Viral Transduction of Human Rod Opsin or Channelrhodopsin Variants to Mouse ON Bipolar Cells Does Not Impact Retinal Anatomy or Cause Measurable Death in the Targeted Cells.

The viral gene delivery of optogenetic actuators to the surviving inner retina has been proposed as a strategy for restoring vision in advanced retinal degeneration. We investigated the safety of ectopic expression of human rod opsin (hRHO), and two channelrhodopsins (enhanced sensitivity CoChR-3M and red-shifted ReaChR) by viral gene delivery in ON bipolar cells of the mouse retina. Adult Grm6Cre mice were bred to be retinally degenerate or non-retinally degenerate (homozygous and heterozygous for the rd1Pde6b mutation, respectively) and intravitreally injected with recombinant adeno-associated virus AAV2/2(quad Y-F) serotype containing a double-floxed inverted transgene comprising one of the opsins of interest under a CMV promoter. None of the opsins investigated caused changes in retinal thickness; induced apoptosis in the retina or in transgene expressing cells; or reduced expression of PKCα (a specific bipolar cell marker). No increase in retinal inflammation at the level of gene expression (IBA1/AIF1) was found within the treated mice compared to controls. The expression of hRHO, CoChR or ReaChR under a strong constitutive promoter in retinal ON bipolar cells following intravitreal delivery via AAV2 does not cause either gross changes in retinal health, or have a measurable impact on the survival of targeted cells.

Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli.

Escherichia coli strains have been modified in a variety of ways to enhance the production of different recombinant proteins, targeting membrane protein expression, proteins with disulphide bonds, and more recently, proteins which require N-linked glycosylation. The addition of glycans to proteins remains a relatively inefficient process and here we aimed to combine genetic modifications within central carbon metabolic pathways in order to increase glycan precursor pools, prior to transfer onto polypeptide backbones. Using a lectin screen that detects cell surface representation of glycans, together with Western blot analyses using an O-antigen ligase mutant strain, the enhanced uptake and phosphorylation of sugars (ptsA) from the media combined with conservation of carbon through the glyoxylate shunt (icl) improved glycosylation efficiency of a bacterial protein AcrA by 69% and over 100% in an engineered human protein IFN-α2b. Unexpectedly, overexpression of a gene involved in the production of DXP from pyruvate (dxs), which was previously seen to have a positive impact on glycosylation, was detrimental to process efficiency and the possible reasons for this are discussed.

Differential expression of cyclin-dependent kinases in the adult human retina in relation to CDK inhibitor retinotoxicity.

Cyclin-dependent kinases (CDKs) are a family of kinases associated predominantly with cell cycle control, making CDK inhibitors interesting candidates for anti-cancer therapeutics. However, retinal toxicity (loss of photoreceptors) has been associated with CDK inhibitors, including the pan-CDK inhibitor AG-012896. The purpose of this research was to use a novel planar sectioning technique to determine CDK expression profiles in the ex vivo human retina with the aim of identifying isoforms responsible for CDK retinotoxicity. Four CDK isoforms (CDK11, 16, 17 and 18) were selected as a result of IC50 data comparing neurotoxic (AG-012986 and NVP-1) and non-neurotoxic (dinaciclib and NVP-2) CDK inhibitors, with IC50s at CDK11 showing a clear difference between the neurotoxic and non-neurotoxic drugs. CDK11 was maximally expressed in the photoreceptor layer, whereas CDK16, 17 and 18 showed maximal expression in the inner nuclear layer. CDK5 (an isoform associated with retinal homeostasis) was maximally expressed in the retinal ganglion cell layer. Apart from CDK18, each isoform showed expression in the photoreceptor layer. The human Müller cell line MIO-M1 expressed CDK5, 11, 16 and 17 and AG-01298 (0.02-60 µM) caused a dose-dependent increase in MIO-M1 cell death. In conclusion, CDK11 appears the most likely candidate for mediation of photoreceptor toxicity. RNA profiling can be used to determine the distribution of genes of interest in relation to retinal toxicity in the human retina.

Lipid profiles of prostate cancer cells.

Lipids are important cellular components which can be significantly altered in a range of disease states including prostate cancer. Here, a unique systematic approach has been used to define lipid profiles of prostate cancer cell lines, using quantitative mass spectrometry (LC-ESI-MS/MS), FTIR spectroscopy and fluorescent microscopy. All three approaches identified significant difference in the lipid profiles of the three prostate cancer cell lines (DU145, LNCaP and 22RV1) and one non-malignant cell line (PNT1a). Specific lipid classes and species, such as phospholipids (e.g., phosphatidylethanolamine 18:1/16:0 and 18:1/18:1) and cholesteryl esters, detected by LC-ESI-MS/MS, allowed statistical separation of all four prostate cell lines. Lipid mapping by FTIR revealed that variations in these lipid classes could also be detected at a single cell level, however further investigation into this approach would be needed to generate large enough data sets for quantitation. Visualisation by fluorescence microscopy showed striking variations that could be observed in lipid staining patterns between cell lines allowing visual separation of cell lines. In particular, polar lipid staining by a fluorescent marker was observed to increase significantly in prostate cancer lines cells, when compared to PNT1a cells, which was consistent with lipid quantitation by LC-ESI-MS/MS and FTIR spectroscopy. Thus, multiple technologies can be employed to either quantify or visualise changes in lipid composition, and moreover specific lipid profiles could be used to detect and phenotype prostate cancer cells.

A Molecular Probe for the Detection of Polar Lipids in Live Cells.

Lipids have an important role in many aspects of cell biology, including membrane architecture/compartment formation, intracellular traffic, signalling, hormone regulation, inflammation, energy storage and metabolism. Lipid biology is therefore integrally involved in major human diseases, including metabolic disorders, neurodegenerative diseases, obesity, heart disease, immune disorders and cancers, which commonly display altered lipid transport and metabolism. However, the investigation of these important cellular processes has been limited by the availability of specific tools to visualise lipids in live cells. Here we describe the potential for ReZolve-L1™ to localise to intracellular compartments containing polar lipids, such as for example sphingomyelin and phosphatidylethanolamine. In live Drosophila fat body tissue from third instar larvae, ReZolve-L1™ interacted mainly with lipid droplets, including the core region of these organelles. The presence of polar lipids in the core of these lipid droplets was confirmed by Raman mapping and while this was consistent with the distribution of ReZolve-L1™ it did not exclude that the molecular probe might be detecting other lipid species. In response to complete starvation conditions, ReZolve-L1™ was detected mainly in Atg8-GFP autophagic compartments, and showed reduced staining in the lipid droplets of fat body cells. The induction of autophagy by Tor inhibition also increased ReZolve-L1™ detection in autophagic compartments, whereas Atg9 knock down impaired autophagosome formation and altered the distribution of ReZolve-L1™. Finally, during Drosophila metamorphosis fat body tissues showed increased ReZolve-L1™ staining in autophagic compartments at two hours post puparium formation, when compared to earlier developmental time points. We concluded that ReZolve-L1™ is a new live cell imaging tool, which can be used as an imaging reagent for the detection of polar lipids in different intracellular compartments.

Imaging nuclear, endoplasmic reticulum and plasma membrane events in real time.

Live cell imaging can provide important information on cellular dynamics; however, the full utilisation of this technology has been hampered by the limitations of imaging reagents. Metal-based complexes have the potential to overcome many of the issues common to many current imaging agents. The rhenium (I)-based complex fac-[Re(CO)3 (1,10-phenanthroline)(4-pyridyltetrazolate)], herein referred to as ReZolve-ER(™) , shows promise as a live cell imaging agent with rapid cell uptake, low cytotoxicity, resistance to photobleaching and compatibility with multicolour imaging. ReZolve-ER(™) localised to the nuclear membrane/endoplasmic reticulum (ER) and allowed the detection of exocytotic events at the plasma membrane. Thus, we present a new imaging agent for monitoring live cell events in real time, which is ideal for imaging either short- or long-time courses.

Reduced keratin expression in colorectal neoplasia and associated fields is reversible by diet and resection.

BACKGROUND: Patients with adenomatous colonic polyps are at increased risk of developing further polyps suggesting field-wide alterations in cancer predisposition. The current study aimed to identify molecular alterations in the normal mucosa in the proximity of adenomatous polyps and to assess the modulating effect of butyrate, a chemopreventive compound produced by fermentation of dietary residues. METHODS: A cross-sectional study was undertaken in patients with adenomatous polyps: biopsy samples were taken from the adenoma, and from macroscopically normal mucosa on the contralateral wall to the adenoma and from the mid-sigmoid colon. In normal subjects biopsies were taken from the mid-sigmoid colon. Biopsies were frozen for proteomic analysis or formalin-fixed for immunohistochemistry. Proteomic analysis was undertaken using iTRAQ workflows followed by bioinformatics analyses. A second dietary fibre intervention study arm used the same endpoints and sampling strategy at the beginning and end of a high-fibre intervention. RESULTS: Key findings were that keratins 8, 18 and 19 were reduced in expression level with progressive proximity to the lesion. Lesional tissue exhibited multiple K8 immunoreactive bands and overall reduced levels of keratin. Biopsies from normal subjects with low faecal butyrate also showed depressed keratin expression. Resection of the lesion and elevation of dietary fibre intake both appeared to restore keratin expression level. CONCLUSION: Changes in keratin expression associate with progression towards neoplasia, but remain modifiable risk factors. Dietary strategies may improve secondary chemoprevention. TRIAL REGISTRATION NUMBER: ISRCTN90852168.

Proton-induced reversible modulation of the luminescent output of rhenium(I), iridium(III), and ruthenium(II) tetrazolate complexes.

One of the distinct features of metal-tetrazolate complexes is the possibility of performing electrophilic additions onto the imine-type nitrogens of the coordinated five-membered ring. These reactions, in particular, provide a useful tool for varying the main structural and electronic properties of the starting tetrazolate complexes. In this paper, we demonstrate how the use of a simple protonation-deprotonation protocol enables us to reversibly change, to a significant extent, the light-emission output and performance of a series of Re(I)-tetrazolate-based phosphors of the general formulation fac-[Re(N(∧)N)(CO)3L], where N(∧)N denotes diimine-type ligands such as 2,2'-bipyridine (bpy) or 1,10-phenanthroline (phen) and L represents a series of different 5-aryl tetrazolates. Indeed, upon addition of triflic acid to these neutral Re(I) complexes, a consistent blue shift (Δλmax ca. 50 nm) of the emission maximum is observed and the protonated species also display increased quantum yield values (4-13 times greater than the starting compounds) and longer decay lifetimes. This alteration can be reversed to the initial condition by further treating the protonated Re(I) complex with a base such as triethylamine. Interestingly, the reversible modulation of luminescent features by the same protonation-deprotonation mechanism appears as a quite general characteristic of photoactive metal tetrazolate complexes, even for compounds in which the 2-pyridyl tetrazolate ligands coordinate the metal center with a bidentate mode, such as the corresponding Ir(III) cyclometalates [Ir(C(∧)N)2L] and the Ru(II) polypyridyl derivatives [Ru(bpy)2L](+). In these cases, the protonation of the starting materials leads to red-shifted and more intense emissions for the Ir(III) complexes, while almost complete quenching is observed in the case of the Ru(II) analogues.

Application of the CIRAD mass spectrometry approach for lysine acetylation site discovery.

Mass spectrometry (MS)-based methods typically assess acetylation by detection of a diagnostic ion at 126.1 m/z, corresponding to the immonium ion of acetyl-lysine -NH(3), which is generated by collisionally induced dissociation. A novel implementation of this approach, based on the accurate mass and retention time technique, couples high mass resolution measurement with rapid cycling between low and elevated collision energies to generate intact and fragment high-resolution mass spectra. This allows acetyl lysine diagnostic ions at 126.1 m/z to be monitored and aligned to the precursor m/z based on retention time profile. The technique is termed Collisionally Induced Release of Acetyl Diagnostic. Sequence information is also obtained for acetylation site assignment. This technique to identify acetylation species is information independent as it does not require the sequence of the protein/peptides to identify acetylation, and thus complementary to data-dependent methods. It is suitable for analysis of acetylated peptides, or proteins enriched by immunoprecipitation with acetyl lysine-specific antibodies.

Comparative study of in vitro expansion of bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BMD-MSCs) are of great interest for tissue engineering, but require expansion before they can be used for therapeutic applications. We compared three different culture techniques for their potential for large scale expansion of rat BMD-MSCs, i.e. monolayer cultures, stirred suspension cultures and pour-off cultures, and found that pour-off cultures supported the biggest expansion in BMD-MSCs as measured by the fibroblastic-colony forming unit assay (CFU-f). BMD-MSCs expanded in stirred suspension cultures stopped proliferating altogether and, although monolayer cultures allowed for expansion of BMD-MSCs, they favoured a differentiated phenotype over uncommitted MSCs. Only BMD-MSCs expanded in pour-off cultures were able to differentiate into both osteoblastic and adipocytic lineages and maintain CFU-f numbers. These data suggest that pour-off cultures are a viable method of BMD-MSC expansion.

An integrated workflow for extraction and solubilization of intermediate filaments from colorectal biopsies for proteomic analysis.

We report a technique for isolation and solubilization of intermediate filament (IF) proteins from colonic biopsies compatible with both gel electrophoresis and liquid chromatography "shotgun" proteomics using mass spectrometry (MS). This is important because changes in the IF proteome, particularly in keratin expression and modification, are noted in colonic mucosa of patients with colorectal cancer. Though keratins have traditionally been dissolved in high concentration of urea, the latter solvent precludes efficient proteolytic digestion by trypsin prior to gel-free LC-MS/MS approaches. The extraction of cytoskeletal proteins was initially evaluated using MCF-7 cancer cell lines using a published, differential detergent solubilization protocol. IF proteins were extracted from colonic biopsies using a combination of homogenization and sonication. Since comparable efficiency of solubilization was noted on the extracted IF from cell lines between urea and guanidine hydrochloride (GuHCl) in triethylammonium bicarbonate buffer, isolated proteins from endoscopic biopsies were solubilized in GuHCl. Using immunoblotting techniques, we successfully demonstrated isolation of keratins and preservation of posttranslational modifications (phosphorylation, acetylation). Dissolved proteins were tryptically digested and peptides analyzed by MS, showing the functionality of the workflow in shotgun proteomic applications, specifically compatibility of the workflow for isobaric tagging relative and absolute quantification based quantitation approaches.

Proteomic analysis of the impact of static culturing on the expansion of rat bone marrow mesenchymal stem cells.

The clinical potential of mesenchymal stem cells (MSC) in tissue engineering and regenerative medicine is due to their self-renewal, proliferation and multi-lineage differentiation potential. Clinical use requires large cell numbers; which can, theoretically, be generated by ex vivo expansion of plastic adherent, MSC subpopulation, of bone marrow cells (BMC). Effects of serial culture on MSC phenotype were investigated using non-gel based quantitative proteomic methodology for static monolayer cultures of rat BMC. In total, 382 proteins were relatively quantified (≥ 2 peptides). Nine proteins were up-regulated and seven down-regulated at passage 4 relative to passage 2 (p ≤ 0.05). We propose that serial culture impacts on MSC expansion (observed decline in colony forming potential and colony size) is through a combination of osteogenic differentiation and ageing/senescence and propose six novel protein biomarkers as candidates for quality control purposes in bioprocessing.

Protein profiling of the secretome of FcεRI activated RBL-2H3.1 cells.

BACKGROUND: Secretory proteins of IgE receptor activated mast cells and basophils play a pivotal role in the generation of immediate and long term immune responses in allergy and type I hypersensitivity. OBJECTIVE: The present study aims to generate a 2-D map and profile of proteins secreted from a high secretory variant of the rat basophilic leukemia cell line, RBL-2H3.1, which in view of the difficulty associated with gaining adequate numbers of pure primary mast cell and basophiles, represents an accepted model system for the study and standardization of the methodology to characterize the secretome of these cell types. METHODS: A 2-D map of secretory proteins was generated by 2-D PAGE and a shotgun mass spectrometric approach carried out for protein identification. RESULTS: Study resulted into identification of 299 proteins released from resting and IgE receptor activated RBL-2H3.1 cells after 90 s, 30 min and 3 h antigen challenge. Further sequence analysis identified ~53% of total proteins as secretory proteins which could be attributed to classical and non-classical secretory pathways. Additionally, functional classification of classic secretory proteins verified the presence of proteins belonged to cytokines, receptors, membrane proteins, lysosomal proteins and proteins associated with specific sub-cellular localizations such as endoplasmic reticulum, mitochondria, nucleus, cytoplasm and ribosome. According to this data the presence of some secretory proteins such as cytokines (e.g. MCP-2, PF-4, CSF-1 and TGF-ß1) are all subject to Ag challenge which may point to their importance toward pathogenesis in allergic diseases. CONCLUSION: In view of both a beneficial and adverse role of mast cell mediators in health and disease, an identification of temporal changes in the secretory pattern may form the basis for future tailor made intervention strategies that may enable us to harvest the therapeutic potential inherent in mast cell exocytosis while inhibiting/attenuating negative outcomes.

iTRAQ identification of candidate serum biomarkers associated with metastatic progression of human prostate cancer.

A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n = 5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer 'BPH', (ii) localised cancer with no evidence of progression, 'non-progressing' (iii) localised cancer with evidence of biochemical progression, 'progressing', and (iv) bone metastasis at presentation 'metastatic'. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and 'panels' of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p = 0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation.

A proteomic analysis of differential cellular responses to the short-chain fatty acids butyrate, valerate and propionate in colon epithelial cancer cells.

The short chain fatty acids (SCFAs) are inhibitors of histone deacetylases (HDACi); they are produced naturally in the colon by fermentation. They affect cellular processes at a molecular and transcriptional level, the mechanisms of which may involve large numbers of proteins and integrated pathways. Butyrate is the most biologically potent of the SCFAs in colon epithelial cells, inhibiting human colon carcinoma cell proliferation and inducing apoptosis in vitro. In order to investigate the hypothesis that propionate and valerate possess unique and independent actions from butyrate, we combined proteomic and cellomic approaches for large-scale comparative analysis. Proteomic evaluation was undertaken using an iTRAQ tandem mass-spectrometry workflow and high-throughput High-content Analysis microscopy (HCA) was applied to generate cellomic information on the cell cycle and the cytoskeletal structure. Our results show that these SCFAs possess specific effects. Butyrate was shown to have more pronounced effects on the keratins and intermediate filaments (IFs); while valerate altered the ß-tubulin isotypes' expression and the microtubules (MTs); propionate was involved in both mechanisms, displaying intermediate effects. These data suggest distinct physiological roles for SCFAs in colon epithelial function, offering new possibilities for cancer therapeutics.

Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae.

Macrophages are central effectors of innate immune responses to bacteria. We have investigated how activation of the abundant macrophage lysosomal protease, cathepsin D, regulates the macrophage proteome during killing of Streptococcus pneumoniae. Using the cathepsin D inhibitor pepstatin A, we demonstrate that cathepsin D differentially regulates multiple targets out of 679 proteins identified and quantified by eight-plex isobaric tag for relative and absolute quantitation. Our statistical analysis identified 18 differentially expressed proteins that passed all paired t-tests (α = 0.05). This dataset was enriched for proteins regulating the mitochondrial pathway of apoptosis or inhibiting competing death programs. Five proteins were selected for further analysis. Western blotting, followed by pharmacological inhibition or genetic manipulation of cathepsin D, verified cathepsin D-dependent regulation of these proteins, after exposure to S. pneumoniae. Superoxide dismutase-2 up-regulation was temporally related to increased reactive oxygen species generation. Gelsolin, a known regulator of mitochondrial outer membrane permeabilization, was down-regulated in association with cytochrome c release from mitochondria. Eukaryotic elongation factor (eEF2), a regulator of protein translation, was also down-regulated by cathepsin D. Using absence of the negative regulator of eEF2, eEF2 kinase, we confirm that eEF2 function is required to maintain expression of the anti-apoptotic protein Mcl-1, delaying macrophage apoptosis and confirm using a murine model that maintaining eEF2 function is associated with impaired macrophage apoptosis-associated killing of Streptococcus pneumoniae. These findings demonstrate that cathepsin D regulates multiple proteins controlling the mitochondrial pathway of macrophage apoptosis or competing death processes, facilitating intracellular bacterial killing.

Rapid fabrication of glass/PDMS hybrid µIMER for high throughput membrane proteomics.

Mass spectrometry (MS) based proteomics has brought a radical approach to systems biology, offering a platform to study complex biological functions. However, key proteomic technical challenges remain, mainly the inability to characterise the complete proteome of a cell due to the thousands of diverse, complex proteins expressed at an extremely wide concentration range. Currently, high throughput and efficient techniques to unambiguously identify and quantify proteins on a proteome-wide scale are in demand. Miniaturised analytical systems placed upstream of MS help us to attain these goals. One time-consuming step in traditional techniques is the in-solution digestion of proteins (4-20 h). This also has other drawbacks, including enzyme autoproteolysis, low efficiency, and manual operation. Furthermore, the identification of α-helical membrane proteins has remained a challenge due to their high hydrophobicity and lack of trypsin cleavage targets in transmembrane helices. We demonstrate a new rapidly produced glass/PDMS micro Immobilised Enzyme Reactor (µIMER) with enzymes covalently immobilised onto polyacrylic acid plasma-modified surfaces for the purpose of rapidly (as low as 30 s) generating peptides suitable for MS analysis. This µIMER also allows, for the first time, rapid digestion of insoluble proteins. Membrane protein identification through this method was achieved after just 4 min digestion time, up to 9-fold faster than either dual-stage in-solution digestion approaches or other commonly used bacterial membrane proteomic workflows.

Eight-plex iTRAQ analysis of variant metastatic human prostate cancer cells identifies candidate biomarkers of progression: An exploratory study.

BACKGROUND: Due to the heterogeneity in the biological behavior of prostate cancer, biomarkers that can reliably distinguish indolent from aggressive disease are urgently needed to inform treatment choices. METHODS: We employed 8-plex isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to profile the proteomes of two distinct panels of isogenic prostate cancer cells with varying growth and metastatic potentials, in order to identify novel biomarkers associated with progression. The LNCaP, LNCaP-Pro5, and LNCaP-LN3 panel of cells represent a model of androgen-responsive prostate cancer, while the PC-3, PC-3M, and PC-3M-LN4 panel represent a model of androgen-insensitive disease. RESULTS: Of the 245 unique proteins identified and quantified (>or=95% confidence; >or=2 peptides/protein), 17 showed significant differential expression (>or=+/-1.5), in at least one of the variant LNCaP cells relative to parental cells. Similarly, comparisons within the PC-3 panel identified 45 proteins to show significant differential expression in at least one of the variant PC-3 cells compared with parental cells. Differential expression of selected candidates was verified by Western blotting or immunocytochemistry, and corresponding mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Immunostaining of prostate tissue microarrays for ERp5, one of the candidates identified, showed a significant higher immunoexpression in pre-malignant lesions compared with non-malignant epithelium (P < 0.0001, Mann-Whitney U-test), and in high Gleason grade (4-5) versus low grade (2-3) cancers (P < 0.05). CONCLUSIONS: Our study provides proof of principle for the application of an 8-plex iTRAQ approach to uncover clinically relevant candidate biomarkers for prostate cancer progression.

Nuclear proteome dynamics in differentiating embryonic carcinoma (NTERA-2) cells.

The use of stem cells for generating cell types suitable for therapy is dependent on understanding the mechanisms, and identifying biomarkers, that control cell fate into different lineages. In this study, we aimed to characterize the nuclear protein dynamics of NTERA-2 cells undergoing retinoic acid-induced differentiation. We focused specifically on the first six days of differentiation, to provide insight into the earliest differentiation events, and employed techniques to specifically monitor the nuclear proteome. Well-characterized gene expression markers were used to precisely stage cell differentiation across the experimental time course. A combination of the novel iTRAQ and ExacTag labeling technologies, together with LC-ESI tandem mass spectrometry, were then used to accurately measure nuclear protein expression changes occurring within these differentiation-staged cells. We report proteins that showed significantly altered expression over the first 6 days of differentiation. Extensive bioinformatic analysis was undertaken, resulting in the construction of a novel interactome network, which revealed the temporal dynamics of the nuclear protein network in the context of neuronal differentiation.

Developmental and tissue-specific expression of NITRs.

Novel immune-type receptors (NITRs) are encoded by large multi-gene families and share structural and signaling similarities to mammalian natural killer receptors (NKRs). NITRs have been identified in multiple bony fish species, including zebrafish, and may be restricted to this large taxonomic group. Thirty-nine NITR genes that can be classified into 14 families are encoded on zebrafish chromosomes 7 and 14. Herein, we demonstrate the expression of multiple NITR genes in the zebrafish ovary and during embryogenesis. All 14 families of zebrafish NITRs are expressed in hematopoietic kidney, spleen and intestine as are immunoglobulin and T cell antigen receptors. Furthermore, all 14 families of NITRs are shown to be expressed in the lymphocyte lineage, but not in the myeloid lineage, consistent with the hypothesis that NITRs function as NKRs. Sequence analyses of NITR amplicons identify known alleles and reveal additional alleles within the nitr1, nitr2, nitr3, and nitr5 families, reflecting the recent evolution of this gene family.