Proteomics in diagnosis of prostate cancer.

Prostate cancer (PCa) is the second most frequently diagnosed malignancy in men worldwide. The introduction of prostate specific antigen (PSA) has greatly increased the number of men diagnosed with PCa but at the same time, as a result of the low specificity, led to overdiagnosis, resulting to unnecessary biopsies and high medical cost treatments. The primary goal in PCa research today is to find a biomarker or biomarker set for clear and effecttive diagnosis of PCa as well as for distinction between aggressive and indolent cancers. Different proteomic technologies such as 2-D PAGE, 2-D DIGE, MALDI MS profiling, shotgun proteomics with label-based (ICAT, iTRAQ) and label-free (SWATH) quantification, MudPIT, CE-MS have been applied to the study of PCa in the past 15 years. Various biological samples, including tumor tissue, serum, plasma, urine, seminal plasma, prostatic secretions and prostatic-derived exosomes were analyzed with the aim of identifying diagnostic and prognostic biomarkers and developing a deeper understanding of the disease at the molecular level. This review is focused on the overall analysis of expression proteomics studies in the PCa field investigating all types of human samples in the search for diagnostics biomarkers. Emphasis is given on proteomics platforms used in biomarker discovery and characterization, explored sources for PCa biomarkers, proposed candidate biomarkers by comparative proteomics studies and the possible future clinical application of those candidate biomarkers in PCa screening and diagnosis. In addition, we review the specificity of the putative markers and existing challenges in the proteomics research of PCa.

Proteomic analysis of infiltrating ductal carcinoma tissues by coupled 2-D DIGE/MS/MS analysis.

There is a growing interest in protein expression profiling aiming to identify novel diagnostic markers in breast cancer. Proteomic approaches such as two-dimensional differential gel electrophoresis coupled with tandem mass spectrometry analysis (2-D DIGE/MS/MS) have been used successfully for the identification of candidate biomarkers for screening, diagnosis, prognosis and monitoring of treatment response in various types of cancer. Identifying previously unknown proteins of potential clinical relevance will ultimately help in reaching effective ways to manage the disease. We analyzed breast cancer tissues from five tumor and five normal tissue samples from ten breast cancer subjects with infiltrating ductal carcinoma (IDC) by 2-D DIGE using two types of immobilized pH gradient (IPG) strips: pH 3-10 and pH 4-7. From all the spots detected, differentially expressed (p < 0.05 and ratio > 2) were 50 spots. Of these, 39 proteins were successfully identified by MS, representing 29 different proteins. Ten proteins were overexpressed in the tumor samples. The 2-D DIGE/MS/MS analysis revealed an increase in the expression levels in tumor samples of several proteins not previously associated with breast cancer, such as: macrophage-capping protein (CAPG), phosphomannomutase 2 (PMM2), ATPase ASN1, methylthioribose-1-phosphate isomerase (MRI1), peptidyl-prolyl cis-trans isomerase FKBP4, cellular retinoic acid-binding protein 2 (CRABP2), lamin B1 and keratin, type II cytoskeletal 8 (KRT8). Ingenuity Pathway Analysis (IPA) revealed highly significant (p = 10(-26)) interactions between the identified proteins and their association with cancer. These proteins are involved in many diverse pathways and have established roles in cellular metabolism. It remains the goal of future work to test the suitability of the identified proteins in samples of larger and independent patient groups.

Proteomic analysis of seminal plasma in men with different spermatogenic impairment.

Seminal plasma is a potential source of biomarkers for many disorders of the male reproductive system including male infertility. The identification and characterisation of differentially expressed proteins in seminal plasma of man with normal and impaired spermatogenesis can help in the elucidation of the molecular basis of male infertility. We compared the protein expression profiles of seminal plasma from four different groups of men as follows: normozoospermic, asthenozoospermic, oligozoospermic and azoospermic groups, using two-dimensional differential gel electrophoresis (2-D DIGE). We found eight proteins with statistically significant increased expression in azoospermia compared with at least one of the other studied groups. The differentially expressed spots were fibronectin, prostatic acid phosphatase (PAP), proteasome subunit alpha type-3, beta-2-microglobulin, galectin-3-binding protein, prolactin-inducible protein and cytosolic nonspecific dipeptidase. Notably, PAP was increased in patients with azoospermia compared with that of all other groups. We have observed no statistically significant differences in protein expression between three of the groups: normozoospermic, oligozoospermic and asthenozoospermic. We suggest that the identified panel of proteins in our study especially PAP have a strong potential to be used as azoospermia markers. However, further investigations will be necessary to validate these markers in samples of larger and independent patient cohorts and to clarify their role in the pathogenesis of male infertility.

Human seminal plasma proteome study: a search for male infertility biomarkers.

Seminal plasma is a potential source of biomarkers for many disorders of the male reproductive system including male infertility. Knowledge of the peptide and protein components of seminal fluid is accumulating especially with the appearance of high-throughput MS-based techniques. Of special interest in the field of male infertility biomarkers, is the identification and characterization of differentially expressed proteins in seminal plasma of men with normal and impaired spermatogenesis. However, the data obtained until now is still quite heterogeneous and with small percentage of overlap between independent studies. Extensive comparative analysis of seminal plasma proteome is still needed in order to establish a potential link between seminal plasma proteins and male infertility.

Microbiological and chemical characteristics of water and sediment from Vrelo Cave, Republic of Macedonia.

Vrelo Cave is the deepest cave in Macedonia, located in the canyon Matka which is home to many endemic species not found anywhere else in Europe. Until now, Vrelo Cave has not been investigated in terms of its composition and biodiversity. The purpose of this study was to offer some preliminary data for physical and chemical parameters of water and sediments from Vrelo Cave, as well as its microbiological diversity. Samples were taken from 5 locations. They were analysed for a wide array of physico-chemical parameters, macro- and microelements and concentration of selected organic pollutants. All samples were investigated for several groups of bacteria, yeasts and moulds by a conventional selective media approach. Molecular identification of the isolated bacterial species was done by sequencing of the bacterial 16S ribosomal RNA gene. Regarding the total dry components, total hardness, dissolved oxygen, biochemical and chemical consumption of oxygen, water from Vrelo Cave belongs to Class I. All of the investigated groups of microorganisms except anaerobic sporogenic bacteria were present in water and sediment samples. Notably, a large number of coliformic bacteria (total and faecal) were isolated from all of the investigated samples which classify this water in Class IV, as ecologically unsuitable drinking water. Most of the identified non-coliformic bacteria belonged to the genus Bacillus. We have also identified representatives from Staphylococcus, Proteus, Brevundimonas and Enterobacter. Overall findings suggest a possible connection between the water from the cave and surface waters. Further investigation should be performed to determine the origin of these waters.

A new thermostable DNA polymerase mixture for efficient amplification of long DNA fragments.

The thermostable DNA polymerases have been used for amplification of DNA fragments since the invention of PCR. The constraint on the maximum size of the amplified fragments can be solved to certain level by the use of unbalanced mixtures of non-proofreading and proofreading thermostable DNA polymerases. In this study, we tested the use of a mixtures of N-terminal deletional variant of Taq polymerase - Klentaq278 and Tne polymerase from Thermotoga neapolitana. Klentaq278 and Tne polymerase genes were cloned and expressed in different expression vectors under tac promoter. The most efficient ratio of Klentaq278/Tne polymerase for amplification was 10 : 1. The polymerase mixture of Klentaq278 and Tne polymerase is very effective in amplification of DNA fragments for up to 8 kb and is useful addition to a DNA polymerases used in long-range PCR.

Substitution of Ile(707) for Leu in Klentaq DNA polymerase reduces the amplification capacity of the enzyme.

The high yield and specificity of PCR amplifications are affected by DNA polymerase activity at room temperature. One way of preventing this unwanted activity is by genetic modifications of the DNA polymerase. For Taq DNA polymerase, mutations in the gene (Glu626Lys, Trp706Arg, Ile707Leu and Glu708Asp), when introduced individually or in certain combinations, were found to contribute to a significant decrease of the enzyme activity at room temperature. The aim of this study was to evaluate the usefulness of the Ile707Leu cold-sensitive mutation in the N-terminal deletional variant of Taq DNA polymerase in PCR reaction. The Ile(707) to Leu substitution was introduced to Klentaq278 by site-directed mutagenesis. Normal and mutant DNA polymerases were expressed under a tac promoter and purified to homogeneity. The mutant polymerase showed reduced polymerase activity at room temperature by up to 12 times and no significant change in thermostability, compared to Klentaq278 DNA polymerase. The major effect of the amino acid substitution was the reduction of the amplification capacity of the polymerase. Mutant polymerase could not amplify fragments over 1 kb. In conclusion, the substitution of Ile707Leu in Klentaq278 DNA polymerase reduces the overall processivity of the enzyme and therefore limits the application of this DNA polymerase in PCR.