Association study of single nucleotide polymorphisms in IL-10 and IL-17 genes with the severity of microbial keratitis

PURPOSE Exploratory analysis to assess the association of single nucleotide polymorphisms (SNPs) in the interleukin (IL) 10 and IL-17 genes with severity of contact lens keratitis. METHODS This was a retrospective case control study of 88 contact lens keratitis cases (25 severe) and 185 healthy contact lens wearers recruited from studies conducted at Moorfields Eye Hospital and in Australia-wide during 2003–2005. Buccal swab samples were collected on Whatman FTA cards and mailed by post for DNA extraction and SNP genotyping. IL-10 (rs1800871; rs1800896; rs1800872) and IL-17 (rs1800871; rs1800896; rs1800872) SNPs were screened by pyrosequencing. Genetic association analyses were performed via Cochran-Armitage trend tests and logistic regression models using PLINK software. RESULTS None of the SNPs tested showed evidence of association with severity of contact lens keratitis at P <  0.05. Nevertheless, minor allele G in SNP rs2397084 of the IL-17F gene was associated with increased risk of severe MK, with OR=2.1 (95% CI=0.9-4.8, P = 0.066). CONCLUSION Our study cannot exclude with confidence that genetic variation in the IL-17 F proinflammatory cytokine is associated with more severe outcomes of MK. However, there is general body of information that the IL-17 pathway is important in the mechanisms of MK. Studies with larger power and the expanded array of laboratory tools will elucidate the exact role of IL-17 in MK

Feasibility of Silicon Quantum Dots as a Biomarker for the Bioimaging of Tear Film

This study investigated the fluorescence and biocompatibility of hydrophilic silicon quantum dots (SiQDs) that are doped with scandium (Sc-SiQDs), copper (Cu-SiQDs), and zinc (Zn-SiQDs), indicating their feasibility for the bioimaging of tear film. SiQDs were investigated for fluorescence emission by the in vitro imaging of artificial tears (TheraTears®), using an optical imaging system. A trypan blue exclusion test and MTT assay were used to evaluate the cytotoxicity of SiQDs to cultured human corneal epithelial cells. No difference was observed between the fluorescence emission of Sc-SiQDs and Cu-SiQDs at any concentration. On average, SiQDs showed stable fluorescence, while Sc-SiQDs and Cu-SiQDs showed brighter fluorescence emissions than Zn-SiQDs. Cu-SiQDs and Sc-SiQDs showed a broader safe concentration range than Zn-SiQDs. Cu-SiQDs and Zn-SiQDs tend to aggregate more substantially in TheraTears® than Sc-SiQDs. This study elucidates the feasibility of hydrophilic Sc-SiQDs in studying the tear film’s aqueous layer

Development of an In Vitro Compartmentalization Screen for High-Throughput Directed Evolution of [FeFe] Hydrogenases

BACKGROUND: [FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library. CONCLUSIONS/SIGNIFICANCE: [FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy

Influence of dietary nitrate supplementation on local sweating and cutaneous vascular responses during exercise in a hot environment.

Purpose We investigated the influence of inorganic nitrate (NO−3) supplementation on local sweating and cutaneous vascular responses during exercise in hot conditions. Method Eight healthy, young subjects were assigned in a randomized, double-blind, crossover design to receive NO−3 -rich beetroot (BR) juice (140 mL/day, containing ~8 mmol of NO−3) and NO−3-depleted placebo (PL) juice (140 mL/day, containing ~0.003 mmol of NO−3) for 3 days. On day 3 of supplementation, subjects cycled at an intensity corresponding to 55% of V̇ O2max for 30 min in hot conditions (30 °C, 50% relative humidity). Chest and forearm sweat rate (SR) and skin blood flow (SkBF), were measured continuously. Cutaneous vascular conductance (CVC) was calculated by SkBF/mean arterial pressure (MAP). Results Prior to exercise, plasma NO− 3 (21±6 and 581±161 µM) and nitrite (NO− 2 , 87±28 and 336±156 nM) concentrations were higher after BR compared to PL supplementation (P≤0.011, n=6). Oesophageal, mean skin, and mean body temperatures during exercise were not different between conditions. In addition, BR supplementation did not affect SR, SkBF, and CVC during exercise. A lower MAP was found after 30 min of exercise following BR supplementation (112±6 and 103±6 mmHg for PL and BR, respectively, P=0.021). Conclusion These results suggest that inorganic NO− 3 supplementation, which increases the potential for O2-independent NO production, does not affect local sweating and cutaneous vascular responses, but attenuates blood pressure in young healthy subjects exercising in a hot environment

GENCODE 2021

© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. The GENCODE project annotates human and mouse genes and transcripts supported by experimental data with high accuracy, providing a foundational resource that supports genome biology and clinical genomics. GENCODE annotation processes make use of primary data and bioinformatic tools and analysis generated both within the consortium and externally to support the creation of transcript structures and the determination of their function. Here, we present improvements to our annotation infrastructure, bioinformatics tools, and analysis, and the advances they support in the annotation of the human and mouse genomes including: the completion of first pass manual annotation for the mouse reference genome; targeted improvements to the annotation of genes associated with SARS-CoV-2 infection; collaborative projects to achieve convergence across reference annotation databases for the annotation of human and mouse protein-coding genes; and the first GENCODE manually supervised automated annotation of lncRNAs. Our annotation is accessible via Ensembl, the UCSC Genome Browser and https://www.gencodegenes.org.National Human Genome Research Institute of the National Institutes of Health [U41HG007234]; the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health; Wellcome Trust [WT108749/Z/15/Z, WT200990/Z/16/Z]; European Molecular Biology Laboratory; Swiss National Science Foundation through the National Center of Competence in Research ‘RNA & Disease’ (to R.J.); Medical Faculty of the University of Bern (to R.J). Funding for open access charge: National Institutes of Health

Differences in Tear Film Biochemistry of Symptomatic and Asymptomatic Lens Wearers

SIGNIFICANCE The concentration of selected proteins and inflammatory mediators in tears of symptomatic and asymptomatic contact lens wearers were quantified. The level of leukotriene B4 was higher in the symptomatic group. This may suggest that inflammation can be the cause of discomfort sensation at the end of day. PURPOSE The present study aims to quantify the concentration of selected tear lipids and proteins in symptomatic and asymptomatic contact lens wearers. METHODS Unstimulated evening tears were collected using glass capillary tubes from 45 healthy, adapted contact lens wearers. Twenty-two had self-described symptoms of dryness and discomfort with contact lenses and 23 were asymptomatic. Tear proteins were assayed using selected reaction monitoring mass spectrometry. Enzyme immunoassay kits were used to measure prostaglandins, leukotriene B4, and cysteinyl leukotrienes. Ocular comfort was rated on a scale of 1 to 100 at the time of tear collection. RESULTS The average evening comfort level was above 70 for the asymptomatic (83.96 ± 9.51, mean ± SE) and equal or below 70 for the symptomatic group (57.28 ± 12.38) (P .1) between symptomatic and asymptomatic subjects. CONCLUSIONS The LTB4 concentration was significantly higher in symptomatic contact lens wearers compared to the asymptomatic group, and this may partly mediate the discomfort response during lens wear in the symptomatic lens wearers. No other differences were found in the level of tear factors of interest between the two groups

A pilot study on corneal Langerhans cells in keratoconus

Purpose: To report the density and morphology of cells that are analogous to corneal Langerhans cells and their associations in keratoconus. Materials and methods: This prospective cross-sectional study included a convenience sample of keratoconus subjects aged between 18-65 years. Corneal topography, assessment of ocular symptoms, tear variables, corneal sensitivity, in-vivo confocal microscopy were performed. The number of Langerhans cells were manually counted and averaged across three central corneal images. Cell morphology was graded on a 0-3 scale, where grade 3 indicates cells with long visible dendrites. Associations of Langerhans cells with other variables were evaluated using Spearman's correlation. Results: Twenty-one keratoconus subjects with a mean age of 43 ± 11 years were included. Eighty-one percent of them were males, 48% had mild keratoconus and 52% were contact lens wearers. Langerhans cells were present in the central cornea in 91% of subjects. Median cell density was 15 cells/mm2(IQR: 3-21). Cell morphology of grades 2 or 3 (with short or long dendrites) was seen in 71% of subjects. There was a significant association between Langerhans cell frequency and density with male gender (rho and p-values: -0.669, 0.001 and -0.441,0.045) and between Langerhans cell density and nerve fibre tortuosity (0.479,0.028). No significant association observed with age, contact lens wear or ocular symptoms. Conclusion: Langerhans cells were present in a significant number of subjects suggesting the possibility of inflammation in keratoconus. Based on the association of Langerhans cells with nerve parameters, we propose inflammation as the underlying cause for corneal nerve changes in keratoconus