Purpose Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood. Methods Four human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs). Results scRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks. Conclusions Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of oc...

HighlightsThis study examined long-term trends in antibiotic resistance on a national scale in India.Colistin-resistant Klebsiella pneumoniae and Escherichia coli strains have emerged in India.In 2014, the prevalence of carbapenem-resistant E. coli was11.5%, the highest reported to date globally.

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( E )- N ′-((2-Hydroxynaphthalen-1-yl)methylene)picolinohydrazide (H-PNAP) shows aggregation-induced emission (AIE) strictly in a 90% water/MeOH (v/v) mixture at 540 nm, and the solid-state emission is blue-shifted to 509 nm upon excitation at 400 nm. The AIE activity of H-PNAP is selectively quenched by 2,4,6-trinitrophenol (TNP) and 2,4-dinitrophenol (DNP) out of different nitroaromatic compounds with a limit of detection (LOD) of 7.79 × 10 –7 and 9.08 × 10 –7 M, respectively. The probe is nonemissive in aqueous medium (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES buffer, pH 7.2); however, it shows a strong emission to Al 3+ (λ em , 490 nm) in the presence of 17 other biological metal ions, and the LOD is 2.09 nM which is far below the WHO recommended value (7.41 mM). The emission of the [Al(PNAP)(NO 3 ) 2 ] complex is quenched by HF 2 – (F – and PO 4 3– are weak quencher), and the LOD is as low as 15 nM. The probable mechanism of the sensing feature of the probe has been authenticated by 1 H nuclear magnetic resonance titration, mass spectrometry, Fourier transform infrared spectroscopy, Benesi–Hildebrand plot, and Job’s plot in each case. The probe has some practical applications such as recovery of Al 3+ from the drinking water sample, construction of the INHIBIT logic gate, and detection kits for Al 3+ and TNP/DNP by simple paper test strips. The probe, H-PNAP, has successfully been applied to the detection of intracellular Al 3+ and HF 2 – ions in the human breast cancer cell, MDA-MB-468.

A single fluorescence probe recognizes multiple ions and grabs the great attention of scientists.