Comparative LM, SEM and EDAX study of chalk glands on leaf and stem of two species of Plumbago Linn.

Stem and leaves of two species of PlumbagoLinn.viz. P. zeylanica Linn.andP. auriculata Lam. were investigated for the structure and chemical composition of chalk glands. Light Microscopy (LM) and Scanning Electron Microscopy (SEM) revealed the presence of chalk glands on both lower as well as upper surface of leaf and stem of both species. Chalk glands are abundant on lower surface and sparse on upper surface of leaf. Chalk glands are approximately hemispherical glands with oval or almost circular outline. It is composed of 8 cells arranged in two circles – central circle of 4 secretory cells and outer circle of 4 adjoining cells. Each secretory cell has depression which corresponds to pore. Each gland is surrounded by 4 subsidiary cells. No significant difference in the structure of chalk glands in both species was noticed. Chalk glands occupy three different positions with regard to epidermal cells –at the same level of the epidermis, slightly sunken in the epidermis and slightly raised above the epidermis. Common elements found in EDAX analysis of all chalk glands are carbon, oxygen, magnesium, sulphur, potassium and calcium. Differences in the presence of elements silicon, chlorine, aluminium, sodium, phosphorus were observed. The presence of significant amount of calcium in chalk glands and their dried deposits and absence of sodium and chlorine from dried deposits and even in some chalk glands appealed to use the term ‘Chalk gland’ instead of ‘Salt gland’ in Plumbago.

Myxospermy Evolution in Brassicaceae: A Highly Complex and Diverse Trait with Arabidopsis as an Uncommon Model

The ability to extrude mucilage upon seed imbibition (myxospermy) occurs in several Angiosperm taxonomic groups, but its ancestral nature or evolutionary convergence origin remains misunderstood. We investigated seed mucilage evolution in the Brassicaceae family with comparison to the knowledge accumulated in Arabidopsis thaliana. The myxospermy occurrence was evaluated in 27 Brassicaceae species. Phenotyping included mucilage secretory cell morphology and topochemistry to highlight subtle myxospermy traits. In parallel, computational biology was driven on the one hundred genes constituting the so-called A. thaliana mucilage secretory cell toolbox to confront their sequence conservation to the observed phenotypes. Mucilage secretory cells show high morphology diversity; the three studied Arabidopsis species had a specific extrusion modality compared to the other studied Brassicaceae species. Orthologous genes from the A. thaliana mucilage secretory cell toolbox were mostly found in all studied species without correlation with the occurrence of myxospermy or even more sub-cellular traits. Seed mucilage may be an ancestral feature of the Brassicaceae family. It consists of highly diverse subtle traits, probably underlined by several genes not yet characterized in A. thaliana or by species-specific genes. Therefore, A. thaliana is probably not a sufficient reference for future myxospermy evo–devo studies.

Struktur Sel Sekresi Daun Jeruk Kalamansi (Citrus microcarpa Bunge.) di Pulau Ambon

Perbedaan lokasi tumbuh dapat mengakibatkan perbedaan penampilan fenotipik tanaman yang dapat diamati secara morfologi dan anatomi seperti struktur anatomi sel sekretori. Tujuan penelitian ini adalah untuk mengetahui struktur sel sekretori daun jeruk kalamansi di pulau Ambon. Metode jelajah dilakukan pada 13 lokasi di Pulau Ambon untuk koleksi sampel, dan pada setiap lokasi diambil 3 tanaman sebagai 3 ulangan. Tiap tanaman diambil 5 daun pada setiap sisi pohon tanaman jeruk kalamansi dengan ukuran panjang 5-7 cm dan warna hijau tua. Pembuatan preparat mengikuti metode free hand section. Pengamatan menggunakan kamera Optilab pada mikroskop Olympus dengan perbesaran 400x. Pengukuran diameter sel menggunakan fitur measure pada software Image Ruster. Data kualitatif berupa struktur sel sekresi daun jeruk Kalamansi ditampilkan dalam bentuk gambar dan dideskripsikan sesuai hasil yang terlihat, sedangkan data hasil pengukuran diameter sel sekresi adalah rerata 3 ulangan dan ditampilkan sebagai mean ± standar deviasi (SD). Hasil penelitian menunjukkan adanya sel sekretori yang berjumlah satu sel. Struktur sel sekretori terdiri dari sel epitel, sel selubung, dan rongga sekretori. Bentuk sel sekresi ada yang bulat dan lonjong. Diameter rongga sekretori berkisar antara 106,08-167,60 µm. Berdasarkan hasil penelitian, maka dapat disimpulkan bahwa sel sekresi pada daun jeruk kalamansi pada lokasi-lokasi berbeda di Pulau Ambon bervariasi baik bentuk maupun ukurannya. Differences in habitat can induce differences in the phenotypic appearance of plants that can be observed morphologically and anatomically such as the anatomical structure of secretory cells. The purpose of this study was to determine the structure of the secretory cells in the leaves of Calamansy citrus in Ambon island. Tracking method was done for sample collections, and at each location 3 plants were taken as replicates. Each plant was taken 5 leaves with a length of 5-7 cm and dark green color. Prior to be observed, the fresh sample was done with free-hand section method. Microscopy observations were done by a light microscope at 400x magnification. Measurement of cell diameter was done by the measure feature in Image Ruster software. Qualitative data such as secretory cell structures of Calamansy citrus leaves were shown in form of images and described according to the results, while the data of the measurement of secretory cell diameters is the average of 3 replications and was shown as mean ± standard deviation (SD). The results showed the presence of secretory cells which amounted to one cell. The secretory cell structure is composed of epithelial cells, sheath cells, and secretory cavities. Cell shapes vary, including round and oval. The diameter of the secretory cell cavity ranges from 106.08-167.60 µm.

Cigarette Smoke Specifically Affects Small Airway Epithelial Cell Populations and Triggers the Expansion of Inflammatory and Squamous Differentiation Associated Basal Cells

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and causes remodeling of the small airways. However, the exact smoke-induced effects on the different types of small airway epithelial cells (SAECs) are poorly understood. Here, using air–liquid interface (ALI) cultures, single-cell RNA-sequencing reveals previously unrecognized transcriptional heterogeneity within the small airway epithelium and cell type-specific effects upon acute and chronic cigarette smoke exposure. Smoke triggers detoxification and inflammatory responses and aberrantly activates and alters basal cell differentiation. This results in an increase of inflammatory basal-to-secretory cell intermediates and, particularly after chronic smoke exposure, a massive expansion of a rare inflammatory and squamous metaplasia associated KRT6A+ basal cell state and an altered secretory cell landscape. ALI cultures originating from healthy non-smokers and COPD smokers show similar responses to cigarette smoke exposure, although an increased pro-inflammatory profile is conserved in the latter. Taken together, the in vitro models provide high-resolution insights into the smoke-induced remodeling of the small airways resembling the pathological processes in COPD airways. The data may also help to better understand other lung diseases including COVID-19, as the data reflect the smoke-dependent variable induction of SARS-CoV-2 entry factors across SAEC populations.

Identification of Type 2-pediatric Asthma Based on Single-cell Transcriptomic Analysis

Type 2-pediatric asthma characterized by T2 cytokine-driven airway inflammation is the most common type of asthma. Recently, T2 cytokine inhibitors have reduced the exacerbation rates of asthma, but their ability to improve lung function is limited. Screening novel therapeutic strategies for Type 2-pediatric asthma patients is imperative. We obtained single-cell RNA sequencing (scRNA-seq) describing the chronic stimulation GSE145013 dataset with IL-13. Consensus clustering was performed to classify pediatric asthmatic patients from validation datasets GSE65204 and GSE40888, based on the cell marker genes. We found three cellular subtypes including ciliated cells, secretory cell 1, and secretory cell 2. The expression of CCL26, PRB1, and SLC9B2 was higher in secretory cell 1, while SCGB3A1 and BPIFA1 were higher in secretory cell 2. Consensus clustering based on the five cell marker genes produced two patient subtypes (C1 and C2). The expression of SCGB3A1 and BPIFA1 was higher in C2 subtypes, while CCL26, PRB1, and SLC9B2 was higher in C1 subtypes. Patients in C2 subtypes may more secretory cell 2, while the patients in C1 may have higher secretory cell 1 in the infiltrate. More Type 2 T helper cells were in the infiltrate in the C2 subtype, while type 1 T helper cells were higher in the C1 subtype. T2 cytokines (IL-13, IL-33, IL-3, IL-4, and TSLP) were expressed more in the C2 subtype, corresponding to Type 2-pediatric asthma. This study identified five cell marker genes to screen Type 2-pediatric asthma that could potentially be therapeutic targets for Type 2-pediatric asthma.

A149 TRP53 LOSS IN KRT15+ INTESTINAL STEM CELLS SEVERELY AFFECTS SECRETORY CELL FATE AND SELF-RENEWAL

Background Intestinal epithelium homeostasis is maintained by two main populations of stem cells: Lgr5+ and reserve stem cells. Under injury, cell plasticity has been observed in progenitor and differentiated cells. We recently reported that Krt15+ cells are present in small intestinal and colon epithelia, and harbor self-renewal, multipotent and regenerative capacities. As in Lgr5+and reserve stem cells, hyperactivation of Wnt/b-catenin pathway in Krt15+ stem cells lead to tumor formation in the intestinal epithelium. While these intestinal stem cell populations can act as tumor-initiating cells in sporadic colon cancer, little is known about the cell-of-origin of colitis-associated colon cancer (CAC). TP53 alteration is reported as an early event in CAC. Therefore, we hypothesize that Trp53 loss specifically in Krt15+ stem cells will perturb the epithelial homeostasis and lead to tumor formation. Aims Identify if Krt15+ cells may act as the cell-of-origin in colitis-associated colorectal cancer Methods To induce Trp53 loss specifically in Krt15+ cells, we generated Krt15-CrePR1;Trp53fl/fl (Krt15△Trp53) mice, induced Cre recombination by injecting RU486 (PR agonist) and euthanized the mice at different time points following recombination. Results Results Twelve-month following Cre recombination, adenoma formation was observed in a small proportion of Krt15△Trp53 mice. Though, Trp53 loss in Krt15+ cells severely perturbed the small intestinal morphology in every mouse studied. Increased crypt length and villi width was observed in Krt15△Trp53 vs control mice without any changes in cell proliferation. We also observed an increased number of Tuft cells and goblet cells in the villi of experimental mice. In the crypt, higher number of Paneth cells and aberrant presence of goblet cells were noted in Krt15△Trp53mice. Interestingly, we also observed crypt cells expressing goblet and Paneth cell markers and decreased Notch pathway activation suggesting dysregulation of secretory cell fate. Krt15△Trp53 mice display higher number of fibroblasts in the villi and the submucosa, as well as thickening of the muscularis layer. Interestingly, similar observations (accumulation of secretory cells and fibrosis) have been reported in IBD patients, supporting the possible role of Krt15+ cells in CAC. Furthermore, crypts isolated from Krt15△Trp53 mice rapidly die when seeded as organoids vs crypts from control mice, suggesting that the alterations observed in vivo in Paneth cells might interfere with the stem cell niche and therefore reduce self-renewal of Krt15+ cells. Conclusions Trp53 loss specifically in Krt15+ cells impaired cell fate decision, induced secretory cell hyperplasia, affected self-renewal ability, and initiated adenoma formation supporting the possible role of Krt15+ cells in gut inflammation and cancer. Funding Agencies Canada Research Chair, Cancer Research Society, CFI