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  1. Article ; Online: Collection of cells for single-cell RNA sequencing using high-resolution fluorescence microscopy

    Hendrika A. Segeren / Kiki C. Andree / Lisa Oomens / Bart Westendorp

    STAR Protocols, Vol 2, Iss 3, Pp 100718- (2021)

    2021  

    Abstract: Summary: FACS sorting followed by single-cell RNA-sequencing (SORT-Seq) is a popular procedure to select cells of interest for single-cell transcriptomics. However, FACS is not suitable for measurement of subcellular distribution of fluorescence or for ... ...

    Abstract Summary: FACS sorting followed by single-cell RNA-sequencing (SORT-Seq) is a popular procedure to select cells of interest for single-cell transcriptomics. However, FACS is not suitable for measurement of subcellular distribution of fluorescence or for small samples (<1,000 cells). The VYCAP puncher system overcomes these limitations. Here, we describe a workflow to capture, image, and collect fluorescent human retina pigment epithelium cells for SORT-Seq using this system. The workflow can be used for any cell type with a diameter of ∼5–50 μm.For complete details on the use and execution of this protocol, please refer to Segeren et al. (2020).
    Keywords Cell Biology ; Single Cell ; Genomics ; Microscopy ; Science (General) ; Q1-390
    Language English
    Publishing date 2021-09-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Glycosylated extracellular mucin domains protect against SARS-CoV-2 infection at the respiratory surface.

    Maitrayee Chatterjee / Liane Z X Huang / Anna Z Mykytyn / Chunyan Wang / Mart M Lamers / Bart Westendorp / Richard W Wubbolts / Jos P M van Putten / Berend-Jan Bosch / Bart L Haagmans / Karin Strijbis

    PLoS Pathogens, Vol 19, Iss 8, p e

    2023  Volume 1011571

    Abstract: Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by ... ...

    Abstract Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of terminal mucin glycans sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry.
    Keywords Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2023-08-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Excessive E2F Transcription in Single Cancer Cells Precludes Transient Cell-Cycle Exit after DNA Damage

    Hendrika A. Segeren / Lotte M. van Rijnberk / Eva Moreno / Frank M. Riemers / Elsbeth A. van Liere / Ruixue Yuan / Richard Wubbolts / Alain de Bruin / Bart Westendorp

    Cell Reports, Vol 33, Iss 9, Pp 108449- (2020)

    2020  

    Abstract: Summary: E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer ... ...

    Abstract Summary: E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer biopsy specimens that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we delete the atypical E2F repressors (E2F7/8) or overexpress the E2F3 activator in untransformed cells. Cells with elevated E2F activity during S/G2 phase fail to exit the cell cycle after DNA damage and undergo mitosis. In contrast, wild-type cells complete S phase and then exit the cell cycle by activating the APC/CCdh1 via repression of the E2F target Emi1. Many arrested wild-type cells eventually inactivate APC/CCdh1 to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F transcription fail to exit the cell cycle after DNA damage, which potentially causes genomic instability, promotes malignant progression, and reduces drug sensitivity.
    Keywords cell cycle ; anaphase-promoting complex/cyclosome ; E2F transcription ; live-cell imaging ; single-cell RNA sequencing ; DNA damage ; Biology (General) ; QH301-705.5
    Language English
    Publishing date 2020-12-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Dietary sodium restriction specifically potentiates left ventricular ACE inhibition by zofenopril, and is associated with attenuated hypertrophic response in rats with myocardial infarction

    Bart Westendorp / Regien G Schoemaker / Hendrik Buikema / Dick de Zeeuw / Dirk J van Veldhuisen / Wiek H van Gilst

    Journal of the Renin-Angiotensin-Aldosterone System, Vol

    2004  Volume 5

    Abstract: Introduction In patients with myocardial infarction (MI)-induced heart failure, angiotensin-converting enzyme (ACE) inhibitors are proven effective therapy in inhibiting the progression towards overt heart failure. However, the prognosis in these ... ...

    Abstract Introduction In patients with myocardial infarction (MI)-induced heart failure, angiotensin-converting enzyme (ACE) inhibitors are proven effective therapy in inhibiting the progression towards overt heart failure. However, the prognosis in these patients is still very poor, and optimisation of therapy is warranted. The antihypertensive and renoprotective effects of ACE inhibitors (ACE-Is) can be substantially enhanced by dietary sodium restriction. In line with the latter, the aim of the present study was to explore whether dietary sodium restriction enhances the efficacy of ACE-I after MI. Methods Rats with MI-induced left ventricular (LV) dysfunction received ACE-I therapy with zofenopril (5.5 mg/kg/day orally), with or without dietary sodium restriction. ACE activity was measured in non-infarcted LV tissue, kidneys and plasma. Effects on cardiac hypertrophy were examined by means of organ weight/body weight ratios. After blood pressure (BP) measurements, functional consequences of therapy were evaluated as LV pressure development in isolated perfused hearts. Results Dietary sodium restriction alone had no effect on any of the measured parameters, whereas zofenopril alone significantly reduced plasma and kidney ACE activity, but not LV ACE activity, nor LV weight/body weight ratio. However, only when ACE-I therapy was combined with dietary sodium restriction was LV ACE activity significantly reduced. This effect was paralleled by inhibition of LV hypertrophy. BP was reduced after infarction, and further reduced by zofenopril, but not affected by dietary sodium. Neither treatment was associated with effects on the MI-induced reduction of LV function in vitro . Conclusions Effects of ACE inhibition with zofenopril can be potentiated by additional dietary sodium restriction. However, these effects were tissue-specific, since LV, but not kidney or plasma, ACE activity was affected by the additional dietary sodium restriction. Effects on LV ACE activity were paralleled by reduced LV hypertrophy. Since the measured ...
    Keywords Medicine (General) ; R5-920
    Subject code 610
    Language English
    Publishing date 2004-03-01T00:00:00Z
    Publisher Hindawi - SAGE Publishing
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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