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Ananthi S.,Dr G Venkataswamy Eye Research Institute | Venkatesh Prajna N.,Cornea Clinic | Lalitha P.,Aravind Eye Hospital | Valarnila M.,Dr G Venkataswamy Eye Research Institute | Dharmalingam K.,Madurai Kamaraj University
PLoS ONE | Year: 2013

Fusarium is the major causative agent of fungal infections leading to corneal ulcer (keratitis) in Southern India and other tropical countries. Keratitis caused by Fusarium is a difficult disease to treat unless antifungal therapy is initiated during the early stages of infection. In this study tear proteins were prepared from keratitis patients classified based on the duration of infection. Among the patients recruited, early infection (n = 35), intermediate (n = 20), late (n = 11), samples from five patients in each group were pooled for analysis. Control samples were a pool of samples from 20 patients. Proteins were separated on difference gel electrophoresis (DIGE) and the differentially expressed proteins were quantified using DeCyder software analysis. The following differentially expressed proteins namely alpha-1-antitrypsin, haptoglobin α2 chain, zinc-alpha-2-glycoprotein, apolipoprotein, albumin, haptoglobin precursor - β chain, lactoferrin, lacrimal lipocalin precursor, cystatin SA III precursor, lacritin precursor were identified using mass spectrometry. Variation in the expression level of some of the proteins was confirmed using western blot analysis. This is the first report to show stage specific tear protein profile in fungal keratitis patients. Validation of this data using a much larger sample set could lead to clinical application of these findings. © 2013 Ananthi et al.

Lechner J.,Queens University of Belfast | Bae H.A.,Flinders University | Guduric-Fuchs J.,Queens University of Belfast | Rice A.,University of Leeds | And 20 more authors.
Investigative Ophthalmology and Visual Science | Year: 2013

PURPOSE. A mutation miR-184({thorn}57C>T) in the seed region of miR-184 (encoded by MIR184 [MIM*613146]) results in familial severe keratoconus combined with early-onset anterior polar cataract and endothelial dystrophy, iris hypoplasia, congenital cataract, and stromal thinning (EDICT) syndrome (MIM#614303). In order to investigate the phenotypic spectrum resulting from MIR184 mutation, MIR184 was sequenced in a keratoconus cohort of mixed ethnicity and a Chinese axial myopia cohort. METHODS. Sequencing of MIR184 was performed in 780 unrelated keratoconus patients and 96 unrelated Han southern Chinese subjects with axial myopia. Effects of identified mutations on RNA secondary structure were predicted computationally using mFold and RNAFold algorithms. MIR184 amplicons from patients harboring mutations were cloned and transfected into human embryonic kidney 293T (HEK293T) cells, and mature mutant miR184 expression was analyzed by stem-loop real-time quantitative PCR (RT-qPCR). RESULTS. Two novel heterozygous substitution mutations in MIR184 were identified in the two patients with isolated keratoconus: miR-184({thorn}8C>A) and miR-184({thorn}3A>G). Computational modeling predicted that these mutations would alter the miR-184 stem-loop stability and secondary structure. Ex vivo miR-184 expression analysis demonstrated that miR184({thorn}8C>A) almost completely repressed the expression of miR-184 (P = 0.022), and miR-184({thorn}3A>G) reduced the expression of miR-184 by approximately 40% (P = 0.002). There was no significant association of rs41280052, which lies within the stem-loop of miR184, with keratoconus. No MIR184 mutations were detected in the axial myopia cohort. CONCLUSIONS. Two novel heterozygous substitution mutations in MIR184 were identified in two patients with isolated keratoconus: miR-184({thorn}8C>A) and miR-184({thorn}3A>G). Mutations in MIR184 are a rare cause of keratoconus and were found in 2 of 780 (0.25%) cases. ©: 2013 The Association for Research in Vision and Ophthalmology, Inc.

Ananthi S.,Dr G Venkataswamy Eye Research Institute | Santhosh R.S.,Dr G Venkataswamy Eye Research Institute | Nila M.V.,Dr G Venkataswamy Eye Research Institute | Prajna N.V.,Cornea Clinic | And 2 more authors.
Experimental Eye Research | Year: 2011

The proteomic profile of tear fluid is of fundamental interest in eye research. In this study we optimized the tear sample preparation method for two-dimensional (2D) analysis and determined the protein profile of tear fluid from healthy males and females. To find the most efficient method for tear sample preparation, four widely applied precipitation methods and ultrafiltration were compared. Of these, TCA precipitation & ultrafiltration resulted in efficient sample concentration and desalting. Use of a nonionic wetting agent, Tergitol NP7, in rehydration solution during isoelectric focusing improves protein separation in 2D gel electrophoresis considerably. Using this optimized method, tear protein profile was analyzed from healthy males and females. Of the thirty six tear proteins identified by LC-MS/MS, seven tear proteins were found to be significantly up regulated in the healthy female tear samples when compared to the male tear samples. These results indicate that the tear protein profile differs with respect to the sex. Mostly, the up regulated proteins are involved in the local immune defense; implying that there may be a sex difference in the ability to defend against infection at the anterior segment of the eyes of normal individuals. © 2011 Elsevier Ltd.

Verma A.,Dr G Venkataswamy Eye Research Institute | Das M.,Cornea Clinic | Srinivasan M.,Cornea Clinic | Prajna N.V.,Cornea Clinic | Sundaresan P.,Dr G Venkataswamy Eye Research Institute
BMC Research Notes | Year: 2013

Background: The involvement of VSX1 gene for the genetic basis of keratoconus is unclear and controversial. The genetic screening of VSX1 from different ethnic populations can enlighten this subject. The aim of the present study is to investigate the role of VSX1 gene in patients with sporadic cases of keratoconus from South India. Methods. The VSX1 gene coding regions, including exon-intron boundaries were screened by direct sequencing analysis in 117 sporadic cases of keratoconus. The identified variations were also analyzed in 108 ethnic matched healthy blood donors. Results: In the VSX1 gene screening, no pathogenic mutation was identified, whereas we could find the presence of four reported single nucleotide polymorphisms; c.546A>G (rs12480307), c.627+23G>A (rs6138482), c.627+84T>A (rs56157240) and c.504-24C>T (IVS3-24C). These variations were observed in similar frequency between cases and controls. Conclusions: The lack of VSX1 pathogenic variations in a large number of unrelated sporadic keratoconus patients tend to omit its role, and corroborate the involvement of other genetic, environmental or behavioural factors in the development of this complex disorder. © 2013 Verma et al.; licensee BioMed Central Ltd.

Selvam R.M.,Dr G Venkataswamy Eye Research Institute | Selvam R.M.,Bharathidasan University | Nithya R.,Dr G Venkataswamy Eye Research Institute | Devi P.N.,Dr G Venkataswamy Eye Research Institute | And 10 more authors.
Journal of Proteomics | Year: 2015

Aspergillus flavus infects the human eye leading to keratitis. Extracellular proteins, the earliest proteins that come in contact with the host and virulence related exoproteins, were identified in the fungus isolated from infected cornea. Virulence of the corneal isolates was tested in the Galleria mellonella larvae model and those isolates showing higher virulence were taken for subsequent exoproteome analysis. High resolution two-dimensional electrophoresis and mass spectrometry were used to generate A. flavus exoproteome reference map as well as to profile most of the exoproteins. Analysis of the identified proteins clearly shows the major biological processes that they are involved in. Nearly 50% of the exoproteins possess catalytic activity and one of these, an alkaline serine protease (Alp1) is present in high abundance as well as multiple proteoforms. Many proteins in the A. flavus exoproteome have been shown to be virulence factors in other pathogens indicating the probable role for these proteins in the corneal infection as well. Interestingly, the majority of the exoproteins do not have secretory signal indicating that they are secreted through the non-classical pathway. Thus, this study provides a clue to the early strategies employed by the pathogen to establish an infection in an immunocompetent host. Biological significance: The outcome of a fungal infection in an immunocompetent human eye depends on the ability of the fungus to overcome the host defense and propagate itself. In this process, the earliest events with respect to the fungal proteins involved include the secretory proteins of the invading organism. As a first step towards understanding the role of the extracellular proteins, exoproteome profile of the fungal isolates was generated. The fungal isolates from cornea showed a distinct pattern of the exoproteome when compared to the saprophyte. Since corneal isolates also showed higher virulence in the insect larval model, presumably the proteins elaborated by the corneal isolates are virulence related. One of the abundant proteins is an alkaline serine protease and this protein exists as multiple proteoforms. This study reports the comprehensive profile of exoproteome and reveals proteins that are potential virulence factors. © 2014 Elsevier B.V.

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