the lack of uniformity in scientific writing, stated that there were four central questions: 1. “Why did you start?” – Introduction 2. “What did you do?” – Methods 3. “What answer did you get?” – Results 4. “What does it mean?” – Discussion These are usually formulated into the IMRaD (Introduction, Methods, Results, and Discussion) format of papers that has been adopted by most journals. In parallel, the IMRaD format is the most widely used approach for writing an abstract. This is known as a structured abstract with the Introduction, Methods, Results, and Discussion forming subheadings within the abstract. abstracts often follow a wineglass structure with the opening and closing sentences very general and the detailed content in the methods and results sections. Typically, a structured abstract has 1-2 sentences for the Introduction, 1-2 for Methods, 3-4 for Results, and 1-2 for Discussion. not all journals have adopted the IMRaD approach, even many high-profile traditional journals, such as Nature and Science. However, the parallel structure can still be clearly seen despite the lack of subheading. often in these cases, a series of related studies have been carried out to address a common problem, and therefore combining methods with results is a clear way to express these findings. below you can find two examples published from high-impact journals, i.e., Nature and Neuron. In both, the abstracts open and close with broadly understood concepts understandable to non-specialists. The main section provides sufficient detail for specialists to grasp the novelty and sufficient details of the work. activation of microglia and inflammationmediated neurotoxicity are suggested to play a decisive role in the pathogenesis of several neurodegenerative disorders. activated microglia release pro-inflammatory factors that may be neurotoxic. Here we show that the orderly activation of caspase-8 and caspase-3/7, known executioners of apoptotic cell death, regulate microglia activation through a protein kinase C (PKC)-δ-dependent pathway. We find that stimulation of microglia with various inflammogens activates caspase-8 and caspase-3/7 in microglia without triggering cell death in vitro and in vivo. Knockdown or chemical inhibition of each of these caspases hindered microglia activation and consequently reduced neurotoxicity. We observe that these caspases are activated in microglia in the ventral mesencephalon of Parkinson’s disease (PD) and the frontal cortex of individuals with alzheimer’s disease (aD). Taken together, we show that caspase-8 and caspase-3/7 are involved in regulating microglia activation. We conclude that inhibition of these caspases could be neuroprotective by targeting the microglia rather than the neurons themselves. (burguillos et al., 2011) The olfactory bulbs (obs) are the first site of odor representation in the mammalian brain, and their unique ultrastructure is considered a necessary substrate for spatiotemporal coding of smell. given this, we were struck by the serendipitous observation at MRI of two otherwise healthy young left-handed women, yet with no apparent obs. Standardized tests revealed normal odor awareness, detection, discrimination, identification, and representation. functional MRI of these women’s brains revealed that odorant-induced activity in piriform cortex, the primary ob target, was similar in its extent to that of intact controls. finally, review of a public brain-MRI database with 1,113 participants (606 women) also tested for olfactory performance, uncovered olfaction without anatomically defined obs in ~0.6% of women and ~4.25% of left-handed women. Thus, humans can perform the basic facets of olfaction without canonical obs, implying extreme plasticity in the functional neuroanatomy of this sensory system. (Weiss et al., 2020) Introduction Methods and Results Discussion 59 How to write a good abstract for a biomedical paper Medical Science Pulse 2020 (14) 1 Example of a badly structured abstract Introduction: Huntington’s (HD) disease is an autosomal dominant neurodegenerative disease with no known cure. It is caused by progressive nerve cell death primarily affecting the dorsal striatum and leads to motor and cognitive symptoms. The

Dawid Wesolowski

Papers

Dermal exposure to immunostimulants induces changes in activity and proliferation of coelomocytes of Eisenia andrei

How to write a good abstract for a biomedical paper