Lymphoma associated antigen (LAA)--a unique biomarker for lymphomas.

A lymphoma associated antigen (LAA) isolated from pooled lymph nodes of confirmed Hodgkin's and non-Hodgkin's lymphomas has been purified and characterized. Using a xenogenic rabbit anti-serum, enzyme-linked immunosorbent assay (ELISA) and RIA were developed for LAA. LAA was detected in the sera of all confirmed lymphomas, the test being negative for normals, for patients with benign lymphadenitis and various other types of cancers. Except for a very few false positive results, no false negative was observed. LAA was identified in urine, CSF, saliva and gastric juice of a few lymphoma patients, and the test proved to be of diagnostic potential, as for a few patients it had a lead time of a few months over the histological diagnosis. In order to render the LAA test more precise and specific, monoclonal antibodies were generated by both in vitro and in vivo immunization procedures. Seven monoclonals were generated, viz. 7D6, 7D2, 7G2, 7C5, 6G2, 23B7 and 23G11, which exhibited cytoplasmic staining of frozen sections of malignant lymphoid tissues of B cell derived non-Hodgkin's lymphomas. Two of these monoclonal antibodies, 7D6 and 23B7, revealed strong cytoplasmic staining of frozen sections, impression smears and cytospin specimens of B cell non-Hodgkin's lymphomas. The reactivity was very weak or negative for T cell lymphomas. The test was negative for Hodgkin's disease and controls. These results were confirmed by dot blotting, immunoprecipitation and immunofluorescence study. By ELISA with a sensitivity of 15 ng/ml, serum LAA levels for lymphomas were in the range 72-1250 ng/ml. LAA could not be detected in the sera of normals and controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimized blood cell profiling method for genomic biomarker discovery using high-density microarray

High-quality biomarkers for disease progression, drug efficacy and toxicity liability are essential for improving the efficiency of drug discovery and development. The identification of drug-activity biomarkers is often limited by access to and the quantity of target tissue. Peripheral blood has increasingly become an attractive alternative to tissue samples from organs as source for biomarker discovery, especially during early clinical studies. However, given the heterogeneous blood cell population, possible artifacts from ex vivo activations, and technical difficulties associated with overall performance of the assay, it is challenging to profile peripheral blood cells directly for biomarker discovery. In the present study, Applied BioSystems' blood collection system was evaluated for its ability to isolate RNA suitable for use on the Affymetrix microarray platform. Blood was collected in a TEMPUS tube and RNA extracted using an ABI-6100 semi-automated workstation. Using human and rat whole blood samples, it was demonstrated that the RNA isolated using this approach was stable, of high quality and was suitable for Affymetrix microarray applications. The microarray data were statistically analysed and compared with other blood protocols. Minimal haemoglobin interference with RNA labelling efficiency and chip hybridization was found using the TEMPUS tube and extraction method. The RNA quality, stability and ease of handling requirement make the TEMPUS tube protocol an attractive approach for expression profiling of whole blood to support target and biomarker discovery.     

Lectin precipitation using phytohemagglutinin-L(4) coupled to avidin-agarose for serological biomarker discovery in colorectal cancer

N-acetylglucosaminyltransferase V (GnT-V) has been reported to be upregulated in malignant cancer cells, and its targets have been sought after with regard to biomarker identification. The low capacity and high false positive rates of 2-DE gel-based lectin blots using phytohemagglutinin-L(4) (L-PHA) prompted us to develop a novel protocol for identifying GnT-V targets, in which serum proteins were subjected to immunodepletion, alkylation, and lectin precipitation using L-PHA coupled to avidin-agarose bead complexes, and tryptic digestion. Proteins captured by L-PHA conjugates were analyzed by a nano-LC-FT-ICR/LTQ MS. Here, we report 26 candidate biomarkers for colorectal cancer (CRC) that show 100% specificity and sensitivities of greater than 50%. Not only can these candidate proteins be used as analytes for validation, but the novel protocol described herein can be applied to biomarker discovery in nonCRCs.     

Humoral response to cancer as a tool for biomarker discovery

There is an important need to find relevant biomarkers that show high sensitivity and specificity for early diagnosis and prognosis of cancer. An immune response to cancer is elicited in humans, as demonstrated in part by the identification of autoantibodies against a number of tumor-associated antigens in sera from patients with different types of cancer. Identification of tumor-associated antigens and their cognate autoantibodies is a promising strategy for the discovery of relevant biomarkers. During the past few years, proteomic approaches, including SEREX, SERPA and, more recently, protein microarrays, have been the dominant strategies used to identify tumor-associated antigens and their cognate autoantibodies. In this review, we aim to describe advantages, drawbacks, and recent improvements of these approaches for the study of humoral responses.     

LUMA (LUminometric Methylation Assay)--a high throughput method to the analysis of genomic DNA methylation

Changes in genomic DNA methylation are recognized as important events in normal and pathological cellular processes, contributing both to normal development and differentiation as well as cancer and other diseases. Here, we report a novel method to estimate genome-wide DNA methylation, referred to as LUminometric Methylation Assay (LUMA). The method is based on combined DNA cleavage by methylation-sensitive restriction enzymes and polymerase extension assay by Pyrosequencing. The method is quantitative, highly reproducible and easy to scale up. Since no primary modification of genomic DNA, such as bisulfite treatment, is needed, the total assay time is only 6 h. In addition, the assay requires only 200-500 ng of genomic DNA and incorporates an internal control to eliminate the problem of varying amounts of starting DNA. The accuracy and linearity of LUMA were verified by in vitro methylated lambda DNA. In addition, DNA methylation levels were assessed by LUMA in DNA methyltransferase knock-out cell lines and after treatment with the DNA methyltransferase inhibitor (5-AzaCytidine). The LUMA assay may provide a useful method to analyze genome-wide DNA methylation for a variety of physiological and pathological conditions including etiologic, diagnostic and prognostic aspects of cancer.     

Isotope-differentiated binding energy shift tags (IDBEST) for improved targeted biomarker discovery and validation

Mass spectrometry has proved to be an important tool for protein biomarker discovery, identification and characterization. However, global proteomic profiling strategies often fail to identify known low-abundance biomarkers as a result of the limited dynamic range of mass spectrometry (two to three orders of magnitude) compared with the large dynamic range of protein concentrations in biologic fluids (11 to 12 orders of magnitude for serum). In addition, the number of peptides generated in such methods vastly overwhelms the resolution capacity of mass spectrometers, requiring extensive sample clean-up (e.g., affinity tag, retentate chromatography and/or high-performance liquid chromatography) before mass spectrometry analysis. Baiting and affinity pre-enrichment strategies, which overcome the dynamic range and sample complexity issues of global proteomic strategies, are very difficult to couple to mass spectrometry. This is due to the fact that it is nearly impossible to sort target peptides from those of the bait since there will be many cases of isobaric peptides. IDBEST (Target Discovery, Inc.) is a new tagging strategy that enables such pre-enrichment of specific proteins or protein classes as the resulting tagged peptides are distinguishable from those of the bait by a mass defect shift of approximately 0.1 atomic mass units. The special characteristics of these tags allow: resolution of tagged peptides from untagged peptides through incorporation of a mass defect element; high-precision quantitation of up- and downregulation by using stable isotope versions of the same tag; and potential analysis of protein isoforms through more complete peptide coverage from the proteins of interest.     

Perspective: a program to improve protein biomarker discovery for cancer

Biomarkers for cancer risk, early detection, prognosis, and therapeutic response promise to revolutionize cancer management. Protein biomarkers offer tremendous potential in this regard due to their great diversity and intimate involvement in physiology. An effective program to discover protein biomarkers using existing technology will require team science, an integrated informatics platform, identification and quantitation of candidate biomarkers in disease tissue, mouse models of disease, standardized reagents for analyzing candidate biomarkers in bodily fluids, and implementation of automation. Technology improvements for better fractionation of the proteome, selection of specific biomarkers from complex mixtures, and multiplexed assay of biomarkers would greatly enhance progress.     

Combination of abundant protein depletion and multi-lectin affinity chromatography (M-LAC) for plasma protein biomarker discovery

We report on the development of a robust and relatively high-throughput method for in-depth proteomic analysis of human plasma suitable for biomarker discovery. The method consists of depletion of albumin and IgG and multi-lectin affinity chromatography (M-LAC), followed by nanoLC-MS/MS analysis of digested proteins and label-free comparative quantitation of proteins. The performance of the method is monitored by multiple quality control points to ensure reproducibility of the analysis. The method identifies proteins that are reported to be present in normal plasma at concentrations of 10-100 ng/mL and that may be of particular interest when studying a variety of disease conditions. Numerous tissue leakage proteins of potentially even lower concentrations are also identified. When the method was used in a study to identify potential biomarkers of psoriasis, the differential abundance of proteins present at low mug/mL level was quantitated and later verified by ELISA measurements.     

Comprehensive approaches to molecular biomarker discovery for detection and identification of Cronobacter spp. (Enterobacter sakazakii) and Salmonella spp

Cronobacter spp. (formerly Enterobacter sakazakii) and Salmonella spp. are increasingly implicated internationally as important microbiological contaminants in low-moisture food products, including powdered infant formula. Estimates indicate that 40 to 80% of infants infected with Cronobacter sakazakii and/or Salmonella in the United States may not survive the illness. A systematic approach, combining literature-based data mining, comparative genome analysis, and the direct sequencing of PCR products of specific biomarker genes, was used to construct an initial collection of genes to be targeted. These targeted genes, particularly genes encoding virulence factors and genes responsible for unique phenotypes, have the potential to function as biomarker genes for the identification and differentiation of Cronobacter spp. and Salmonella from other food-borne pathogens in low-moisture food products. In this paper, a total of 58 unique Salmonella gene clusters and 126 unique potential Cronobacter biomarkers and putative virulence factors were identified. A chitinase gene, a well-studied virulence factor in fungi, plants, and bacteria, was used to confirm this approach. We found that the chitinase gene has very low sequence variability and/or polymorphism among Cronobacter, Citrobacter, and Salmonella, while differing significantly in other food-borne pathogens, either by sequence blasting or experimental testing, including PCR amplification and direct sequencing. This computational analysis for Cronobacter and Salmonella biomarker identification and the preliminary laboratory studies are only a starting point; thus, PCR and array-based biomarker verification studies of these and other food-borne pathogens are currently being conducted.     

tert.-Butyl aminocarbonate (tert.-butyloxycarbonyloxyamine)--a new acylating reagent for amines

tert.-Butyl amino carbonate (1, tert.-butyloxycarbonyloxyamine) was prepared by reaction of hydroxylamine with di-tert.-butyl dicarbonate (2). 1 was used to acylate different amino acids or amines in water or in aqueous dioxane. 1 was also prepared in situ and used to acylate amino acids directly. 1 reacted 1.5-2.5 times faster than 2 with all amines studied either in water or 50% (v/v) dioxane. Remarkably, 1 retained its ability to acylate amines even in acid solution; the rate of acylation of L-Phe at pH 6.5 (15 M-1 X min-1) was about 20% of the rate at pH 10 (72 M-1 X min-1). 2 was not an acylating reagent below pH 7.0 and as expected, the rate at pH 8.3 (4 M-1 X min-1) was about 10% of that at pH 9.3 (39 M-1 X min-1). Pure BOC-amino acids were obtained in high yield and could be quantitatively deprotected by standard procedures.     

Development and validation of a fluorescence polarization-based competitive peptide-binding assay for HLA-A*0201--a new tool for epitope discovery

Various approaches are currently proposed to successfully develop therapies for the prevention and treatment of infectious diseases and cancer. One of the most promising approaches is the development of vaccines that elicit cytotoxic T lymphocyte (CTL) responses. Consequently, identification and exact definition of molecular parameters involved in peptide-MHC class-I interactions of putative CTL epitopes are of prime importance for the development of immunomodulating compounds. To better facilitate epitope discovery, we developed and validated a novel state-of-the-art biochemical HLA-A0201 assay, which is comprised of technologically advanced cutting edge reagents. The technique is based on competition and uses a FITC-labeled reference peptide and highly purified soluble HLA-A0201 molecules to quantitatively measure the binding capacity of nonlabeled peptide candidates. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. During standardization, the problem of assay parameter variation is discussed, showing the dramatic influence of HLA and reference peptide concentrations as well as the choice of the reference peptide itself on IC(50) determinations. For validation, a panel of 15 well-defined HLA-A0201 ligands from various sources covering a broad range of binding affinities was tested. Binding data were used to compare against pre-existing quantitative assay systems. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with recombinant sHLA molecules is highly advantageous for the accurate assessment of peptide binding. Furthermore, the assay also features high-throughput screening capacity, providing uniquely efficient means of identifying and evaluating immune target molecules.     

Anti-arthritis effects of vitamin K(2) (menaquinone-4)--a new potential therapeutic strategy for rheumatoid arthritis

Vitamin K(2) (menaquinone-4, MK-4) has been reported to induce apoptosis in hepatocellular carcinoma, leukemia and myelodysplastic syndrome cell lines. The effects of MK-4 on the development of arthritis have never been addressed thus far. In the present study, we investigated the effect of MK-4 upon the proliferation of rheumatoid synovial cells and the development of arthritis in collagen-induced arthritis. We analyzed the effect of MK-4 on the proliferation of fibroblast-like synoviocytes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The pro-apoptotic effect of MK-4 upon fibroblast-like synoviocytes was investigated with annexin V staining and DNA fragmentation and caspase 3/7 assays. Moreover, we analyzed the effect of MK-4 on the development of collagen-induced arthritis in female dark agouti rats. Our results indicated that MK-4 inhibited the proliferation of fibroblast-like synoviocytes and the development of collagen-induced arthritis in a dose-dependent manner. We conclude that MK-4 may represent a new agent for the treatment of rheumatoid arthritis in the setting of combination therapy with other disease-modifying antirheumatic drugs.     

A new cloud-based classification methodology (CBCM) for efficient semantic web service discovery

Over the last decades, web services are used for performing specific tasks demanded by users. The most important task of service’s classification system is to match an anonymous input service with the stored pre-classified web services. The most challenging issue is that web services are currently organized and classified according to syntax while the context of the requested service is ignored. Due to this motivation, Cloud-based Classification Methodology is proposed as it presents a new methodology based on semantic web service’s classification. Furthermore, cloud computing is used for not only storing but also allocating the high scale of web services with both high availability and accessibility. Fog technology is employed to reduce the latency and to speed up response time. The experimental results using the suggested methodology show a better performance of the proposed system regarding both precision and accuracy in comparison with most of the methods discussed in the literature of the current study.

     

A new algorithm for cluster analysis of genomic methylation: the Helicobacter pylori case

Motivation: The genomic methylation analysis is useful to typebacteria that have a high number of expressed type II methyltransferases.Methyltransferases are usually committed to Restriction andModification (R-M) systems, in which the restriction endonucleaseimposes high pressure on the expression of the cognate methyltransferasethat hinder R-M system loss. Conventional cluster methods donot reflect this tendency. An algorithm was developed for dendrogramconstruction reflecting the propensity for conservation of R-MType II systems. Results: The new algorithm was applied to 52 Helicobacter pyloristrains from different geographical regions and compared withconventional clustering methods. The algorithm works by firstgrouping strains that share a common minimum set of R-M systemsand gradually adds strains according to the number of the R-Msystems acquired. Dendrograms revealed a cluster of Africanstrains, which suggest that R-M systems are present in H.pylorigenome since its human host migrates from Africa. Availability: The software files are available at http://www.ff.ul.pt/paginas/jvitor/Bioinformatics/MCRM_algorithm.zip Contact: filipavale{at}fe.ucp.pt Supplementary information: Supplementary data are availableat Bioinformatics online. Associate Editor: Martin Bishop