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 dental pulp stem/stromal cells of Huntington monkey tooth germs

Background

Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors. High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive.

Results

Here we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca²⁺)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca²⁺ concentration [Ca²⁺] difference between bulk cytosolic and the sub-plasma membrane rim.

Conclusion

This approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane. Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation. Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs affecting the translocation process.     

Single-channel properties of N- and L-subtypes of acetylcholine receptor in Ascaris suum

We are interested in the properties of the target site of cholinergic anti-nematodal drugs for therapeutic reasons. The target receptors are ligand-gated ion channels that have different subtypes, and each subtype may have a different pharmacology. In a contraction assay using the parasitic nematode Ascaris suum, our laboratory has identified several subtypes, including an N-subtype, preferentially activated by nicotine, and an L-subtype, preferentially activated by levamisole. Here we use patch-clamp recordings to test the hypothesis that the single-channel selectivities of nicotine and levamisole are different. Unitary currents evoked by nicotine in this preparation were characterised for the first time. In some patches, both nicotine and levamisole activated small- and large-conductance channels. In other patches, the agonists activated just one channel amplitude. Discriminant analysis allowed classification of the one-conductance patch channels into the small or large categories, based on sets defined by the two-conductance patch data. The small channels had a conductance of 26.1+/-1.5 pS, n=18 (mean+/-SEM); the large conductance channels had a conductance of 38.8+/-1.2 pS, n=23 (mean+/-SEM). Analysis of amplitude histograms of the two-conductance patches showed that nicotine preferentially activated the small-conductance channels and levamisole preferentially activated the large-conductance channels. Our observations suggest that the N-subtype receptor channel has a conductance of 26 pS channel and the L-subtype receptor channel has a conductance of 39 pS.     

High-density transient gene expression in suspension-adapted 293 EBNA1 cells

Large-scale transient gene expression (TGE) in mammalian cells is an attractive method to rapidly produce recombinant proteins for pre-clinical studies, with some processes reported to reach 100 L. However, the yield remains low, hardly over 20 mg protein/L, mainly because the current TGEs have been performed at low cell density (approximately 5 x 10(5) cells/mL). In this study, the strategy to improve TGE focuses on facilitating transfection at high cell density. A high-density perfusion culture of 293 EBNA1 cells was established in 2-L bioreactor using Freestyle 293 expression medium (Invitrogen, Singapore) to grow the cells for transfection. Transfection was then carried out at 1 x 10(7) cells/mL using polyethylenimine (PEI) as DNA carrier, at the optimized conditions of 6 microg DNA/10(7) cells and 1:3 DNA to PEI mass ratio. During the post-transfection phase, 80.8 mg/L of the model protein, EPO was obtained at day 5.5 post-transfection (130 mg total EPO production) using a fed-batch culture mode. In comparison, perfusion cultures using an enriched SFM II medium resulted in a longer post-transfection production phase (8 days), and 227 mg of EPO was produced in 10.7 L medium, showing that high-density TGE enables the production of several hundreds of milligrams of protein in a 2 L bioreactor. In addition, a protocol for economical plasmid preparation based on anion exchange was also established to satisfy TGE's demand in terms of quality and quantity. To the best of our knowledge, this is the first report of transient transfections at a high cell density of up to 1 x 10(7) cells/mL.     

Deep sequencing reveals clonal evolution patterns and mutation events associated with relapse in B-cell lymphomas

Background

Molecular mechanisms associated with frequent relapse of diffuse large B-cell lymphoma (DLBCL) are poorly defined. It is especially unclear how primary tumor clonal heterogeneity contributes to relapse. Here, we explore unique features of B-cell lymphomas - VDJ recombination and somatic hypermutation - to address this question.

Results

We performed high-throughput sequencing of rearranged VDJ junctions in 14 pairs of matched diagnosis-relapse tumors, among which 7 pairs were further characterized by exome sequencing. We identify two distinctive modes of clonal evolution of DLBCL relapse: an early-divergent mode in which clonally related diagnosis and relapse tumors diverged early and developed in parallel; and a late-divergent mode in which relapse tumors developed directly from diagnosis tumors with minor divergence. By examining mutation patterns in the context of phylogenetic information provided by VDJ junctions, we identified mutations in epigenetic modifiers such as KMT2D as potential early driving events in lymphomagenesis and immune escape alterations as relapse-associated events.

Conclusions

Altogether, our study for the first time provides important evidence that DLBCL relapse may result from multiple, distinct tumor evolutionary mechanisms, providing rationale for therapies for each mechanism. Moreover, this study highlights the urgent need to understand the driving roles of epigenetic modifier mutations in lymphomagenesis, and immune surveillance factor genetic lesions in relapse.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0432-0) contains supplementary material, which is available to authorized users.