Dual metal cations coated magnetic mesoporous silica probe for highly selective capture of endogenous phosphopeptides in biological samples.

For the first time, dual metal ions (Ti4+-Zr4+) were successfully modified into the channel of magnetic mesoporous silica to obtain an affinity probe for highly selective capture of endogenous phosphopeptides in biological samples. The newly prepared Fe3O4@mSiO2@Ti4+-Zr4+ composites possessed the advantages of ordered mesoporous channels, superparamagnetism, and enhanced affinity properties of dual metal ions of Ti4+ and Zr4+. The phosphopeptide enrichment efficiency of the Fe3O4@mSiO2@Ti4+-Zr4+ composite was investigated, and the result indicated an ultrahigh size exclusive ability (weight ratio of ß-casein tryptic digests, BSA, and α-casein protein reached up to 1:1000:1000). Compared to magnetic affinity probes with single metal ions (Fe3O4@mSiO2@Ti4+, Fe3O4@mSiO2@Zr4+), the composite possessed stronger specificity, higher sensitivity, and better efficiency; and more importantly, it showed much enhanced enrichment ability towards both mono- and multi-phosphorylated peptides. Additionally, by utilizing the Fe3O4@mSiO2@Ti4+-Zr4+ affinity probe, a total number of 104 endogenous phosphopeptides including 95 mono-phosphopeptides and 9 multi-phosphopeptides were captured and identified from human saliva, indicating the great potential for the application of the novel probe for the peptidome analysis in the future. Graphic abstract.

Imaging phosphorylated peptide distribution in human lens by MALDI MS.

Phosphorylation plays vital roles in complex biological processes such as cellular growth, division and signaling transduction. However, due to the low ionization efficiency of phosphorylated peptides, it is still a huge challenge to obtain region-specific phosphorylated peptide distribution by imaging mass spectrometry. To achieve the on-tissue analysis of phosphorylated peptides, we took advantage of a graphene oxide-immobilized enzyme reactor to conduct the in situ digestion, followed by dephosphorylation treatment that removed the phosphate groups and thereby helped to improve the signal intensity of phosphorylated peptides. A visual representation of the phosphoproteome of a human lens was successfully mapped. Results showed that phosphorylated peptides localized mainly in the nucleus region of a healthy lens while the outer cortex is the dominant region for phosphorylated peptides of a cataractous lens.

Effect of HSF4b on age related cataract may through its novel downstream target Hif1α.

Our previous study identified five new heat shock factor 4 (HSF4) mutations in 150 age-related cataract (ARC) patients which indicated that HSF4 mutations may be associated with this disease. Hypoxia-inducible factor (Hif1α) is an important downstream target of HSF4b. It has been found that Hif1α play also important roles in cataract development. To identify if HSF4b play it role in cataract development through HIF1α, we transfected SRA01/04 lens epithelial cells with small hairpin RNA of HSF4b and measured expressions Hif1α after transfection. Then, we perform chromatin immunoprecipitation quantitative PCR to see the relationship between HSF4b and HIF1α. We found that HSF4 downregulation led to decrease of HIF1α mRNA expression. Furthermore, we demonstrated by ChIP followed by quantitative PCR (ChIP-qPCR) that these HIF-1α is bound by HSF4b near promoters, not gene bodies.

Realization of on-tissue protein identification by highly efficient in situ digestion with graphene-immobilized trypsin for MALDI imaging analysis.

A novel implementation of in situ protein digestion supported by a graphene oxide-immobilized enzyme reactor (GO-IMER) in the MALDI imaging mass spectrometry (IMS) workflow is reported, which enables the simultaneous diagnostic identity and distribution attributes of the proteome on tissue.

Proteomic analysis of SRA01/04 transfected with wild-type and mutant HSF4b identified from a Chinese congenital cataract family.

PURPOSE: Congenital cataracts account for about 10% of cases of childhood blindness. Heat shock transcription factor 4 (HSF4) is related with human autosomal dominant lamellar and Marner cataracts; a T→C transition at nucleotide 348 was found in a large Chinese cataract family. The aim of this study was to analyze the unique role of HSF4b and the mutation of HSF4b. METHODS: The isobaric tags for relative and absolute quantification (iTRAQ), coupled with the two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) technique, was used to identify and quantify differential proteomes in human lens epithelial cell lines SRA 01/04 expressing wild-type and mutant HSF4b. RESULTS: A total of 104 unique proteins were identified from the human lens epithelial cell lines SRA 01/04. Apart from the proteins due to the effect of the pcDNA3.1 vector, the wild-type and mutant HSF4b led to 23 differentially expressed proteins, of which four were histone proteins and three were ribosomal proteins. The T→C transition at nucleotide 348 in HSF4b led to 18 differentially expressed proteins in SRA 01/04, among which serpin H1 precursor, heat shock protein beta-1, and stress-70 protein belong to heat shock protein families. The up- or down-regulated proteins were functionally analyzed using Ingenuity Pathways Analysis (IPA) to interpret the interaction network and predominant canonical pathways involved in these differentially expressed proteins. CONCLUSIONS: A multitude of differentially expressed proteins was found to be associated with HSF4b and a T→C transition at nucleotide 348 in HSF4b. The proteins interacted directly or indirectly with each other, and they may provide clues as to how HSF4b modulates protein expression in the lens epithelial cells of SRA 01/04. Although further investigation is required, the results may provide some new clues to the transcriptional mechanism of HSF4b and cataract formation.

[The progress of proteomics approaches in cataract research].

Researches on lens proteomics are becoming prevalent in recent years. Characterized by their high resolution and efficiency, proteomics approaches provide reliable methodological support for lenses proteomic research, which demonstrates a new orientation for revealing the mechanism of cataractogenesis. This review outlines the separation, identification and some novel approaches of proteomic techniques applied in recent lenses proteomic research.

Precise Detection of Cataracts with Specific High-Risk Factors by Layered Binary Co-Ionizers Assisted Aqueous Humor Metabolic Analysis.

Diabetes and high myopia as well-known high-risk factors can aggravate cataracts, yet clinical coping strategy remains a bottleneck. Metabolic analysis tends to be powerful for precisely detection and mechanism exploration since most of diseases including cataracts are accompanied by metabolic disorder. Herein, a layered binary co-ionizers assisted aqueous humor metabolic analysis tool is proposed for potentially etiological typing and detection of cataracts, including age-related cataracts (ARC), cataracts with diabetes mellitus (CDM), and cataracts with high myopia (CHM). Startlingly, taking advantage of the optimal machine learning algorithm and all metabolic fingerprints, 100% of accuracy, precision, and recall rates are achieved for arbitrary comparison between groups. Moreover, 11, 9, and 7 key metabolites with explicit identities are confirmed as markers of discriminating CDM from ARC, CHM from ARC, and CDM from CHM, and the corresponding area under the curve values of validation cohorts are 0.985, 1.000, and 1.000. Finally, the critical impact of diabetes/high myopia on cataracts is revealed by excavating the change levels and metabolic pathways of key metabolites. This work updates the insights of prevention and treatment about cataracts at metabolic level and throws out huge surprises and progresses metabolic diagnosis toward a reality.

Evaluation of axial length/total corneal refractive power ratio as a potential marker for ocular diagnosis of Marfan's syndrome in children.

AIM: To investigate whether the axial length (AL)/total corneal refractive power (TCRP) ratio is a sensitive and simple factor that can be used for the early diagnosis of Marfan's syndrome (MFS) in children. METHODS: The relationship between the AL/TCRP ratio and the diagnosis of MFS for 192 eyes in 97 children were evaluate. The biological characteristics, including age, sex, AL, and TCRP, were collected from medical records. Receiver operating characteristic (ROC) curve analysis was performed to investigate whether the AL/TCRP ratio effectively distinguishes MFS from other subjects. The Youden index was used to re-divide the whole population into two groups according to an AL/TCRP ratio of 0.59. RESULTS: Of 96 subjects (mean age 7.46±3.28y) evaluated, 56 (110 eyes) had a definite diagnosis of MFS in childhood based on the revised Ghent criteria, 41 (82 eyes) with diagnosis of congenital ectopia lentis (EL) were included as a control group. AL was negatively correlated with TCRP, with a linear regression coefficient of -0.36 (R 2=0.08). A significant correlation was found between age and the AL/TCRP ratio (P=0.023). ROC curve analysis showed that the AL/TCRP ratio distinguished MFS from the other patients at a threshold of 0.59. MFS patients were present in 24/58 (41.38%) patients with an AL/TCRP ratio of ≤0.59 and in 34/39 (87.18%) patients with an AL/TCRP ratio of >0.59. CONCLUSION: An AL/TCRP ratio of >0.59 is significantly associated with the risk of MFS. The AL/TCRP ratio should be measured as a promising marker for the prognosis of children MFS. Changes in the AL/TCRP ratio should be monitored over time.

The Influence of Decentration on Higher-Order Aberrations in Artisan Aphakic Intraocular Lens Implantation Eyes.

PURPOSE: To characterize the influence of decentration on higher-order aberrations of Artisan aphakic intraocular lens implantation eyes. Setting. Department of Ophthalmology and Vision Science, Eye and ENT Hospital of Fudan University, Shanghai, China. DESIGN: Retrospective case series. METHODS: Twenty-three eyes of 18 patients were retrospectively examined. Location of the IOL was imaged using a slit-lamp, and decentration (the distance from the center of a pupil to the geometric center of the IOL) was measured using the AutoCAD 2007 software. Ocular and internal higher-order aberrations were measured using the wavefront analyzer KR-1W (Topcon) and their correlation with decentration were analyzed. Coma, spherical, 3rd-, 4th-order, trefoil, tetrafoil aberrations, ocular, and internal higher-order aberrations were calculated for a 4.0 mm pupil diameter. RESULTS: The mean age of patients was 33.6 ± 21.4 years (ranging from 7 to 72 years). The mean follow-up period was 28.2 ± 10.5 months (ranging from 12 to 52 months). The mean postoperative best-corrected visual acuity (BCVA) was 0.19 ± 0.19 logMAR (range -0.18-0.52). The mean decentration was 0.57 ± 0.28 mm (range 0.15-1.21 mm). There was no correlation between Artisan IOL decentration and ocular higher-order aberrations, internal higher-order aberrations, coma, spherical, 3rd-, 4th-order, trefoil, and tetrafoil aberrations, respectively. There was a positive correlation between Artisan IOL decentration and 2nd-astig aberrations when 2nd-astig aberrations were less than 0.1. CONCLUSIONS: The position of Artisan IOLs showed slight decentration and the amounts of decentration were not large enough to influence higher-order aberrations.

Liquid-chromatographic and mass-spectrometric identification of lens proteins using microwave-assisted digestion with trypsin-immobilized magnetic nanoparticles.

We used a newly developed method combining trypsin-immobilized magnetic nanoparticles (TIMNs) and microwave-assisted protein digestion to study the proteins of human lens tissue. The digested proteins were identified by liquid chromatography and mass spectrometry. The lens proteins were digested under optimized conditions (digestion time 1 min, microwave power 400 W, trypsin-to-protein ratio 1:5) determined using bovine serum albumin as the standard protein, before liquid-chromatographic and mass-spectrometric analysis. Twenty-six proteins were identified with the new digestion method compared with 11 proteins identified with traditional in-solution digestion (12h). gamma-Crystallin, beta-crystallin, and superoxide dismutase 1 proteins, identified with the microwave-assisted method but not the traditional method, are related to cataract development according to some studies. The TIMNs were easily separated from the digestion products. This new digestion method could prove extremely useful for large-scale proteomic analyses.

[Clinical study of diffuse lamellar keratitis after laser in situ keratomileusis].

OBJECTIVE: To investigate a new clinical grading scale of diffuse lamellar keratitis (DLK) following laser in situ keratomileusis (LASIK) and to observe the clinic outcomes of eyes treated with corresponding classified therapy. METHODS: It was a prospective case-control study. Eyes that developed DLK after LASIK were diagnosed by a new criterion of clinical grading scale based on both severity and scope of inflammatory cells infiltrations in the corneal interface. Dose and frequency of topical or systemic corticosteroid were determined by the classified profiles according to each grade. Clinical symptoms, corneal inflammatory cells infiltrations, visual acuity, refractive error and intraocular pressure (IOP) were evaluated on 1, 3, 5, 7, 10 days and 1 month after diagnosis as well as at the time of diagnosis. RESULTS: Among the 35 eyes of 29 patients that developed DLK, mild inflammatory cells infiltrations were mostly seen. Thirty eyes had degree I DLK and 3 had degree II DLK. One eye had degree III DLK and one had degree IV DLK. Inflammatory cells infiltrations in 20 eyes invaded peripheral zone (zone 1) and those in 11 eyes invaded mid-peripheral zone (zone 2). Inflammatory cells infiltrates in 4 eyes invaded central zone (zone 3). Nineteen eyes had degree I zone 1 DLK. Thirty three eyes were diagnosed between the 1st day and the 3rd day after surgery. Uncorrected visual acuity (UCVA) was worse than preoperative best spectacle corrected visual acuity (BSCVA). Inflammatory cells infiltrations retreated within 6.06 2.04 days after classified corticosteroid therapy. One month after the treatment, UCVA had improved to the level of preoperative BSCVA. BSCVA in 30 eyes were equal to or better than those before surgery. Spherical equivalent of the refractive error was (-0.21 +/- 1.16) D. There was no statistic difference in the retreat time of inflammatory cells infiltrations, recovery of visual acuity and refractive error among the different grades of DLK or among the DLK treated with different therapy profiles. No eye suffered from high intraocular pressure during the treatments. CONCLUSIONS: Clinical grading scale and corresponding classified therapy were proved to be effective in the treatment for DLK.

Mutation screening of HSF4 in 150 age-related cataract patients.

PURPOSE: Heat shock transcription factor 4 (HSF4) regulates the expression of several heat shock protein (HSP) genes. HSPs are one of the major components responsible for lens protein organization. Recently, we found that mutations of HSF4 result in hereditary cataract. In this study, we explore the role of HSF4 in the development of age-related cataract. METHODS: We screened sequence variants of HSF4 in age-related cataract patients and the natural population from Shanghai, China. RESULTS: In individuals of natural populations, we detected no single nucleotide polymorphism (SNP) with a frequency higher than 5% in a complete coding region or in their exon-intron boundaries. In 150 age-related cataract patients, we identified seven sequence changes. We found an intronic G-->A transition (c.1020-25G>A) in one patient, a missense mutation (c.1078A>G) in exon 4 in two patients, a silent mutation (c.1223 C>T) in exon 5 in two patients, an intronic C-->T transition (c.1256+25C>T) in one patient, and a silent mutation in exon 6 (c.1286 C>T) in one patient. These five variants were not represented in 220 control individuals. We also identified an intronic C-->T transition (c.1019+9C>T) and a missense mutation (c.1243G>A) in exon 3 in three patients, but these two variants were also present in 100 control subjects. CONCLUSIONS: We identified five new HSF4 mutations in 150 age-related cataract patients, enlarging the spectrum of HSF4 mutations in cataract patients. This result indicates that HSF4 mutations account for only a small fraction of age-related cataracts.

Aspheric intraocular lenses implantation for cataract patients with extreme myopia.

Objective. To evaluate the postoperative visual quality of cataract patients with extreme myopia after implantation of aspheric intraocular lenses (IOLs). Methods. Thirty-three eyes were enrolled in this prospectivestudy. Eighteen eyes with an axial length longer than 28 mm were included in the extreme myopia group, and the other 15 eyes were included in the nonextreme myopia group. Phacoemulsification and aspheric IOL implantation were performed. Six months after cataract surgery, best-corrected visual acuity (BCVA), contrast sensitivity, and wavefront aberrations were measured, and subjective visual quality was assessed. Results. The BCVA improved significantly after surgery for both groups, and patients in the nonextreme myopia group achieved better postoperative BCVA due to better retinal status of the eyes. The evaluation of contrast sensitivity without glare was the same in both groups, whereas patients in the nonextreme myopia group performed better at intermediate spatial frequencies under glare conditions. The two groups did not show a significant difference in high-order aberrations. With regard to subjective visual quality, the composite scores of both groups did not differ significantly. Conclusions. Aspheric IOLs provided good visual outcomes in cataract patients with extreme myopia. These patients should undergo careful evaluation to determine the maculopathy severity level before surgery.

Visual function and subjective quality of life in Chinese cataract patients after implantation with aspheric intraocular lenses.

PURPOSE: To evaluate and compare the objective and subjective visual function after implantation of 2 aspheric intraocular lenses (IOLs) in Chinese cataract patients. METHODS: Forty-one eyes of 28 patients with cataract were randomly assigned to receive either the MC X11 ASP IOL or the AcrySof IQ IOL. Three months postoperatively, best-corrected visual acuity (BCVA), contrast sensitivity, wavefront aberrations, and subjective visual quality were measured. The degree of posterior capsule opacification (PCO) was recorded at last follow-up. RESULTS: Postoperative mean monocular BCVA was 0.05±0.13 logMAR in the MC X11 group and 0.05±0.08 logMAR in the IQ group. There was no significant difference in visual acuity, contrast sensitivity, subjective visual quality, and degree of PCO between the 2 groups. For a 6-mm pupil diameter, the mean spherical aberration (Z40) was 0.075±0.076 µm in the MC X11 group and 0.056±0.111 µm in the IQ group. However, 45.5% and 57.9% of the eyes in the MC X11 and IQ groups had values >0.10 µm, respectively. The mean RMS values of the high-order aberrations were similar between the 2 groups except the higher fifth- and sixth-order aberrations, which were significantly higher in the MC X11 group. CONCLUSIONS: Both IOLs provided similar and good visual outcomes. However, the data suggest that the amount of negative spherical aberration generated by current available aspheric IOLs might not be optimal for Chinese eyes.