Immunogenic cell death in colorectal cancer: a review of mechanisms and clinical utility.

Colorectal cancer (CRC) is a major cause of cancer-related morbidity and mortality worldwide. Despite several clinical advances the survival of patients with advanced colorectal cancer remains limited, demanding newer approaches. The immune system plays a central role in cancer development, propagation, and treatment response. Within the bowel, the colorectal mucosa is a key barrier and site of immune regulation that is generally immunosuppressive. Nonetheless, within this tumour microenvironment, it is evident that anti-neoplastic treatments which cause direct cytotoxic and cytostatic effects may also induce immunogenic cell death (ICD), a form of regulated cell death that leads to an anti-tumour immune response. Therefore, novel ICD inducers and molecular biomarkers of ICD action are urgently needed to advance treatment options for advanced CRC. This article reviews our knowledge of ICD in CRC.

Cytokine profiling of docetaxel-resistant castration-resistant prostate cancer.

BACKGROUND: Docetaxel improves symptoms and survival in metastatic castration-resistant prostate cancer (CRPC). However, ∼50% of patients are chemoresistant. This study examined whether changes in cytokine levels predict for docetaxel resistance in vitro and in a clinical cohort. METHODS: PC3 cells or their docetaxel-resistant subline (PC3Rx) were co-cultured with U937 monocytes, with and without docetaxel treatment, and cytokine levels were measured. The circulating levels of 28 cytokines were measured pre-/post cycle 1 of docetaxel from 55 men with CRPC, and compared with prostate-specific antigen (PSA) response. RESULTS: PC3Rx-U937 co-culture expressed more cytokines, chiefly markers of alternative macrophage differentiation, compared with PC3-U937 co-culture. Docetaxel treatment enhanced cytokine production by PC3Rx-U937 co-culture, while reducing cytokine levels in PC3-U937. In patients, changes in the levels of seven circulating cytokines (macrophage inhibitory cytokine 1 (MIC1), interleukin (IL)-1ra, IL-1ß, IL-4, IL-6, IL-12 and IFNγ) after cycle 1 of docetaxel were associated with progressive disease (all P<0.05). The combination of changes in MIC1, IL-4 and IL-6 most strongly predicted PSA response (P=0.002). CONCLUSIONS: In vitro studies suggest docetaxel resistance is mediated, at least in part, by cytokines induced by the interaction between the docetaxel-resistant tumour cells and macrophages. Early changes in circulating cytokine levels were associated with docetaxel resistance in CRPC patients. When considered together, these data suggest a significant role for the inflammatory response and macrophages in the development of docetaxel resistance in CRPC.

High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.

Digitized mammograms: a preliminary clinical evaluation and the potential for telemammography.

We performed a preliminary clinical evaluation of digitized mammograms to assess whether digital images suitable for telemammography could be obtained. Thirty mammograms were digitized at a resolution of 4000 x 4000 pixels and 12 bit/pixel. The series contained 17 carcinomas in 16 patients. Five consultant radiologists reported both the original mammograms and the digitized images. There was agreement between the reports of the mammograms and the digitized images in relation to whether a suspicious lesion was present or not in 95% of cases. No study considered benign on viewing the film images was interpreted as malignant on reporting the digitized images. This suggests that film digitizers may allow a digital image of a mammogram of acceptable quality for telemammography to be obtained in the absence of a purpose-built digital mammography system.

Characterization of the rat liver membrane proteome using peptide immobilized pH gradient isoelectric focusing.

Membrane proteins are of particular interest in proteomics because of their potential therapeutic utility. Past proteomic approaches used to investigate membrane proteins have only been partially successful at providing a comprehensive analysis due to the inherently hydrophobic nature and low abundance for some of these proteins. Recently, these difficulties have been improved by analyzing membrane protein enriched samples using shotgun proteomics. In addition, the recent application of methanol-assisted trypsin digestion of membrane proteins has been shown to be a method to improve membrane protein identifications. In this study, a comparison of different concentrations of methanol was assessed for assisting membrane protein digestion with trypsin prior to analysis using a gel-based shotgun proteomics approach called peptide immobilized pH gradient isoelectric focusing (IPG-IEF). We demonstrate the use of peptide IEF on pH 3-10 IPG strips as the first dimension of two-dimensional shotgun proteomics for protein identifications from the membrane fraction of rat liver. Tryptic digestion of proteins was carried out in varying concentrations of methanol in 10 mM ammonium bicarbonate: 0% (v/v), 40% (v/v), and 60% (v/v). A total of 800 proteins were identified from 60% (v/v) methanol, which increased the protein identifications by 17% and 14% compared to 0% (v/v) methanol and 40% (v/v) methanol assisted digestion, respectively. In total, 1549 nonredundant proteins were identified from all three concentrations of methanol including 690 (42%) integral membrane proteins of which 626 of these proteins contained at least one transmembrane domain. Peptide IPG-IEF separation of peptides was successful as the peptides were separated into discrete pI regions with high resolution. The results from this study prove utility of 60% (v/v) methanol assisted digestion in conjunction with peptide IPG-IEF as an optimal shotgun proteomics technique for the separation and identification of previously unreported membrane proteins.

Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients.

Two-dimensional electrophoresis (2-DE) is a highly resolving technique for arraying proteins by isoelectric point and molecular mass. To date, the resolving ability of 2-DE for protein separation is unsurpassed, thus ensuring its use as the fundamental separation method for proteomics. When immobilized pH gradients (IPGs) are used for isoelectric focusing in the first dimension, excellent reproducibility and high protein load capacity can be achieved. While this has been beneficial for separations of soluble and mildly hydrophobic proteins, separations of membrane proteins and other hydrophobic proteins with IPGs have often been poor. Stimulated by the growing interest in proteomics, recent developments in 2-DE methodology have been aimed at rectifying this situation. Improvements have been made in the area of protein solubilization and sample fractionation, leading to a revamp of traditional approaches for 2-DE of membrane proteins. This review explores these developments.

Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry.

The development of rapid, global methods for monitoring states of protein phosphorylation would provide greater insight for understanding many fundamental biological processes. Current best practices use mass spectrometry (MS) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifying phosphopeptides from within a complex mixture containing many other unmodified peptides and ionizing phosphopeptides in positive-ion MS. We have modified an approach that uses barium hydroxide to rapidly eliminate the phosphoryl group of serine and threonine modified amino acids, creating dehydroamino acids that are susceptible to nucleophilic derivatization. By derivatizing a protein digest with a mixture of two different alkanethiols, phosphopeptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommodate simple and rapid screening for phosphopeptides in a protein digest, obviating the use of isotopically labeled samples for qualitative phosphopeptide detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to reveal the site of derivatization and, thus, phosphorylation. We demonstrated the technique by identifying phosphopeptides from beta-casein and ovalbumin. The approach was further used to examine in vitro phosphorylation of recombinant human HSP22 by protein kinase C, revealing phosphorylation of Thr-63.

Hickmans in unusual places.

Radiologists now commonly insert long-term indwelling right atrial catheters. The insertion of these catheters in patients with limited venous access is a challenging clinical problem. Our experience in dealing with patients with limited venous access from a series of 500 Hickman catheter insertions is presented, as are the problems encountered during the insertion of catheters in patients with anomalies of the venous system.

The Australian Proteome Analysis Facility (APAF): assembling large scale proteomics through integration and automation.

The field of proteomics opens new possibilities for the mass screening of proteins from many different sources. While genomics is well understood to be a big science field, proteomics is just emerging as such. This paper describes the setting up of the first national proteomics facility. The facility has been funded by the Australian government and this funding has allowed the design of purpose built, integrated laboratories with state of the art equipment for large scale proteome research.

Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis.

Compared to soluble proteins, hydrophobic proteins, in particular membrane proteins, are an underrepresented protein species on two-dimensional (2-D) gels. One possibility is that many hydrophobic proteins are simply not extracted from the sample prior to 2-D gel separation. We attempted to isolate hydrophobic proteins from Escherichia coli by extracting with organic solvents, then reconstituting the extracted proteins in highly solubilising sample solution amenable to 2-D electrophoresis using immobilized pH gradients (IPGs). This was conducted by an extraction with a mixture of chloroform and methanol, followed by solubilisation using a combination of urea, thiourea, sulfobetaine detergents and tributyl phosphine. Peptide mass fingerprinting assisted in the identification of 13 proteins, 8 of which have not previously been reported on 2-D gels. Five of these new proteins possess a positive hydropathy plot. These results suggest that organic solvent extractions may be useful for selectively isolating some proteins that have previously been missing from proteome maps.

Percutaneous catheter and guidewire fragmentation with local administration of recombinant tissue plasminogen activator as a treatment for massive pulmonary embolism.

The purpose of this article is to report four patients with massive pulmonary embolism treated with percutaneous catheter and guidewire fragmentation and local administration of recombinant tissue plasminogen activator (r-TPA). Four patients with massive pulmonary embolism initially underwent pulmonary angiography. Thrombus fragmentation was performed with both standard angiographic guidewires and catheters followed by local infusion of 41-200 mg of r-TPA. Pulmonary angiography was repeated after treatment. All patients survived with improvement in their clinical status and eventual discharge from hospital. Angiography in all patients post treatment demonstrated improvement in pulmonary perfusion (mean Miller score before treatment 22.5; mean Miller score after treatment 5.75). No patient had a significant complication. Mechanical fragmentation of the thrombus followed by local infusion of r-TPA was an effective treatment for massive pulmonary embolism in these four patients with no significant complications.

Gadopentetate dimeglumine as a contrast agent in peripheral angioplasty. A case report.

Gadopentetate dimeglumine (Gd-DTPA) is widely used as a contrast agent in MR imaging. We report on a case in which Gd-DTPA was used as the contrast agent during angioplasty in a patient who had recently had an adverse reaction to a non-ionic iodinated contrast medium. Gd-DTPA allowed a diagnostic angiogram to be performed with no side effects, and may thus be a useful contrast agent at angioplasty in patients with contra-indications to iodinated contrast media.

Two-dimensional electrophoresis and peptide mass fingerprinting of bacterial outer membrane proteins.

Many bacterial outer membrane proteins (OMPs) are missing from two-dimensional (2-D) gel proteome maps. Recently, we developed a technique for 2-D electrophoresis (2-DE) of Escherichia coli OMPs using alkaline pH incubation for isolation of OMPs, followed by improved solubilization conditions for array by 2-DE using immobilized pH gradients. In this report, we expanded our study, examining protein components from the outer membranes of two enteric bacteria, Salmonella typhimurium and Klebsiella pneumoniae (also known as Klebsiella aerogenes), as well as the unrelated, free-living alpha-proteobacteria Caulobacter crescentus. Patterns of OMPs expression appeared remarkably conserved between members of the Enterobacteriaceae, while C. crescentus was unique, displaying a greater number of clusters of higher-molecular-weight proteins (>80 kDa). Peptide mass fingerprinting (PMF) was used for protein identification, and despite matching across-species boundaries, proved useful for first-pass protein assignment of enteric OMPs. In contrast, identification of C. crescentus OMPs was successful only when searching against its recently completed genome. For all three microorganisms examined, the majority of proteins identified on the 2-D gel appear localized to the outer membrane, a result consistent with our previous finding in Escherichia coli. In addition, we discuss some of the benefits and limitations of PMF in cross-species searching.

Identification of wallaby milk whey proteins separated by two-dimensional electrophoresis, using amino acid analysis and sequence tagging.

Micropreparative two-dimensional polyacrylamide gel electrophoresis has been used to separate milk whey proteins from the Tammar wallaby (Macropus eugenii). We have used a combination of amino acid analysis and N-terminal sequence tagging as a rapid and sensitive method to identify the major whey proteins. Using these techniques, we confidently identified alpha-lactalbumin and late lactation protein. While these are the only two M. eugenii whey proteins with a corresponding SWISS-PROT entry, we demonstrate that by using amino acid analysis and matching across species boundaries, we can identify previously unsequenced conserved wallaby whey proteins including beta-lactoglobulin and serum albumin.

Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as reducing agent.

In this study, dithiothreitol was replaced by tributyl phosphine as the reducing agent in both the sample solution for the first-dimensional isoelectric focusing and during the immobilised pH gradient (IPG) equilibration procedure. Tributyl phosphine improves protein solubility during isoelectric focusing, which results in shorter run times and increased resolution. Tributyl phosphine is nonionic and thus does not migrate in the IPG, therefore maintaining reducing conditions during the course of the first-dimensional separation. The increased solubility provided by the maintenance of reducing conditions gives improved focusing and decreased horizontal streaking on the subsequent second-dimension gel. The use of tributyl phosphine in the equilibration step allows the procedure to be simplified, incorporating reduction and alkylation in a single step. This is possible because, in direct contrast to dithiothreitol (DTT), tributyl phosphine does not contain a free thiol and therefore does not react with thiol-specific alkylating reagents.

Development of mini-gel technology in two-dimensional electrophoresis for mass-screening of samples: application to tears.

Despite the extensive literature available on tear proteins and lipids, very little has been reported on the tear fluid as a whole and it's changes in contact lens wear or ocular diseased patients. Initially a human reflex tear two-dimensional map was created by Molloy et al. (Electrophoresis 1997, 18, 2811-2815), using this information a process for mass-screening was established. The large format two-dimensional technique was evaluated, using a basal tear reference map, and modified to describe a fast, efficient and cost effective method of protein separation. The use of one pH 3-10 18 cm nonlinear immobilised pH gradient (IPG) strip and two mini-gels for the second-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) results in an effective separation of tear proteins which will be applied in diagnostic studies of tear samples.

Prefractionation of protein samples prior to two-dimensional electrophoresis.

Thousands of proteins may be visualised on a two-dimensional (2-D) gel, but only hundreds are present at levels sufficient for chemical analysis. Therefore, prefractionation of protein samples prior to 2-D polyacrylamide gel electrophoresis (PAGE) will be important for the investigation of proteins that are present at sub-picogram levels in physiological samples. We describe an approach to prefractionate protein samples prior to 2-D PAGE using the Gradiflow, which is a new (preparative) electrokinetic membrane apparatus designed to fractionate proteins in a number of different ways. We have fractionated human serum under nonreducing conditions using the 'reflux' mode, in which proteins are fractionated according to their relative mobility under controlled electrophoretic conditions, where the current is periodically reversed. We describe how fractionation occurs and present examples of enrichment of specific proteins.

Characterisation of wool intermediate filament proteins separated by micropreparative two-dimensional electrophoresis.

Wool intermediate filament proteins (IFP) are a subclass of the cytokeratins, a group of structural proteins which form intermediate filaments in many cell types. Post-translational modifications, such as phosphorylation, play an important role in the control of intermediate filament assembly. Two-dimensional electrophoresis has previously been used to study the IFP distribution in wools with different physical characteristics. Charge heterogeneity has been observed in Type I and Type II IFP. In a previous study, two-dimensional electrophoresis of alkaline phosphatase-treated wool protein extracts was used to show that Type II IFP are phosphorylated. To facilitate post-separation analysis, micropreparative two-dimensional electrophoresis was used to separate milligram quantities of wool protein. Direct phosphoamino acid analysis has confirmed the presence of phosphorylation on serine residues on Type II IFP, whose identity was confirmed by amino acid compositional analysis. The isoelectric points of Type I IFP are very similar and they do not separate completely on the commercially available pH 4-7 immobilized pH gradients (IPG) used in this study. In situ tryptic digestion followed by automated Edman sequencing of the high performance liquid chromatography (HPLC)-separated peptides was used to confirm the identity of this group as Type I IFP. To improve the separation of the Type I IFP it will be necessary to use narrow range IPGs such as Immobiline DryPlates which are available from Pharmacia Biotech, in the pH ranges 4.2-4.9, 4.5-5.4, 5.0-6.0 and 5.6-6.6.

Analysis of the outer membrane proteome of Caulobacter crescentus by two-dimensional electrophoresis and mass spectrometry.

Caulobacter crescentus, a Gram negative alpha-purple bacterium that displays an invariant asymmetric cell division pattern, has become a key model system for the study of bacterial development. Membrane proteins play key roles in cell cycle events, both as components of landmark morphological structures and as critical elements in regulation of the cell cycle. Recent advances for the isolation and solubilization of bacterial membrane proteins prior to isoelectric focusing have significantly improved the separation of outer membrane proteins by two-dimensional (2-D) electrophoresis. In this work we describe the analysis of the outer membrane proteome of Caulobacter crescentus. Proteins were identified using 2-D gel electrophoresis and peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. We identified 54 unique proteins out of which 41 were outer membrane proteins. Of the outer membrane proteins, 16 were identified as TonB-dependent receptor proteins. These studies were executed simultaneously with the Caulobacter genome sequencing project and advantages and limitations of proteomic analysis of a nonannotated genome are discussed. Finally, protein levels between cells grown in rich and minimal media are compared which demonstrates that many of the TonB-dependent receptor proteins are found at higher levels in minimal medium.

Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa PAO1.

With the completion of many genome projects, a shift is now occurring from the acquisition of gene sequence to understanding the role and context of gene products within the genome. The opportunistic pathogen Pseudomonas aeruginosa is one organism for which a genome sequence is now available, including the annotation of open reading frames (ORFs). However, approximately one third of the ORFs are as yet undefined in function. Proteomics can complement genomics, by characterising gene products and their response to a variety of biological and environmental influences. In this study we have established the first two-dimensional gel electrophoresis reference map of proteins from the membrane fraction of P. aeruginosa strain PA01. A total of 189 proteins have been identified and correlated with 104 genes from the P. aeruginosa genome. Annotated membrane proteins could be grouped into three distinct categories: (i) those with functions previously characterised in P. aeruginosa (38%); (ii) those with significant sequence similarity to proteins with assigned function or hypothetical proteins in other organisms (46%); and (iii) those with unknown function (16%). Transmembrane prediction algorithms showed that each identified protein sequence contained at least one membrane-spanning region. Furthermore, the current methodology used to isolate the membrane fraction was shown to be highly specific since no contaminating cytosolic proteins were characterised. Preliminary analysis showed that at least 15 gel spots may be glycosylated in vivo, including three proteins that have not previously been functionally characterised. The reference map of membrane proteins from this organism is now the basis for determining surface molecules associated with antibiotic resistance and efflux, cell-cell signalling and pathogen-host interactions in a variety of P. aeruginosa strains.