Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Characterization of a novel xylanase from Armillaria gemina and its immobilization onto SiO2 nanoparticles

Enhanced catalytic activities of different lignocellulases were obtained from Armillaria gemina under statistically optimized parameters using a jar fermenter. This strain showed maximum xylanase, endoglucanase, cellobiohydrolase, and β-glucosidase activities of 1,270, 146, 34, and 15 U mL^?1, respectively. Purified A. gemina xylanase (AgXyl) has the highest catalytic efficiency (k _cat/K _m?=?1,440 mg?mL^?1?s^?1) ever reported for any fungal xylanase, highlighting the significance of the current study. We covalently immobilized the crude xylanase preparation onto functionalized silicon oxide nanoparticles, achieving 117 % immobilization efficiency. Further immobilization caused a shift in the optimal pH and temperature, along with a fourfold improvement in the half-life of crude AgXyl. Immobilized AgXyl gave 37.8 % higher production of xylooligosaccharides compared to free enzyme. After 17 cycles, the immobilized enzyme retained 92 % of the original activity, demonstrating its potential for the synthesis of xylooligosaccharides in industrial applications.     

Total phenolics, flavonoids and antioxidant activity of Saptarangi (Salacia chinensis L.) fruit pulp

Salacia chinensis L. has various beneficial properties including antidiabetic and antioxidant activity. The S. chinensis fruit pulp (SCFP) was extracted with four different solvents (Methanol, ethanol, acetone and water) and was screened for total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity (AOA). The AOA was assessed by evaluating the 1, 1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and metal chelating assay. Methanolic SCFP extract exhibited the highest phenolic content (3.20?±?0.12 mg GAE/g FW) whereas, ethanolic extract showed highest flavonoid content (0.31?±?0.68 mg RE/g FW). The methanolic extract possesses highest antioxidant activity towards DPPH (92.44 %), FRAP (1.939 O.D) and metal chelating activity (74.16 %). AOA (DPPH and FRAP) was significantly correlated with TPC. The results indicated that SCFP is a good natural source of antioxidant compounds for use in food and pharmaceutical industry.     

Antifungal activity of some marine organisms from India,against food spoilage Aspergillus strains

Crude aqueous methanol extracts obtained from 31 species of various marine organisms (including flora land faunal), were screened for their antifungal activity against food poisoning strains of Aspergillus. Seventeen species exhibited mild (+ =zone of inhibition 1–2 mm) to significant (+++ =zone of inhibition 3–5 mm) activity against one or the other strain under experiment. However, extracts of 12 species were active against all the three strains. Organisms like Salicornia brachiata(obligate halophyte), Sinularia leptocladus(Soft coral), Elysia grandifolia (Mollusks),Gorgonian sp. 2 and Haliclona sp. exhibited significant (inhibition zone of 3–5 mm) antifungal activity against one or the other strains. However,extracts of A. ilicifolius, Amphiroa sp.,Poryphyra sp., Unidentified sponge, Suberites vestigium, Sinularia compressa,Sunularia sp., Sinularia maxima, Subergorgia suberosa, Echinogorgia pseudorassopo and Sabellaria cementifera were mild (inhibition zone of 1–2 mm) to moderate(inhibition zone of 2–3 mm) active against the respective strains. The growth of A. japonicuswas significantly inhibited by the extracts ofS. leptocladus (r = 0.992, p < 0.0001)and E. grandifolia (r = 0.989, p < 0.0001).This revised version was published online in October 2005 with corrections to the Cover Date.     

Diabetic endothelial dysfunction: the role of poly(ADP-ribose) polymerase activation

Diabetic patients frequently suffer from retinopathy, nephropathy, neuropathy and accelerated atherosclerosis. The loss of endothelial function precedes these vascular alterations. Here we report that activation of poly(ADP-ribose) polymerase (PARP) is an important factor in the pathogenesis of endothelial dysfunction in diabetes. Destruction of islet cells with streptozotocin in mice induced hyperglycemia, intravascular oxidant production, DNA strand breakage, PARP activation and a selective loss of endothelium-dependent vasodilation. Treatment with a novel potent PARP inhibitor, starting after the time of islet destruction, maintained normal vascular responsiveness, despite the persistence of severe hyperglycemia. Endothelial cells incubated in high glucose exhibited production of reactive nitrogen and oxygen species, consequent single-strand DNA breakage, PARP activation and associated metabolic and functional impairment. Basal and high-glucose-induced nuclear factor-kappaB activation were suppressed in the PARP-deficient cells. Our results indicate that PARP may be a novel drug target for the therapy of diabetic endothelial dysfunction.     

Optical quantitative pathology of cervical intraepithelial neoplasia in human tissues using spatial frequency analysis

An optical quantitative histological method in human tissues using spatial frequencies is demonstrated. Optical spatial frequency spectra from different stages of human Cervical Intraepithelial Neoplasia (CIN) tissue are evaluated as a potential quantitative pathological tool. The degree of randomness of tissue structures from normal to different stages of CIN tissue can be recognized by spatial frequency analysis. The standard deviation, σ of human normal and CIN tissue, is obtained by assuming the spatial frequency spectra as a Gaussian distribution. A support vector machine classifier (SVM) is trained in the subspace of σ. Twenty‐eight normal and CIN samples of varying grades are examined and compared with current diagnostic outcomes. Our results suggest that an excellent accuracy for diagnostic purposes can be achieved. This approach offers a simple, efficient and objective way to supplement histopathology in recognizing alterations from normal to different stages of cervical pre‐cancer, which are reflected by spatial information contained within the aperiodic and random structures of the different types of tissue. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.     

Non-ventral lateral neuron-based, non-PDF-mediated clocks control circadian egg-laying rhythm in Drosophila melanogaster

The authors report the results of their study aimed at investigating the consequence of targeted ablation of ventral lateral neurons (LN(v)s--neurons regulating eclosion and locomotor activity rhythms) and genetic disruption of pigment-dispersing factor (PDF--an important output of circadian clocks) on the egg-laying rhythm of Drosophila melanogaster. The results clearly suggest that genetic ablation of LN(v)s and loss of function mutation of PDF abolish eclosion and locomotor activity rhythms, whereas the egg-laying rhythm continues unabated. Furthermore, the results also demonstrate that the period of egg-laying rhythm remains unchanged under different ambient temperatures and nutrition levels, suggesting that the egg-laying rhythm of D. melanogaster is temperature and nutrition compensated. Based on these results, the authors conclude that the egg-laying rhythm in D. melanogaster is regulated by non-LN(v)-based, non-PDF-mediated circadian clocks.     

Microbial conversion of xylose into useful bioproducts

Microorganisms can produce a number of different bioproducts from the sugars in plant biomass. One challenge is devising processes that utilize all of the sugars in lignocellulosic hydrolysates. D-xylose is the second most abundant sugar in these hydrolysates. The microbial conversion of D-xylose to ethanol has been studied extensively; only recently, however, has conversion to bioproducts other than ethanol been explored. Moreover, in the case of yeast, D-xylose may provide a better feedstock for the production of bioproducts other than ethanol, because the relevant pathways are not subject to glucose-dependent repression. In this review, we discuss how different microorganisms are being used to produce novel bioproducts from D-xylose. We also discuss how D-xylose could be potentially used instead of glucose for the production of value-added bioproducts.

     

Diglyceride prodrug strategy for enhancing the bioavailability of norfloxacin

Prodrug approach using diglyceride as a promoiety is a promising strategy to improve bioavailability of poorly absorbed drugs and the same was explored in the present work to improve oral bioavailability of norfloxacin; a second generation fluoroquinolone antibacterial. The prodrug was synthesized by standard procedures using dipalmitine as a carrier and the structure was confirmed by spectral analysis. Higher Log P indicated improved lipophilicity. The ester linkage between norfloxacin and dipalmitine would be susceptible to hydrolysis by lipases to release the parent drug and carrier in the body. In vivo kinetic studies in rats indicated 53% release of norfloxacin in plasma at the end of 8 h. The prodrug exhibited improved pharmacological profile than the parent compound at equimolar dose that indirectly indicated improved bioavailability.