O-083 Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo

Study question Can we separate between control and reversine-treated cells within the inner cell mass (ICM) of the mouse preimplantation embryo by using label-free and non-invasive hyperspectral microscopy? Summary answer Hyperspectral microscopy is able to discern between control and reversine-treated cells using cellular autofluorescence in the complete absence of fluorescence tags. What is known already Embryo mosaicism (containing cells that are euploid (46 chromosomes) and aneuploid (deviation from the expected number of chromosomes)) affects up to 17.3% of human blastocyst embryos. Current diagnosis of aneuploidy in the IVF clinic involves a biopsy of trophectoderm (TE) cells or spent media followed by sequencing. In some blastocyst embryos these approaches will fail to diagnose of the proportion of aneuploid cells within the fetal lineage (ICM). Study design, size, duration The impact of aneuploidy on cellular metabolism was assessed by using cellular autofluoresence and hyperspectral microscopy (broad spectral profile). Two models were employed: (i) Primary human fibroblast cells with known karyotypes (4-6 independent replicates, euploid n = 467; aneuploid n = 969) and reversine induced aneuploidy in mouse embryos (5-8 independent replicates, 30-44 cells per group). Both models were subjected to hyperspectral imaging to quantify native cell fluorescence. Participants/materials, setting, methods The human model is comprised of euploid (male and female) and aneuploid (triploid and trisomies: 13, 18, 21, XXX, and XXY) primary human fibroblast cells. For the mouse model, we treated embryos with reversine, a reversible spindle assembly checkpoint inhibitor, during the 4- to 8-cell division. Individual blastomeres were dissociated from control and reversine treated 8-cell embryos. Blastomeres were either imaged directly or used to generate chimeric blastocysts with differing ratios of control:reversine-treated cells. Main results and the role of chance Following unsupervised linear unmixing, the relative abundance of metabolic cofactors was quantified: reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavins with the subsequent calculation of the optical redox ratio (ORR: Flavins/[NAD(P)H + Flavins]). Primary human fibroblast cells displayed an increase in the relative abundance of NAD(P)H with a decrease in flavins, leading to a significant reduction in the ORR for aneuploid cells (P < 0.05). The mouse embryos displayed an identical trend as the human model between control and reversine-treated embryos. Mathematical algorithms were applied and able to distinguish between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres and (iii) chimeric blastocysts with differing ratios of control and reversine-treated cells. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve. We also showed that hyperspectral imaging of the preimplantation embryo does not impact on embryo developmental competence, pregnancy outcome and offspring health in a mouse model. We believe the role of chance is low as both human somatic cells and mouse embryos showed a consistent shift in cellular metabolism in response to human fibroblast cells that are aneuploid and reversine treated mouse embryos. Limitations, reasons for caution Further validation of our approach could include sequencing of the ICM of individual blastocysts to determine the proportion of aneuploid cells in ICM and correlate this with the metabolic profile obtained through hyperspectral imaging. Wider implications of the findings With hyperspectral imaging able to discriminate between (i) euploid and aneuploid human fibroblast cells and (ii) control and reversine-treated mouse embryos, this could be an accurate, non-invasive and label-free optical imaging approach to assess mosaicism within the ICM of mouse embryos, potentially leading to a new diagnostic tool for embryos. Trial registration number Not applicable

Modulation of αVβ6 integrin in osteoarthritis-related synovitis and the interaction with VTN(381–397 a.a.) competing for TGF-β1 activation

Osteoarthritis is characterized by structural alteration of joints. Fibrosis of the synovial tissue is often detected and considered one of the main causes of joint stiffness and pain. In our earlier proteomic study, increased levels of vitronectin (VTN) fragment (amino acids 381–397) were observed in the serum of osteoarthritis patients. In this work, the affinity of this fragment for integrins and its putative role in TGF-β1 activation were investigated. A competition study determined the interaction of VTN(381–397 a.a.) with αVβ6 integrin. Subsequently, the presence of αVβ6 integrin was substantiated on primary human fibroblast-like synoviocytes (FLSs) by western blot and flow cytometry. By immunohistochemistry, β6 was detected in synovial membranes, and its expression showed a correlation with tissue fibrosis. Moreover, β6 expression was increased under TGF-β1 stimulation; hence, a TGF-β bioassay was applied. We observed that αVβ6 could mediate TGF-β1 bioavailability and that VTN(381–397 a.a.) could prevent TGF-β1 activation by interacting with αVβ6 in human FLSs and increased α-SMA. Finally, we analyzed serum samples from healthy controls and patients with osteoarthritis and other rheumatic diseases by nano-LC/Chip MS–MS, confirming the increased expression of VTN(381–397 a.a.) in osteoarthritis as well as in lupus erythematosus and systemic sclerosis. These findings corroborate our previous observations concerning the overexpression of VTN(381–397 a.a.) in osteoarthritis but also in other rheumatic diseases. This fragment interacts with αVβ6 integrin, a receptor whose expression is increased in FLSs from the osteoarthritic synovial membrane and that can mediate the activation of the TGF-β1 precursor in human FLSs.

Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo

Study questionCan label-free, non-invasive optical imaging by hyperspectral microscopy discern between euploid and aneuploid cells within the inner cell mass of the mouse preimplantation embryo?Summary answerHyperspectral microscopy shows a variance in metabolic activity which enables discrimination between euploid and aneuploid cells.What is known alreadyEuploid/aneuploid mosaicism affects up to 17.3% of human blastocyst embryos with trophectoderm biopsy or spent media currently utilised to diagnose aneuploidy and mosaicism in clinical in vitro fertilisation. Based on their design, these approaches will fail to diagnose the presence or proportion of aneuploid cells within the fetal lineage (inner cell mass (ICM)) of some blastocyst embryos.Study design, size, durationThe impact of aneuploidy on cellular metabolism of primary human fibroblast cells and mouse embryos was assessed by a fluorescence microscope adapted for imaging with multiple spectral channels (hyperspectral imaging). Primary human fibroblast cells with known ploidy were subjected to hyperspectral imaging to record native cell fluorescence (euploid n= 467; aneuploid n= 969). For mouse embryos, 50-70 individual euploid and aneuploid blastomeres (8-cell stage embryo) and chimeric blastocysts (40-50 per group: euploid; aneuploid; or 1:1 and 1:3 ratio of euploid:aneuploid) were utilised for hyperspectral imaging.Participants/materials, setting, methodsTwo models were employed: (i) Primary human fibroblasts with known karyotype and (ii) a mouse model of embryo aneuploidy where mouse embryos were treated with reversine, a reversible spindle assembly checkpoint inhibitor, during the 4-to 8-cell division. Individual blastomeres were dissociated from reversine treated (aneuploid) and control (euploid) 8-cell embryos and either imaged directly or used to generate chimeric blastocysts with differing ratios of euploid:aneuploid cells. Individual blastomeres and embryos were subjected to hyperspectral imaging. Changes in cellular metabolism were determined by quantification of metabolic cofactors (inferred from their autofluorescence signature): reduced nicotinamide adenine dinucleotide (NAD(P)H), flavins with the subsequent calculation of the optical redox ratio (ORR: Flavins/[NAD(P)H + Flavins]). Mathematical algorithms were applied to extract features from the autofluorescence signals of each cell/blastomere/inner cell mass to discriminate between euploid and aneuploid.Main results and the role of chanceAn increase in the relative abundance of NAD(P)H with a decrease in flavins led to a significant reduction in the ORR for aneuploid cells in both primary human fibroblasts and individual mouse blastomeres (P < 0.05). Mathematical algorithms were able to achieve good separation between (i) euploid and aneuploid primary human fibroblast cells, (ii) euploid and aneuploid mouse blastomeres cells and (iii) euploid and aneuploid chimeric blastocysts and (iv) 1:1 and 1:3 chimeric blastocysts. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve of 0.85, 0.99, 0.87 and 0.88, respectively. We believe that the role of chance is low as multiple cellular models (human somatic cells and mouse embryos) demonstrated a consistent shift in cellular metabolism in response to aneuploidy as well as the robust capacity of mathematical features to separate euploid and aneuploid cells in a statistically significant manner.Limitations, reasons for cautionThere would be added value in determining the degree of embryo mosaicism by sequencing the inner cell mass (ICM) of individual blastocysts to correlate with metabolic profile and level of discrimination achieved using the mathematical features approach.Wider implications of the findingsHyperspectral imaging was able to discriminate between euploid and aneuploid human fibroblasts and mouse embryos. This may lead to the development of an accurate and non-invasive optical approach to assess mosaicism within the ICM of human embryos in the absence of fluorescent tags.Study funding/competing interest(s)K.R.D. is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58-2019). This study was funded by the Australian Research Council Centre of Excellence for Nanoscale Biophotonics (CEI40100003). The authors declare that there is no conflict of interest.

Antitumoral Effects of Curcumin (Curcuma longa L.) and Thymoquinone (Nigella sativa L.) on Neuroblastoma Cell Lines

<b><i>Introduction:</i></b> Overall survival of high-risk neuroblastoma patients is still poor, emphasizing the need for novel therapeutic options. There is evidence for anti-cancer properties of the herbal substances thymoquinone and curcumin. These substances are isolated from <i>Nigella sativa</i> L. and <i>Curcuma longa</i> L., respectively, which are used in traditional medicine. <b><i>Objective:</i></b> We investigated cytotoxic effects of thymoquinone and curcumin on neuroblastoma cell lines NLF, NB69, and SK-N-BE(2), in vitro. <b><i>Methods:</i></b> Cytotoxicity of compounds was investigated by MTT cell viability assays. For analyzing effects on cell proliferation BrdU assays were employed and induction of apoptosis was detected by Cell Death ELISA assays. <b><i>Results:</i></b> Both substances showed cytotoxic effects in all three neuroblastoma cell lines, whereby primary human fibroblast cells reacted less sensitively. Overall, lower IC50 values could be calculated for curcumin (3.75–7.42 µM) than for thymoquinone (5.16–16.3 µM). Decreased proliferation and increased apoptosis rates were observed under treatment. <b><i>Conclusions:</i></b> Both substances showed anti-tumoral properties on neuroblastoma cell lines and should be further investigated as therapeutic agents.

In Vitro Activity of Beauvericin against All Developmental Stages of Sarcoptes scabiei

ABSTRACT Scabies is a frequent cutaneous infection caused by the mite Sarcoptes scabiei in a large number of mammals, including humans. As the resistance of S. scabiei against several chemical acaricides has been previously documented, the establishment of alternative and effective control molecules is required. In this study, the potential acaricidal activity of beauvericin was assessed against different life stages of S. scabiei var. suis and in comparison with dimpylate and ivermectin, two commercially available molecules used for the treatment of S. scabiei infection in animals and/or humans. The toxicity of beauvericin against cultured human fibroblast skin cells was evaluated using an MTT proliferation assay. In our in vitro model, developmental stages of S. scabiei were placed in petri dishes filled with Columbia agar supplemented with pig serum and different concentrations of the drugs. Cell sensitivity assays demonstrated low toxicity of beauvericin against primary human fibroblast skin cells. At 0.5 and 5 mM, beauvericin showed higher activity against adults and eggs of S. scabiei compared to dimpylate and ivermectin. These results revealed that the use of beauvericin is promising and might be considered for the treatment of S. scabiei infection.

In vitro activity of beauvericin against all developmental stages of Sarcoptes scabiei

BackgroundScabies is a frequent cutaneous infection caused by the mite Sarcoptes scabiei in a large number of mammals including humans. As the resistance of S. scabiei against several chemical acaricides has been previously documented, the establishment of alternative and effective control molecules is required.ObjectivesIn this study, the potential acaricidal activity of beauvericin was assessed against different life stages of S. scabiei var. suis and, in comparison with dimpylate and ivermectin, two commercially available molecules used for the treatment of S. scabiei infection in animals and/or humans.MethodsIn our in vitro model, developmental stages of S. scabiei have been placed in Petri dishes filled with Columbia agar supplemented with pig serum and different concentrations of the drugs. Moreover, the toxicity of beauvericin against cultured human fibroblast skin cells was evaluated using an MTT proliferation assayResultsBeauvericin showed higher activity against adults and eggs of S. scabiei when compared to dimpylate and ivermectin. In addition, cell sensitivity assays demonstrated low toxicity of beauvericin against primary human fibroblast skin cells.ConclusionThese results revealed that the use of beauvericin is promising and might be considered for the treatment of S. scabiei infection.