Myopia : why study the mechanisms of myopia? Novel approaches to risk factors Signaling eye growth- how could basic biology be translated into clinical insights? Where are genetic and proteomic approaches leading? How does visual function contribute to an

On July 26–29, 2010 the 13th International Myopia Conference was held in Tübingen, Germany and included 17 separate symposia, each with 3–5 presentations. Here, in a single paper, the chairs of those Symposia describe the scientific advances noted at the conference and include the full abstracts of the individual myopia papers presented in each symposium along with the authors and their institutions. The 17 Symposia covered 7 topics: Why Study the Mechanisms of Myopia?; Novel Approaches to Risk Factors; Signaling Eye Growth- How Could Basic Biology Be Translated into Clinical Insights?; Where Are Genetic and Proteomic Approaches Leading?; How Does Visual Function Contribute to and Interact with Ametropia?; Does Eye Shape Matter?; Why Ametropia at All?

Novel targeting of PEGylated liposomes for codelivery of TGF-β1 siRNA and four antitubercular drugs to human macrophages for the treatment of mycobacterial infection: a quantitative proteomic study

Tuberculosis (TB) is still a major public health issue in developing countries, and its chemotherapy is compromised by poor drug compliance and severe side effects. This study aimed to synthesize and characterize new multimodal PEGylated liposomes encapsulated with clinically commonly used anti-TB drugs with linkage to small interfering RNA (siRNA) against transforming growth factor-β1 (TGF-β1). The novel NP-siRNA liposomes could target THP-1-derived human macrophages that were the host cells of mycobacterium infection. The biological effects of the NP-siRNA liposomes were evaluated on cell cycle distribution, apoptosis, autophagy, and the gene silencing efficiency of TGF-β1 siRNA in human macrophages. We also explored the proteomic responses to the newly synthesized NP-siRNA liposomes using the stable isotope labeling with amino acids in cell culture approach. The results showed that the multifunctional PEGylated liposomes were successfully synthesized and chemically characterized with a mean size of 265.1 nm. The novel NP-siRNA liposomes functionalized with the anti-TB drugs and TGF-β1 siRNA were endocytosed efficiently by human macrophages as visualized by transmission electron microscopy and scanning electron microscopy. Furthermore, the liposomes showed a low cytotoxicity toward human macrophages. There was no significant effect on cell cycle distribution and apoptosis in THP-1-derived macrophages after drug exposure at concentrations ranging from 2.5 to 62.5 μg/mL. Notably, there was a 6.4-fold increase in the autophagy of human macrophages when treated with the NP-siRNA liposomes at 62.5 μg/mL. In addition, the TGF-β1 and nuclear factor-κB expression levels were downregulated by the NP-siRNA liposomes in THP-1-derived macrophages. The Ingenuity Pathway Analysis data showed that there were over 40 signaling pathways involved in the proteomic responses to NP-siRNA liposome exposure in human macrophages, with 160 proteins mapped. The top five canonical signaling pathways were eukaryotic initiation factor 2 signaling, actin cytoskeleton signaling, remodeling of epithelial adherens junctions, epithelial adherens junction signaling, and Rho GDP-dissociation inhibitor signaling pathways. Collectively, the novel synthetic targeting liposomes represent a promising delivery system for anti-TB drugs to human macrophages with good selectivity and minimal cytotoxicity.

Mass spectrometry-based proteomic data for cancer diagnosis using interval type-2 fuzzy system

An interval type-2 fuzzy logic system is introduced for cancer diagnosis using mass spectrometry-based proteomic data. The fuzzy system is incorporated with a feature extraction procedure that combines wavelet transform and Wilcoxon ranking test. The proposed feature extraction generates feature sets that serve as inputs to the type-2 fuzzy classifier. Uncertainty, noise and outliers that are common in the proteomic data motivate the use of type-2 fuzzy system. Tabu search is applied for structure learning of the fuzzy classifier. Experiments are performed using two benchmark proteomic datasets for the prediction of ovarian and pancreatic cancer. The dominance of the suggested feature extraction as well as type-2 fuzzy classifier against their competing methods is showcased through experimental results. The proposed approach therefore is helpful to clinicians and practitioners as it can be implemented as a medical decision support system in practice.

Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component, PTEX150

The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex.

CIDB: Chlamydia Interactive Database for cross-querying genomics, transcriptomics and proteomics data

Chlamydiae are important pathogens of humans, birds and a wide range of animals. They are a unique group of bacteria, characterized by their developmental cycle. Chlamydia has been difficult to study because of their obligate intracellular growth habit and lack of a genetic transformation system. However, the past 5 years has seen the full genome sequencing of seven strains of Chlamydia and a rapid expansion of genomic, transcriptomic (RT-PCR, microarray) and proteomic analysis of these pathogens. The Chlamydia Interactive Database (CIDB) described here is the first database of its type that holds genomic, RT-PCR, microarray and proteomics data sets that can be cross-queried by researchers for patterns in the data. Combining the data of many research groups into a single database and cross-querying from different perspectives should enhance our understanding of the complex cell biology of these pathogens. The database is available at: http://www3.it.deakin.edu.au:8080/CIDB/.

Insights into exazaphosphorine resistance and possible approaches to its circumvention

The oxazaphosphorines cyclophosphamide, ifosfamide and trofosfamide remain a clinically useful class of anticancer drugs with substantial antitumour activity against a variety of solid tumors and hematological malignancies. A major limitation to their use is tumour resistance, which is due to multiple mechanisms that include increased DNA repair, increased cellular thiol levels, glutathione S-transferase and aldehyde dehydrogenase activities, and altered cell-death response to DNA damage. These mechanisms have been recently re-examined with the aid of sensitive analytical techniques, high-throughput proteomic and genomic approaches, and powerful pharmacogenetic tools. Oxazaphosphorine resistance, together with dose-limiting toxicity (mainly neutropenia and neurotoxicity), significantly hinders chemotherapy in patients, and hence, there is compelling need to find ways to overcome it. Four major approaches are currently being explored in preclinical models, some also in patients: combination with agents that modulate cellular response and disposition of oxazaphosphorines; antisense oligonucleotides directed against specific target genes; introduction of an activating gene (CYP3A4) into tumor tissue; and modification of dosing regimens. Of these approaches, antisense oligonucleotides and gene therapy are perhaps more speculative, requiring detailed safety and efficacy studies in preclinical models and in patients. A fifth approach is the design of novel oxazaphosphorines that have favourable pharmacokinetic and pharmacodynamic properties and are less vulnerable to resistance. Oxazaphosphorines not requiring hepatic CYP-mediated activation (for example, NSC 613060 and mafosfamide) or having additional targets (for example, glufosfamide that also targets glucose transport) have been synthesized and are being evaluated for safety and efficacy. Characterization of the molecular targets associated with oxazaphosphorine resistance may lead to a deeper understanding of the factors critical to the optimal use of these agents in chemotherapy and may allow the development of strategies to overcome resistance.

Drug bioactivation, covalent binding to target proteins and toxicity relevance

A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients. Further studies using proteomic and genomic approaches with high throughput capacity are needed to identify the protein targetsof reactive drug metabolites, and to elucidate the structure-activity relationships of drug's covalent binding to proteins and their clinical outcomes.

A clustering based hybrid system for mass spectrometry data analysis

Recently, much attention has been given to the mass spectrometry (MS) technology based disease classification, diagnosis, and protein-based biomarker identification. Similar to microarray based investigation, proteomic data generated by such kind of high-throughput experiments are often with high feature-to-sample ratio. Moreover, biological information and pattern are compounded with data noise, redundancy and outliers. Thus, the development of algorithms and procedures for the analysis and interpretation of such kind of data is of paramount importance. In this paper, we propose a hybrid system for analyzing such high dimensional data. The proposed method uses the k-mean clustering algorithm based feature extraction and selection procedure to bridge the filter selection and wrapper selection methods. The potential informative mass/charge (m/z) markers selected by filters are subject to the k-mean clustering algorithm for correlation and redundancy reduction, and a multi-objective Genetic Algorithm selector is then employed to identify discriminative m/z markers generated by k-mean clustering algorithm. Experimental results obtained by using the proposed method indicate that it is suitable for m/z biomarker selection and MS based sample classification.

Zinc and DHA have opposing effects on the expression levels of histones H3 and H4 in human neuronal cells

Zn and DHA have putative neuroprotective effects and these two essential nutrients are known to interact biochemically. We aimed to identify novel protein candidates that are differentially expressed in human neuronal cell line M17 in response to Zn and DHA that would explain the molecular basis of this interaction. Two-dimensional gel electrophoresis and MS were applied to identify major protein expression changes in the protein lysates of human Ml7 neuronal cells that had been grown in the presence and absence of Zn and DHA. Proteomic findings were further investigated using Western immunoblot and real-time PCR analyses. Four protein spots, which had significant differential expression, were identified and selected for in-gel trypsin digestion followed by matrix-assisted laser desorption ionisation MS analysis. The resultant peptide mass fingerprint for each spot allowed their respective identities to be deduced. Two human histone variants H3 and H4 were identified. Both H3 and H4 were downregulated by Zn in the absence of DHA (Zn effect) and upregulated by DHA (DHA effect) in the presence of Zn (physiological condition). These proteomic findings were further supported by Western immunoblot and real-time PCR analyses using H3- and H4-specific monoclonal antibodies and oligonucleotide primers, respectively. We propose that dietary Zn and DHA cause a global effect on gene expression, which is mediated by histones. Such novel information provides possible clues to the molecular basis of neuroprotection by Zn and DHA that may contribute to the future treatment, prevention and management of neurodegenerative diseases such as Alzheimer's disease.

Phospholipase activity in human mesenteric ischaemia and infarction

Currently, diagnostic tests for mesenteric ischaemia and infarction are inadequate due to poor sensitivity and specificity. In addition, many potential markers appear too late to be clinically useful. At present, definitive diagnosis can only be made at the time of surgery, which is not ideal as surgery is often to be avoided in critically ill and elderly patients. A clinically useful, minimally invasive test is likely to decrease the currently very high mortality rate and allow monitoring of 'at risk' patients during their hospital stay. A two-dimensional electrophoresis based proteomic approach was undertaken to assess plasma protein differences between patients with surgically confirmed bowel infarction and control Intensive Care patients. The major protein differences were found to be members or variants of acute phase proteins. Serum amyloid A showed the largest difference between the two patient groups, and this protein was investigated in greater depth. An analysis was performed to compare the diagnostic ability of several commonly used indicators of critical illness and bowel infarction with serum amyloid A and phospholipase A2. Although none of the variables were ideal for clinical use, plasma phospholipase A2 activity showed the best discriminatory power, as determined by Receiver Operating Characteristic curves. From a review of the literature, phospholipase AI (PLA2) appeared to be increased in the bowel as a result of ischaemia and infarction. In one patient, matched tissues were obtained, and PLA2 activity was found to be significantly higher in infarcted bowel tissue compared to ischaemic bowel tissue. PLA2 activity was significantly greater in bowel lumen than tissue, suggesting that the protein was being released, and may enter the circulation. PLA2 activity was increased in the plasma of bowel infarction patients compared with control patients, though the difference was not significant. The phospholipase activity exhibited a number of similarities to typical phospholipase A2 proteins, but also showed a number of inconsistent characteristics. For this reason, we wished to identify the protein responsible for the increased phospholipase activity in infarcted human bowel. The PLA2 activity in human bowel could not be abolished by immunoprecipitation of the PLA2 isoforms IIA (well described in bowel) and V (a closely related isoform). To investigate these proteins, a native urea protein gel devised for snake venom phospholipase A2 was modified for use with mammalian phospholipase AI. The modified gel was used to show that the protein with phospholipase activity from infarcted gut was different from normal gut PLA2 and type IIA PLA2. A number of extensions were devised for these native gels and were found to be useful both in this investigation and for venom investigations. Protein purification was undertaken to identify the protein responsible for the increased phospholipase activity in infarcted bowel. Protein was purified from infarcted human bowel using a number of techniques that exploited unusual characteristics of the protein. The purification techniques each retained the native activity of the protein and the purification could therefore be monitored with a phospholipid hydrolysis assay at each stage. The protein identified by mass spectrometry was an excellent match for cyclophilin B, an inflammatory protein that had previously been identified in rat bowel at the mRNA level (Hasel et al, 1991, Kainer & Doris, 2000). As the purification progress had been monitored throughout with a phospholipid hydrolysis assay, cyclophilin B was an unexpected identification, as it is not known to have phospholipase activity. Cyclophilin B was removed from the highly purified samples via immunoprecipitation and this process abolished all phospholipase activity. The addition of cyclosporin A, (the pharmaceutical ligand of cyclophilin B), did not effect the phospholipase activity. Cyclophilin B protein was found in normal and infarcted human bowel using Western blotting. Cyclophilin B protein also appeared to be present in the bowel lumen and plasma of several patients with bowel infarction, but not in control patients. Immunohistochemistry confirmed the ubiquitous nature of cyclophilin B that had been reported by other groups. This project has investigated the use of two dimensional gel electrophoresis based proteomics to identify proteins present in the plasma of patients with confirmed bowel infarction and control intensive care patients. The major protein classes observed were members of the acute phase proteins, which highlights the need for pre-fractionation of plasma to identify lower abundance, disease associated proteins. A series of potential plasma markers were compared using Receiver Operating Characteristic Curves. Although no ideal marker was clear from this analysis, phospholipase activity appeared to warrant further investigation. Phospholipase activity was investigated in human infarcted bowel. Protein purification identified cyclophilin B as a bowel protein that showed unusual phospholipid hydrolysing activity. Cyclophilin B is a ubiquitous protein in intestinal cell types in both normal and infarcted tissue. There appears to be release of cyclophilin B into bowel lumen and plasma under conditions of mesenteric ischaemia and infarction.

Gene profiling reveals hydrogen sulphide recruits death signaling via the N-methyl-D-aspartate receptor identifying commonalities with excitotoxicity

Recently the role of hydrogen sulphide (H₂S) as a gasotransmitter stimulated wide interest owing to its involvement in Alzheimer's disease and ischemic stroke. Previously we demonstrated the importance of functional ionotropic glutamate receptors (GluRs) by neurons is critical for H₂S-mediated dose- and time-dependent injury. Moreover N-methyl-D-aspartate receptor (NMDAR) antagonists abolished the consequences of H₂S-induced neuronal death. This study focuses on deciphering the downstream effects activation of NMDAR on H₂S-mediated neuronal injury by analyzing the time-course of global gene profiling (5, 15, and 24 h) to provide a comprehensive description of the recruitment of NMDAR-mediated signaling. Microarray analyses were performed on RNA from cultured mouse primary cortical neurons treated with 200 µM sodium hydrosulphide (NaHS) or NMDA over a time-course of 5–24 h. Data were validated via real-time PCR, western blotting, and global proteomic analysis. A substantial overlap of 1649 genes, accounting for over 80% of NMDA global gene profile present in that of H₂S and over 50% vice versa, was observed. Within these commonly occurring genes, the percentage of transcriptional consistency at each time-point ranged from 81 to 97%. Gene families involved included those related to cell death, endoplasmic reticulum stress, calcium homeostasis, cell cycle, heat shock proteins, and chaperones. Examination of genes exclusive to H₂S-mediated injury (43%) revealed extensive dysfunction of the ubiquitin-proteasome system. These data form a foundation for the development of screening platforms and define targets for intervention in H₂S neuropathologies where NMDAR-activated signaling cascades played a substantial role. J. Cell. Physiol. 226: 1308–1322, 2011.

The pseudomonas aeruginosa secreted protein PA2934 decreases apical membrane expression of the cystic fibrosis transmembrane conductance regulator

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl− ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in α/β hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS-5 stromal cell line

Lineage-specific expansion of haematopoietic stem/progenitor cells (HSPCs) from human umbilical cord blood (UCB) is desirable because of their several applications in translational medicine, e.g. treatment of cancer, bonemarrowfailure and immunodeficiencies. The currentmethods forHSPC expansion use either cellular feeder layers and/or soluble growth factors and selected matrix components coated on different surfaces. The use of cell-free extracellular matrices from bone marrow cells for this purpose has not previously been reported. We have prepared insoluble, cell- free matrices from a murine bone marrow stromal cell line (MS-5) grown under four different conditions, i.e. in presence or absence of osteogenic medium, each incubated under 5% and 20% O2 tensions. These acellularmatrices were used as biological scaffolds for the lineage-specific expansion of magnetically sorted CD34+ cells and the results were evaluated by flow cytometry and colony-forming assays. We could get up to 80-fold expansion of some HSPCs on one of the matrices and our results indicated that oxygen tension played a significant role in determining the expansion capacity of the matrices. A comparative proteomic analysis of the matrices indicated differential expression of proteins, such as aldehyde dehydrogenase and gelsolin, which have previously been identified as playing a role in HSPC maintenance and expansion. Our approach may be of value in identifying factors relevant to tissue engineering-based ex vivo HSPC expansion, and itmay also provide insights into the constitution of the niche in which these cells reside in the bone marrow.

Sporobolus stapfianus, a model desiccation-tolerant grass

Sporobolus stapfianus Gandoger, one of ~40 known ‘anabiotic’grass species (i.e. ‘able to regain vital activity from a state of latent life’), is the most versatile tool for research into desiccation tolerance in vegetative grass tissue. Current knowledge on this species is presented, including the features that suit it for investigations into the plant’s ability to survive dehydration of its leaf protoplasm. The main contributors to desiccation tolerance in S. stapfianus leaves appear to be: accumulation during dehydration of protectants of membranes and proteins; mechanisms limiting oxidative damage; a retention of protein synthetic activity in late stages of drying that is linked with changes in gene expression and in the proteomic array; and an ability to retain net synthesis of ATP during drying. S. stapfianus exemplifies an advanced stage of an evolutionary trend in desiccation tolerant plants towards increased importance of the dehydration phase (for induction of tolerance, for synthesis of protectants and for proteomic changes).