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  • A simple method for the isolation of leaf epidermis from graminaceous species for studying stomatal physiology

    Brett, S; Ng, C;
    School of Biology and Environmental Science, UCD, O’Brien Centre for Science (West), University College Dublin, Dublin 4, Ireland
    Brachypodium distachyon_ has been gaining traction as the model annual species for economically important temperate grasses such as wheat and barley. Methods for tissue culture and genetic transformation have been developed for this model grass as a resource for functional genomics and reverse genetics. More recently, _B. distachyon_ has been used to elucidate the underlying mechanisms regulating stomatal development in monocotyledons. However, there is a paucity of methods for facilitating the use of _B. distachyon_ for studying stomatal physiology. We developed a simple and easy method for the isolation of intact leaf epidermis from _B. distachyon_ with minimal mesophyll contamination. Our results showed that stomatal guard cells remain viable in the isolated leaf epidermis and that stomata can open in the presence of potassium chloride and light, and that this opening response can be inhibited by abscisic acid in a dose-dependent manner. Additionally, we also showed that the method we have developed can be used for isolating leaf epidermis from a range of graminaceous species with minimal mesophyll contamination, including Triticum aestivum, T. macha, T. uratu, and T. aethiopicum, Hordeum vulgare, Avena sativa, and Aegilops tauschii.
  • Reactivation of the Hedgehog pathway in esophageal progenitors turns on an embryonic-like program to initiate columnar metaplasia

    Vercauteren Drubbel, A; Pirard, S; Kin, S; Dassy, B; Lefort, A; Libert, F; Nomura, S; Beck, B
    IRIBHM, ULB/Faculty of Medicine, 808 route de Lennik, 1070 Brussels, Belgium
    Columnar metaplasia of the esophagus is the main risk factor for esophageal adenocarcinoma. There is a lack of evidence to demonstrate that esophageal progenitors can be the source of columnar metaplasia. In this study, using transgenic mouse models, lineage tracing, single-cell RNA sequencing, and transcriptomic and epigenetic profiling, we found that the activation of the Hedgehog pathway in esophageal cells modifies their differentiation status in vivo. This process involves an initial step of dedifferentiation into embryonic-like esophageal progenitors. Moreover, a subset of these cells undergoes full squamous-to-columnar conversion and expresses selected intestinal markers. These modifications of cell fate are associated with remodeling of the chromatin and the appearance of Sox9. Using a conditional knockout mouse, we show that Sox9 is required for columnar conversion but not for the step of dedifferentiation. These results provide insight into the mechanisms by which esophageal cells might initiate columnar metaplasia.
  • Mechanistic understanding of the combined immunodeficiency in complete human CARD11 deficiency

    Lu, HY; Sharma, M; Sharma, AA; Lacson, A; Szpurko, A; Luider, J; Dharmani-Khan, P; Shameli, A; Bell, PA; Guilcher, GMT; Lewis, VA; Vasquez, MR; Desai, S; McGonigle, L; Murguia-Favela, L; Wright, NAM; Sergi, C; Wine, E; Overall, CM; Suresh, S; Turvey, SE
    Department of Pediatrics, British Columbia Children's Hospital, The University of British Columbia, Vancouver, BC, Canada; Experimental Medicine Program, Faculty of Medicine, The University of British Columbia, Vancouver, BC, Canada
    Product(s): FK506 (Tacrolimus)
    Germline pathogenic variants impairing the caspase recruitment domain family member 11 (CARD11)-B cell CLL/lymphoma 10 (BCL10)-MALT1 paracaspase (MALT1) [CBM] complex are associated with diverse human diseases including combined immunodeficiency (CID), atopy, and lymphoproliferation. However, the impact of CARD11 deficiency on human B cell development, signalling, and function is incompletely understood. We sought to determine the cellular, immunological, and biochemical basis of disease for two unrelated patients who presented with profound CID associated with viral and fungal respiratory infections, interstitial lung disease, and severe colitis. Patients underwent next-generation sequencing, immunophenotyping by flow cytometry, signalling assays by immunoblot, and transcriptome profiling by RNA-Seq. Both patients carried identical novel pathogenic biallelic loss-of-function variants in CARD11 (c.2509C>T; p.Arg837*) leading to undetectable protein expression. This variant prevented CBM complex formation, severely impairing the activation of NF-κB, JNK, and MALT1 paracaspase activity in B and T cells. This functional defect resulted in a developmental block in B cells at the naïve and type 1 (T1) transitional B cell stage and impaired circulating T follicular helper cell (cTfh) development, which was associated with impaired antibody responses and absent germinal centre structures on lymph node histology. Transcriptomics indicated that CARD11-dependent signalling is essential for immune signalling pathways involved in the development of these cells. Both patients underwent hematopoietic stem cell transplantations (HSCT), which led to functional normalization. Complete human CARD11 deficiency causes profound CID by impairing naïve/T1 B cell and cTfh cell development, and abolishing activation of MALT1 paracaspase, NF-κB, and JNK activity. HSCT functionally restores impaired signalling pathways.
  • The Portal Vertex of KSHV Promotes Docking of Capsids at the Nuclear Pores

    Dünn-Kittenplon, D; Ashkenazy-Titelman, A; Kalt, I; Lellouche, JP; Shav-Tal, Y; Sarid, R;
    The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel.||Department of Chemistry, Bar-Ilan University, Ramat Gan 5290002, Israel.||Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat G
    Product(s): G-418 Sulfate
    Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-related herpesvirus. Like other herpesviruses, the KSHV icosahedral capsid includes a portal vertex, composed of 12 protein subunits encoded by open reading frame (ORF) 43, which enables packaging and release of the viral genome into the nucleus through the nuclear pore complex (NPC). Capsid vertex-specific component (CVSC) tegument proteins, which directly mediate docking at the NPCs, are organized on the capsid vertices and are enriched on the portal vertex. Whether and how the portal vertex is selected for docking at the NPC is unknown. Here, we investigated the docking of incoming ORF43-null KSHV capsids at the NPCs, and describe a significantly lower fraction of capsids attached to the nuclear envelope compared to wild-type (WT) capsids. Like WT capsids, nuclear envelope-associated ORF43-null capsids co-localized with different nucleoporins (Nups) and did not detach upon salt treatment. Inhibition of nuclear export did not alter WT capsid docking. As ORF43-null capsids exhibit lower extent of association with the NPCs, we conclude that although not essential, the portal has a role in mediating the interaction of the CVSC proteins with Nups, and suggest a model whereby WT capsids can dock at the nuclear envelope through a non-portal penton vertex, resulting in an infection 'dead end'.
  • Determination of Free Solanesol Levels in Cigarette Filters by Liquid Chromatography-Mass Spectrometry

    Bravo Cardenas, R; Ngac, P; Watson, C; Valentin-Blasini, L
    Tobacco Volatiles Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, N.E., Mailstop F-19, Atlanta, GA 30341, USA
    Product(s): Geranylgeraniol
    Solanesol, a naturally occurring constituent of tobacco, has been utilized as a good marker for environmental tobacco smoke particulate and as a non-invasive predictor of mainstream cigarette smoke tar and nicotine intake under naturalistic smoking conditions. A fast and accurate method for measuring free solanesol to assess tobacco smoke exposure is highly desirable. We have developed and validated a new environmentally friendly, high throughput method for measuring solanesol content in discarded cigarette filter butts. The solanesol deposited in the used filters can be correlated with mainstream smoke deliveries of nicotine and total particle matter (TPM) to estimate constituent delivery to smokers. A portion of filter material is removed from cigarette butts after machine smoking, spiked with internal standard solution, extracted, and quantitatively analyzed using reverse phase liquid chromatography coupled to a triple quadrupole mass spectrometer. The new method incorporates a 48-well plate format for automated sample preparation that reduces sample preparation time and solvent use and increases sample throughput 10-fold compared to our previous method. Accuracy and precision were evaluated by spiking known amounts of solanesol on both clean and smoked cigarette butts. Recoveries exceeded 93% at both low and high spiking levels. Linear solanesol calibration curves ranged from 1.9 to 367 µg/butt with a 0.05 µg/butt limit of detection.

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