A next generation setup for pre-fractionation of non-denatured proteins reveals diverse albumin proteoforms each carrying several post-translational modifications.

Proteomic biomarker search requires the greatest analytical reproducibility and detailed information on altered proteoforms. Our protein pre-fractionation applies orthogonal native chromatography and conserves important features of protein variants such as native molecular weight, charge and major glycans. Moreover, we maximized reproducibility of sample pre-fractionation and preparation before mass spectrometry by parallelization and automation. In blood plasma and cerebrospinal fluid (CSF), most proteins, including candidate biomarkers, distribute into a multitude of chromatographic clusters. Plasma albumin, for example, divides into 15-17 clusters. As an example of our technique, we analyzed these albumin clusters from healthy volunteers and from dogs and identified cluster-typical modification patterns. Renal disease further modifies these patterns. In human CSF, we found only a subset of proteoforms with fewer modifications than in plasma. We infer from this example that our method can be used to identify and characterize distinct proteoforms and, optionally, enrich them, thereby yielding the characteristics of proteoform-selective biomarkers.

Structure-Function Relationship between Flicker-Defined Form Perimetry and Spectral-Domain Optical Coherence Tomography in Glaucoma Suspects.

PURPOSE: To evaluate the relationship between functional parameters of repeated flicker-defined form perimetry (FDF) and structural parameters of spectral-domain optical coherence tomography (SD-OCT) in glaucoma suspects with normal findings in achromatic standard automated perimetry (SAP). METHODS: Patients with optic nerve heads (ONH) clinically suspicious for glaucoma and normal SAP findings were enrolled in this prospective study. Each participant underwent visual field (VF) testing with FDF perimetry, using the Heidelberg Edge Perimeter (HEP, Heidelberg Engineering, Heidelberg, Germany) at two consecutive visits. Peripapillary RNFL thickness was obtained by SD-OCT (Spectralis, Heidelberg Engineering, Heidelberg, Germany). Correlations and regression analyses of global and sectoral peripapillary RNFL thickness with corresponding global and regional VF sensitivities were investigated. RESULTS: A consecutive series of 65 study eyes of 36 patients were prospectively included. The second FDF test (HEP II) was used for analysis. Cluster-point based suspicious VF defects were found in 34 eyes (52%). Significant correlations were observed between mean global MD (PSD) of HEP II and SD-OCT-based global peripapillary RNFL thickness (r = 0.380, p = 0.003 for MD and r = -0.516, p < 0.001 for PSD) and RNFL classification scores (R2 = 0.157, p = 0.002 for MD and R2 = 0.172, p = 0.001 for PSD). Correlations between mean global MD and PSD of HEP II and sectoral peripapillary RNFL thickness and classification scores showed highest correlations between function and structure for the temporal superior and temporal inferior sectors whereas sectoral MD and PSD correlated weaker with sectoral RNFL thickness. Correlations between linear RNFL values and untransformed logarithmic MD values for each segment were less significant than correlations between logarithmic MD values and RNFL thickness. CONCLUSIONS: In glaucoma suspects with normal SAP, global and sectoral peripapillary RNFL thickness is correlated with sensitivity and VF defects in FDF perimetry.

Flicker defined form perimetry in glaucoma suspects with normal achromatic visual fields.

BACKGROUND: To evaluate if repeated flicker-defined form (FDF) perimetry can detect visual field (VF) defects in glaucoma suspects with normal findings in achromatic standard automated perimetry (SAP). METHODS: Patients with optic nerve heads (ONHs) or retinal nerve fiber layer (RNFL) findings clinically suspicious for glaucoma and normal SAP were enrolled. Patients underwent VF testing with FDF perimetry (Heidelberg Edge Perimetry, HEP) at two consecutive visits (HEP I and HEP II) and confocal scanning laser ophthalmoscopy with the Heidelberg Retina Tomograph (HRT). Abnormal HEP was defined by cluster-point analysis (CPA) and by the HEP specific glaucoma hemi-field test (GHT). Results were compared with an age-matched control group of healthy individuals. RESULTS: In 65 eyes of 36 glaucoma suspects, the mean deviation (MD) in SAP was -0.9 ± 1.3 dB. In HEP I and HEP II, mean MD was -3.6 ± 3.0 and -3.3 ± 3.7 dB, respectively (p = 0.276). The HRT assessed CDR was significantly correlated with the MD in HEP II (r = -0.281, p = 0.04). In HEP I, VF defects on CPA testing were found in 38 study eyes (58.5%). In HEP II, 34 eyes (51.8%) had VF defects on CPA testing. In 46 eyes of 46 age-matched healthy individuals in the control group, the mean MD was -0.2 ± 1.1 and -1.6 ± 2.3 dB in SAP and HEP testing, respectively. The FDF was abnormal in 21.7% in the control group compared to 58.5% in the glaucoma suspect group in HEP I. CONCLUSIONS: In more than half of the patients with ONHs or RNFLs clinically suspicious for glaucoma and normal SAP second FDF perimetry depicts VF defects.

The impact of structural and functional parameters in glaucoma patients on patient-reported visual functioning.

BACKGROUND: To evaluate the impact of structural changes of the retinal nerve fiber layer (RNFL), and visual field loss, on functional impairment assessed by patient-reported visual functioning in glaucoma. METHODS: Patients with glaucomatous optic nerve damage were enrolled in this cross-sectional study. Peripapillary RNFL thickness was obtained with spectral-domain optical coherence tomography (SD-OCT). Function was assessed by patient-reported visual functioning using the Rasch-calibrated Glaucoma Activity Limitation 9 (GAL-9) questionnaire and standard automated perimetry. The impact of peripapillary RNFL loss on functional impairment was analyzed with correlation and linear regression analyses. RESULTS: A total of 176 eyes from 88 glaucoma patients were included. The SD-OCT assessed temporal-superior and temporal-inferior RNFL sector of the worse eye revealed significant correlation with the GAL-9 scores (r=-0.298, p=0.011 and r=-0.251, p=0.033, respectively). In mutivariate regression analysis, the best predictors for patient-reported visual functioning were visual acuity of the better eye and mean defect of the worse eye (R(2)=0.334), while structural parameters could not enhance the prediction of GAL-9 scores. CONCLUSIONS: Self-reported visual functioning of patients with glaucoma is better predicted by visual performance data than structural parameters. However, some structural changes of the worse eye are significantly correlated with patient-reported visual functioning.

Pars plana vitrectomy and internal limiting membrane peeling in epimacular membranes: correlation of function and morphology across the macula.

PURPOSE: To analyze the correlation between morphological and functional results 12 months after epiretinal membrane (ERM) surgery. METHODS: 31 eyes from 31 consecutive patients with metamorphopsia and best corrected visual acuity (BCVA) below 20/32 underwent a transconjunctival 23- gauge vitrectomy with ERM and internal limiting membrane peeling. Preoperatively and 3, 6 and 12 months postoperatively, we assessed BCVA, microperimetry (MP-1) and spectral domain optical coherence tomography (SD-OCT). Photoreceptor inner and outer segment (IS/OS) was graded on SD-OCT images and correlated with microperimetry measurements in the fovea and parafoveal region. RESULTS: The postoperative BCVA was significantly better in eyes with an intact IS/OS junction (p < 0.01). In addition, the mean defect depth was postoperatively decreased in the foveal and parafoveal area in eyes with an intact IS/OS junction. A correlation of SD-OCT IS/OS images and microperimetry in eyes with improvement in BCVA of at least 2 lines revealed a statistically significant result for the parafoveal quadrants (p < 0.011 for SD-OCT and p < 0.005 for microperimetry) but not for the foveal area alone. CONCLUSIONS: The IS/OS regeneration in the parafoveal quadrants contributes significantly to the recovery of BCVA following ERM surgery. Consequently, functional and morphological tests of the macular area should not be limited to the fovea but should be extended to the parafoveal region.

Characterization of an O-demethylase of Desulfitobacterium hafniense DCB-2.

Besides acetogenic bacteria, only Desulfitobacterium has been described to utilize and cleave phenyl methyl ethers under anoxic conditions; however, no ether-cleaving O-demethylases from the latter organisms have been identified and investigated so far. In this study, genes of an operon encoding O-demethylase components of Desulfitobacterium hafniense strain DCB-2 were cloned and heterologously expressed in Escherichia coli. Methyltransferases I and II were characterized. Methyltransferase I mediated the ether cleavage and the transfer of the methyl group to the superreduced corrinoid of a corrinoid protein. Desulfitobacterium methyltransferase I had 66% identity (80% similarity) to that of the vanillate-demethylating methyltransferase I (OdmB) of Acetobacterium dehalogenans. The substrate spectrum was also similar to that of the latter enzyme; however, Desulfitobacterium methyltransferase I showed a higher level of activity for guaiacol and used methyl chloride as a substrate. Methyltransferase II catalyzed the transfer of the methyl group from the methylated corrinoid protein to tetrahydrofolate. It also showed a high identity (∼70%) to methyltransferases II of A. dehalogenans. The corrinoid protein was produced in E. coli as cofactor-free apoprotein that could be reconstituted with hydroxocobalamin or methylcobalamin to function in the methyltransferase I and II assays. Six COG3894 proteins, which were assumed to function as activating enzymes mediating the reduction of the corrinoid protein after an inadvertent oxidation of the corrinoid cofactor, were studied with respect to their abilities to reduce the recombinant reconstituted corrinoid protein. Of these six proteins, only one was found to catalyze the reduction of the corrinoid protein.

Reengineering the glaucoma quality of life-15 questionnaire with rasch analysis.

PURPOSE: To investigate, using Rasch analysis, whether the 15-item Glaucoma Quality of Life-15 (GQL-15) forms a valid scale and to optimize its psychometric properties. METHODS: One hundred eighteen glaucoma patients (mean age, 65.7 years) completed the German-version of the GQL-15. Rasch analysis was performed to assess category function (how respondents differentiated between the response options), measurement precision (discriminative ability), unidimensionality (whether items measure a single construct), targeting (whether items are of appropriate difficulty for the sample), and differential item functioning (whether comparable subgroups respond differently to an individual item). Where any of these attributes were outside acceptable ranges, steps were taken to improve the instrument. RESULTS: The five-response categories of the GQL-15 were well differentiated by respondents, as demonstrated by ordered and well-spaced category thresholds. The GQL-15 had an excellent measurement precision but demonstrated poor targeting of item difficulty to person ability and multidimensionality, indicating that it was measuring more than one construct. Removal of six misfitting items created a nine-item unidimensional instrument with good measurement precision and no differential item functioning but poor targeting. A new name, the Glaucoma Activity Limitation (GAL-9) questionnaire, is proposed for the short version, which better reflects the construct under measurement. CONCLUSIONS: The GAL-9 has superior psychometric properties over the GQL-15. Its only limitation is poor targeting of item difficulty to person ability, which is an inevitable attribute of a vision-related activity limitation instrument for glaucoma patients, most of whom have only peripheral visual field defects and little difficulty with daily activities.

Propionibacterium acnes in capsular bag distension syndrome.

We report a case of capsular bag distension syndrome that developed 6 years after uneventful phacoemulsification with implantation of a foldable, single-piece acrylic intraocular lens (IOL) (AcrySof MA60BM). Slitlamp microscopy revealed a deep anterior chamber with no flare or cells. The posterior capsular bag was distended by a homogeneous milky substance between the back of the IOL and the capsular bag. Using a pars plana approach, a 23-gauge bimanual capsulotomy and anterior vitrectomy were performed. Microbiological analysis revealed Propionibacterium acnes in the material inside the capsular bag. The postoperative period was uneventful. Four weeks after surgery, visual acuity was restored and there were no signs of intraocular inflammation. The origin of late capsular bag distension is not fully understood; it may involve an infectious component with propionibacteria. A surgical approach and removal of the potentially infectious material can be considered as an alternative to neodymium:YAG capsulotomy.