Learning to segment clustered amoeboid cells from brightfield microscopy via multi-task learning with adaptive weight selection

Detecting and segmenting individual cells from microscopy images is critical to various life science applications. Traditional cell segmentation tools are often ill-suited for applications in brightfield microscopy due to poor contrast and intensity heterogeneity, and only a small subset are applicable to segment cells in a cluster. In this regard, we introduce a novel supervised technique for cell segmentation in a multi-task learning paradigm. A combination of a multi-task loss, based on the region and cell boundary detection, is employed for an improved prediction efficiency of the network. The learning problem is posed in a novel min-max framework which enables adaptive estimation of the hyper-parameters in an automatic fashion. The region and cell boundary predictions are combined via morphological operations and active contour model to segment individual cells. The proposed methodology is particularly suited to segment touching cells from brightfield microscopy images without manual interventions. Quantitatively, we observe an overall Dice score of 0.93 on the validation set, which is an improvement of over 15.9% on a recent unsupervised method, and outperforms the popular supervised U-net algorithm by at least $5.8\%$ on average

An ex-vivo Human Intestinal Model to Study Entamoeba histolytica Pathogenesis

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Host Tissue Invasion by Entamoeba histolytica Is Powered by Motility and Phagocytosis

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PI Kinase-EhGEF2-EhRho5 axis contributes to LPA stimulated macropinocytosis in Entamoeba histolytica

International audienceEntamoeba histolytica is a protozoan responsible for several pathologies in humans. Trophozoites breach the intestinal site to enter the bloodstream and thus traverse to a secondary site. Macropinocytosis and phagocytosis, collectively accounting for heterophagy, are the two major processes responsible for sustenance of Entamoeba histolytica within the host. Both of these processes require significant rearrangements in the structure to entrap the target. Rho GTPases play an indispensable role in mustering proteins that regulate cytoskeletal remodelling. Unlike phagocytosis which has been studied in extensive detail, information on machinery of macropinocytosis in E. histolytica is still limited. In the current study, using site directed mutagenesis and RNAi based silencing, coupled with functional studies, we have demonstrated the involvement of EhRho5 in constitutive and LPA stimulated macropinocytosis. We also report that LPA, a bioactive phospholipid present in the bloodstream of the host, activates EhRho5 and translocates it from cytosol to plasma membrane and endomembrane compartments. Using biochemical and FRAP studies, we established that a PI Kinase acts upstream of EhRho5 in LPA mediated signalling. We further identified EhGEF2 as a guanine nucleotide exchange factor of EhRho5. In the amoebic trophozoites, EhGEF2 depletion leads to reduced macropinocytic efficiency of trophozoites, thus phenocopying its substrate. Upon LPA stimulation, EhGEF2 is found to sequester near the plasma membrane in a wortmannin sensitive fashion, explaining a possible mode for activation of EhRho5 in the amoebic trophozoites. Collectively, we propose that LPA stimulated macropinocytosis in E. histolytica is driven by the PI Kinase-EhGEF2-EhRho5 axis

Crosstalk between Entamoeba histolytica and the human intestinal tract during amoebiasis

International audienceThe protozoan parasite Entamoeba histolytica is the microbial agent of amoebiasis-an infection that is endemic worldwide and is associated with high morbidity and mortality rates. As the disease develops, virulent E. histolytica deplete the mucus layer, interact with the intestinal epithelium, and then degrade the colonic mucosa and disrupt the extracellular matrix (ECM). Our research demonstrated that virulent parasites with an invasive phenotype display rapid, highly specific changes in their transcriptome (notably for essential factors involved in carbohydrate metabolism and the processing of glycosylated residues). Moreover, combined activation of parasite and host lytic enzymes leads to the destruction of the intestinal parenchyma. Together, these enzymes degrade the mucus layer and the ECM, and trigger the inflammatory response essential to the development of amoebiasis

Cloning and expression of the calmodulin-sensitive Bacillus anthracis adenylate cyclase in Escherichia coli

International audienceThe adenylate cyclase gene of Bacillus anthracis, encoding the edema factor, a component of anthrax toxin, has been cloned and expressed in Escherichia coli. Clones were selected by their capacity to complement the cyclase deficiency (cya−) of an E. coli strain expressing the eukaryotic protein calmodulin, an essential activator of B. anthracis adenylate cyclase. The protein expressed in E. coli was shown to exhibit adenylate cyclase activity only in the presence of calmodulin. Experiments using a coupled in vitro transcription-translation system revealed that the protein synthesized from the cloned DNA fragment was enzymatically active, upon addition of calmodulin, and could be immunoprecipitated by antibodies directed against purified Bordetella pertussis adenylate cyclase toxin. This indicates that the two calmodulin-dependent adenylate cyclase toxins are immunologically related

EhPAK, a member of the p21-activated kinase family, is involved in the control of Entamoeba histolytica migration and phagocytosis

International audienceEntamoeba histolytica migration is essential for the development of amoebiasis, a human disease characterised by invasion and destruction of tissues. Amoebic motility requires both polarisation of the cell and formation of a predominant pseudopod. As p21-activated kinases PAKs are known to regulate eukaryotic cell motility and morphology, we investigated the role of PAK in E. histolytica. We showed that the C-terminal domain of EhPAK comprised a constitutive kinase activity in vitro and that overproduction of this fragment, in E. histolytica, caused a significant reduction in amoeboid migration, as measured by dynamic image analysis, indicating an involvement of EhPAK in this process. A dramatic loss of polarity, as indicated by the increased number of membrane extensions all around E. histolytica, was also observed, suggesting that the N-terminal domain of EhPAK was necessary for maintenance of cell polarity. To support this view, we showed that despite the absence of the consensus motif to bind to Rac and Cdc42, the N-terminal domain of EhPAK bound to Rac1, suggesting that the N-terminal region was a regulatory domain. In addition, we also found an increased rate of human red blood cell phagocytosis, suggesting for the first time an active role for a PAK protein in this process. Taking together, the results suggest strongly that EhPAK is a key regulatory element in polarity, motility and phagocytosis of E. histolytica

Calcium binding protein 1 of the protozoan parasite Entamoeba histolytica interacts with actin and is involved in cytoskeleton dynamics

International audienceBlocking expression of EhCaBP1, a calmodulin-like, four EF-hand protein from the protozoan parasite Entamoeba histolytica, resulted in inhibition of cellular proliferation. In this paper we report that EhCaBP1 is involved in dynamic changes of the actin cytoskeleton. Both endocytosis and phagocytosis were severely impaired in cells where EhCaBP1 expression was blocked by inducible expression of the antisense RNA. In wild-type cells both actin and EhCaBP1 were found to co-localize in phagocytic cups and in pseudopods. However, in antisense-blocked cells the phagocytic cup formation is affected. Analysis of the staining patterns in the presence and absence of actin dynamics inhibitors, jasplakinolide and cytochalasin D suggested that EhCaBP1 and polymerized F-actin co-localize on membrane protrusions. Direct interaction between soluble EhCaBP1 and F-actin was further demonstrated by a co-sedimentation assay. A variant of EhCaBP1 did not bind F-actin showing the specificity of the interaction between EhCaBP1 and actin. There is no significant change in the kinetics of in vitro polymerization of actin in presence of EhCaBP1, indicating that EhCaBP1 does not affect filament treadmilling. In addition, using atomic force microscopy; it was found that filaments of F-actin, polymerized in presence of EhCaBP1, were thinner. These results indicate that EhCaBP1 may be involved in dynamic membrane restructuring at the time of cell pseudopod formation, phagocytosis and endocytosis in a process mediated by direct binding of EhCaBP1 to actin, affecting the bundling of actin filaments

In Entamoeba histolytica, a BspA family protein is required for chemotaxis toward tumour necrosis factor

Background: [i]Entamoeba histolytica[/i] cell migration is essential for the development of human amoebiasis (an infectious disease characterized by tissue invasion and destruction). The tissue inflammation associated with tumour necrosis factor (TNF) secretion by host cells is a well-documented feature of amoebiasis. Tumour necrosis factor is a chemoattractant for E. histolytica, and the parasite may have a TNF receptor at its cell surface. Methods: confocal microscopy, RNA Sequencing, bioinformatics, RNA antisense techniques and histological analysis of human colon explants were used to characterize the interplay between TNF and E. histolytica. Results: an antibody against human TNF receptor 1 (TNFR1) stained the E. histolytica trophozoite surface and (on immunoblots) binds to a 150-kDa protein. Proteome screening with the TNFR1 sequence revealed a BspA family protein in E. histolytica that carries a TNFR signature domain and six leucine-rich repeats (named here as “cell surface protein”, CSP, in view of its cellular location). Cell surface protein shares structural homologies with Toll-Like receptors, colocalizes with TNF and is internalized in TNF-containing vesicles. Reduction of cellular CSP levels abolished chemotaxis toward TNF and blocked parasite invasion of human colon. Conclusions: there is a clear link between TNF chemotaxis, CSP and pathogenesis