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.     

Engineering a therapeutic IgG molecule to address cysteinylation,aggregation and enhance thermal stability and expression

Antibodies can undergo a variety of covalent and non-covalent degradation reactions that have adverse effects on efficacy, safety, manufacture and storage. We had identified an antibody to Angiopoietin 2 (Ang2 mAb) that neutralizes Ang2 binding to its receptor in vitro and inhibits tumor growth in vivo. Despite favorable pharmacological activity, the Ang2 mAb preparations were heterogeneous, aggregated rapidly and were poorly expressed. Here, we report the engineering of the antibody variable and constant domains to generate an antibody with reduced propensity to aggregate, enhanced homogeneity, 11°C elevated T_m, 26-fold improved level of expression and retained activity. The engineered molecule, MEDI-3617, is now compatible with the large scale material supply required for clinical trials and is currently being evaluated in Phase 1 in cancer patients. This is the first report to describe the stability engineering of a therapeutic antibody addressing non canonical cysteine residues and the design strategy reported here is generally applicable to other therapeutic antibodies and proteins.     

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.     

ATM Influences the Efficiency of TCRβ Rearrangement,Subsequent TCRβ-Dependent T Cell Development,and Generation of the Pre-Selection TCRβ CDR3 Repertoire

Generation and resolution of DNA double-strand breaks is required to assemble antigen-specific receptors from the genes encoding V, D, and J gene segments during recombination. The present report investigates the requirement for ataxia telangiectasia-mutated (ATM) kinase, a component of DNA double-strand break repair, during TCRβ recombination and in subsequent TCRβ-dependent repertoire generation and thymocyte development. CD4⁻CD8⁻ double negative stage 2/3 thymocytes from ATM-deficient mice have both an increased frequency of cells with DNA break foci at TCRβ loci and reduced Vβ-DJβ rearrangement. Sequencing of TCRβ complementarity-determining region 3 demonstrates that ATM-deficient CD4⁺CD8⁺ double positive thymocytes and peripheral T cells have altered processing of coding ends for both in-frame and out-of-frame TCRβ rearrangements, providing the unique demonstration that ATM deficiency alters the expressed TCRβ repertoire by a selection-independent mechanism. ATMKO thymi exhibit a partial developmental block in DN cells as they negotiate the β-selection checkpoint to become double negative stage 4 and CD4⁺CD8⁺ thymocytes, resulting in reduced numbers of CD4⁺CD8⁺ cells. Importantly, expression of a rearranged TCRβ transgene substantially reverses this defect in CD4⁺CD8⁺ cells, directly linking a requirement for ATM during endogenous TCRβ rearrangement to subsequent TCRβ-dependent stages of development. These results demonstrate that ATM plays an important role in TCRβ rearrangement, generation of the TCRβ CDR3 repertoire, and efficient TCRβ-dependent T cell development.     

An Invasive Fish and the Time-Lagged Spread of Its Parasite across the Hawaiian Archipelago

Efforts to limit the impact of invasive species are frustrated by the cryptogenic status of a large proportion of those species. Half a century ago, the state of Hawai''i introduced the Bluestripe Snapper, Lutjanus kasmira, to O''ahu for fisheries enhancement. Today, this species shares an intestinal nematode parasite, Spirocamallanus istiblenni, with native Hawaiian fishes, raising the possibility that the introduced fish carried a parasite that has since spread to naïve local hosts. Here, we employ a multidisciplinary approach, combining molecular, historical, and ecological data to confirm the alien status of S. istiblenni in Hawai''i. Using molecular sequence data we show that S. istiblenni from Hawai''i are genetically affiliated with source populations in French Polynesia, and not parasites at a geographically intermediate location in the Line Islands. S. istiblenni from Hawai''i are a genetic subset of the more diverse source populations, indicating a bottleneck at introduction. Ecological surveys indicate that the parasite has found suitable intermediate hosts in Hawai''i, which are required for the completion of its life cycle, and that the parasite is twice as prevalent in Hawaiian Bluestripe Snappers as in source populations. While the introduced snapper has spread across the entire 2600 km archipelago to Kure Atoll, the introduced parasite has spread only half that distance. However, the parasite faces no apparent impediments to invading the entire archipelago, with unknown implications for naïve indigenous Hawaiian fishes and the protected Papahānaumokuākea Marine National Monument.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Marine mammal culling programs: review of effects on predator and prey populations

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