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PubMed1997

Improved analysis of microsatellites using mass spectrometry.

The primer oligo base extension reaction combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, recently introduced by our group for detection of single-point mutations and small insertions/deletions, has been applied to the reliable quantification of nucleotide repeat units in microsatellites. The AluVpA DNA marker within intron 5 of the interferon-alpha receptor gene was chosen as the model system. By varying the dNTP/ddNTP mixtures used, the assay could also be directed to detect the location of second-site mutations within the repeats, resulting in identification of alleles not detectable by electrophoretic sizing methods and thus an increase of the polymorphism information content for a sampling of 28 unrelated individuals. The method results in highly informative mass signals and has the potential to increase the polymorphism information content for systems containing second-site mutations; thus it is a very attractive alternative technique in statistics-based gene mapping, cancer diagnostics, and forensic applications.

Base Sequence↗

PubMed2005

In vitro measurements of optical properties of porcine brain using a novel compact device.

Knowledge of the optical properties of tissues can be applied in numerous medical and scientific fields, including cancer diagnostics and therapy. There are many different ways of determining the optical properties of turbid media. The paper describes measurements of the optical properties of porcine brain tissue using novel instrumentation for simultaneous absorption and scattering characterisation of small turbid samples. Integrating sphere measurements are widely used as a reference method for determination of the optical properties of relatively thin turbid samples. However, this technique is associated with bulky equipment, complicated measuring techniques, interference compensation techniques and inconvenient sample handling. It is believed that the sphere for some applications can be replaced by a new, compact device, called the combined angular and spatially resolved head sensor, to measure the optical properties of thin turbid samples. The results compare very well with data obtained with an integrating sphere for well-defined samples. The instrument was shown to be accurate to within 12% for microa and 1% for micro's in measurements of intralipid-ink samples. The corresponding variations of data were 17% and 2%, respectively. The reduced scattering coefficient for porcine white matter was measured to be 100 cm(-1) at 633 nm, and the value for coagulated brain tissue was 65 cm(-1). The corresponding absorption coefficients were 2 and 3 cm(-1), respectively.

Animals↗

PubMed1991

Laser surgery in superficial penile tumours.

The first results of laser surgery in superficial penile tumours are reported. In 2 patients with condylomata acuminata, 2 cases of giant condylomata Buschke-Löwenstein and in 2 patients suffering from early stage penile cancer diagnostic and/or therapeutic treatments using carbon dioxide or neodymium-YAG laser have been performed. In one patient with giant condyloma a recurrent tumour was found 5 weeks after the initial treatment and a second one using CO2 laser was done. All other patients are tumour-free with a follow up of 6-24 months and erectile function is normal. Therefore, in superficial penile tumours laser treatment is an excellent alternative therapeutic approach.

Condylomata Acuminata↗

PubMed2000

Mitochondrial ATP synthase 6 as an endogenous control in the quantitative RT-PCR analysis of clinical cancer samples.

BACKGROUND: Real-time polymerase chain reaction (PCR) is a powerful new technique in the evolution of quantitative reverse transcription-PCR assays. With the increased sensitivity and resolution of real-time techniques, the requirements for constitutive expression of endogenous controls have become increasingly stringent. METHODS AND RESULTS: We compare the expression of the mitochondrial gene, adenosine triphosphate synthase 6 (ATPsy6), to the expression of other routinely used endogenous control genes (e.g., beta-actin, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ribosomal RNA 18S [18S rRNA], and cyclophilin). In a diverse assortment of tissues and across a wide range of disease stages, ATPsy6 shows a relative steady state of expression compared with other endogenous controls. ATPsy6 gene expression has been used as an endogenous control in a quantitative real-time PCR assay designed to evaluate the expression of potential cancer diagnostic leads across a diverse tissue panel. CONCLUSION: Mitochondrial ATPsy6 serves as a good endogenous control to measure target gene expression independent of the tissue- or disease-specific variation inherent with many housekeeping genes.

Deoxyribonucleases↗

PubMed2000

Methods in clinical molecular genetics.

UNLABELLED: Mutation detection is fundamental throughout biology, but especially so in medicine and medical research, particularly in gene discovery and cancer diagnostics. Current methods are unsatisfactory due to a range of reasons, but particularly due to cost and lack of convenience. The current methods are reviewed and their advantages and disadvantages discussed. The high profile chip technology whilst making an enormous impact in expression studies is more problematic for mutation detection, especially for unknown mutations and the problems are discussed. Finally, once mutations are discovered they need documenting accurately and the efforts of the Human Genome Organisation mutation database initiative due this direction are discussed. CONCLUSION: Current methods of mutation analysis, although unsatisfactory, are reviewed and the impact of new technologies assessed.

Databases, Factual↗

PubMed2006

In vivo imaging of integrin alpha v beta 3 expression using fluorescence-mediated tomography.

PURPOSE: Optical imaging would be desirable for cancer diagnostics since it can potentially resolve relevant oncological target structures in vivo. We therefore synthesised an alpha v beta(3) targeted fluorochrome and imaged tumour xenografts with different alpha v beta(3) expression levels using both planar and tomographic optical imaging methods. METHODS: An alpha v beta(3)-targeted RGD peptide was labelled with a cyanine dye (Cy 5.5). Binding of the optical tracer was tested on M21 melanoma (n=5), HT-1080 fibrosarcoma (n=6) and MCF-7 adenocarcinoma (n=5) cells and their tumour xenografts. All optical imaging studies were performed using two-dimensional planar fluorescence reflectance imaging (FRI) technology and three-dimensional fluorescence-mediated tomography (FMT). RESULTS: In vitro, the peptide-dye conjugate showed a clear binding affinity to alpha v beta(3)-positive M21 and HT-1080 cells while alpha v beta(3)-negative MCF-7 cells and pre-dosing with the free RGD peptide revealed little to no fluorescence. In vivo, tumour xenografts were clearly visualised by FRI and FMT up to 24 h post injection. FMT allowed quantification of the fluorochrome distribution in deeper tissue sections showing an average fluorochrome concentration of 417.61 +/- 105.82 nM Cy 5.5 (M21), 353.68 +/- 54.02 nM Cy 5.5 (HT-1080) and 262.83 +/- 155.36 nM Cy 5.5 (MCF-7) in the target tissue 60 min after tracer administration. Competition with the free RGD peptide resulted in a reduction in the fluorochrome concentration in M21 tumour tissue (294.35 +/- 84.27 nM). CONCLUSION: RGD-Cy 5.5 combined with novel tomographic optical imaging methods allows non-invasive imaging of tumour-associated alpha v beta(3) expression and may thus be a promising strategy for sensitive evaluation of tumour target expression.

Animals↗

PubMed2006

DNAse I pre-treatment markedly enhances detection of nuclear cyclin-dependent kinase inhibitor p57Kip2 and BrdU double immunostaining in embryonic rat brain.

As a member of the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs), p57Kip2 binds tightly to G1 cyclin/cyclin-dependent kinase complexes to block cell cycle progression. CKIs play critical roles in regulating the transition from proliferation to differentiation in many tissues, including the nervous system. Conversely, CKI dys-regulation contributes to neoplasia and cancer progression. While the combined detection of CKI immunoreactivity and S phase entry using bromodeoxyuridine (BrdU) incorporation may be particularly informative, successful immunostaining may be limited due to "masked" antigen epitopes and acid-induced signal degradation. We now report an improved double immunofluorescent method for detecting p57Kip2 and BrdU in paraformaldehyde-fixed frozen sections of embryonic rat brain. We substituted deoxyribonuclease I (DNAse I) for HCl pre-treatment to expose antigenic sites in frozen sections, and employed a biotinylated tyramide-based system to enhance p57Kip2 visualization. We identified a time- and dose-dependent relationship between DNAse I treatment and double labeling of p57Kip2 and BrdU, increasing both the numbers and intensities of immunopositive nuclei. With excess DNAse I treatment, however, there was signal degradation for both BrdU and total DNA, as reflected by DAPI staining. The use of DNAse I pre-treatment significantly increases the reliability and sensitivity of immunodetection of CKI nuclear factors, and should be useful for both developmental neurobiology studies as well as cancer diagnostic applications.

Animals↗

PubMed2005

Enzymatic DNA methylation is an epigenetic control for genetic functions of the cell.

In eukaryotic cells nuclear DNA is subjected to enzymatic methylation resulting in formation of 5-methylcytosine residues mainly in CG and CNG sequences. In plants and animals, this DNA methylation is species-, tissue-, and organelle-specific. It changes (diminishes) with age and is regulated by hormones. On the other hand, genome methylation can control hormonal signal. There are replicative and post-replicative DNA methylations. They are served by multiple DNA-methyltransferases with different site specificity. Replication is accompanied by appearance of hemi-methylated sites in DNA; pronounced asymmetry of DNA chain methylation disappears at the end of the cell cycle; a model of regulation of replication by DNA methylation is suggested. DNA methylation controls all genetic processes in the cell (replication, transcription, DNA repair, recombination, gene transposition) and it is a mechanism of cell differentiation, gene discrimination, and silencing. Prohibition of DNA methylation stops development (embryogenesis), switches on apoptosis, and is usually lethal. Distortions in DNA methylations result in cancerous cell transformation, and the DNA methylation pattern is one of the safe cancer diagnostics at early stages of carcinogenesis. The malignant cell has a different DNA methylation pattern and a set of DNA-methyltransferase activities expressed as compared with normal cells. Inhibition of DNA methylation in plants is accompanied by induction of genes of seed storage proteins and flowering. In eukaryotes one and the same gene can be methylated both on cytosine and adenine residues; thus, there are, at least, two different and probably interdependent systems of DNA methylation in the cell. First higher eukaryotic adenine DNA-methyltransferase was isolated from plants; this enzyme methylates DNA with formation of N6-methyladenine residues in the sequence TGATCA --> TGm6ATCA. Plants have AdoMet-dependent endonucleases sensitive to DNA methylation status; therefore, like microorganisms, plants seem to have a restriction-modification (R-S) system. Revelation of an essential role of DNA methylation in the regulation of genetic processes has laid a foundation for and materialized epigenetics and epigenomics.

Animals↗

PubMed1994

DNA sequences encoding enolase are remarkably conserved from yeast to mammals.

Enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11), particularly isoform neuron-specific enolase (NSE), is primarily localized in neurons and neuroendocrine cells and is a cancer diagnostic marker for brain tumors. Homology of enolase-coding DNA sequences from human, dog, cow, rat, mouse, rabbit, chicken, and yeast cells was investigated using hybridization techniques, percent sequence divergence, and amino acid analysis. Because enolase is a significant enzyme of the glycolytic pathway, enolase-coding DNA sequences have been found in all organisms tested so far. The human enzyme was found to be more like those of monkey and dog in structure than to those of chicken and yeast. The implications of the existence of the genetic conservation of enolase-coding DNA sequences in understanding concerted evolution as well as post-transcriptional regulation during differentiation are discussed. This is the first report is which sequence divergence in the coding region for enolase has been determined in a variety of organisms.

Amino Acid Sequence↗

PubMed1988

Immunochemical studies of human placental alkaline phosphatase in normal and neoplastic tissues.

Human placental alkaline phosphatase (hPLAP) is normally present in plasma membranes of syncytiotrophoblast microvilli. hPLAP is expressed by many malignant tumors and is considered to be useful as a tumor marker. The objective of the work was to develop immunochemical methods for detection of this marker. hPLAP was isolated from placenta using a procedure with preparative isoelectric focusing as a final purification step, and was used to obtain rabbit antisera. Anti-hPLAP antibodies were obtained by affinity chromatography on the immobilized hPLAP antigen and were used in immunoenzyme tests for detection of hPLAP in sera and tissues of cancer patients with different malignancies. Anti-hPLAP antibodies were also used for localization of hPLAP on the histological level. The described methods may be of use in cancer diagnostics and therapy.

Alkaline Phosphatase↗

PubMed1987

The role of proto-oncogenes in human cancer: implications for diagnosis and treatment.

Proto-oncogenes are normal genes which affect normal cell growth and proliferation, but which have the potential to contribute to cancer development if their expression is altered. A variety of events may activate proto-oncogenes and convert them from benign genes to cancer genes. These events include mutations of nucleic acid sequences, chromosomal rearrangements, and amplification of the number of genes. The observations that alterations in specific gene sequences frequently accompany neoplasia have provided the first insights into the pathogenesis of human cancer at the molecular level. These insights offer new opportunities for developing cancer diagnostics and therapeutics.

Gene Amplification↗

PubMed2005

Mixed self-assembled monolayers (SAMs) consisting of methoxy-tri(ethylene glycol)-terminated and alkyl-terminated dimethylchlorosilanes control the non-specific adsorption of proteins at oxidic surfaces.

Monolayers from the newly synthesized compound methoxy-tri(ethylene glycol)-undecenyldimethylchlorosilane (CH3O(CH2CH2O)3(CH2)11Si(CH3)2Cl, MeO(EG)3C11DMS) and dodecyldimethylchlorosilane (DDMS), both pure and mixed, were prepared by self-assembly from organic solution in the presence of an organic base. The films obtained were characterized by advancing and receding contact angle measurements and ellipsometry to confirm the formation of self-assembled monolayers (SAMs). The resulting data on the covalently attached dimethylsilanes were compared to known oligo(ethylene glycol) (OEG)-terminated SAM systems based on terminal alkenes, thiolates or trihydrolyzable silanes. The composition of the mixed SAMs was found to depend directly and linearly on the composition of the silanization solution. Enhanced protein repellent properties were found for the SAMs using a variety of proteins, including the Ras Binding Domain (RBD), a protein with high relevance for cancer diagnostics. Roughly a RBD protein monolayer amount was adsorbed to silicon oxide surfaces silanized with DDMS or non-silanized silicon wafers, and in contrast, no RBD was adsorbed to surfaces silanized with MeO(EG)3C11DMS or to mixed monolayers consisting of DDMS and MeO(EG)3C11DMS if the content of OEG-silane overcame a critical content of X(EG) approximately 0.9.

Adsorption↗

PubMed2024

Bridging the Gap From Proteomics Technology to Clinical Application: Highlights From the 68th Benzon Foundation Symposium.

The 68th Benzon Foundation Symposium brought together leading experts to explore the integration of mass spectrometry-based proteomics and artificial intelligence to revolutionize personalized medicine. This report highlights key discussions on recent technological advances in mass spectrometry-based proteomics, including improvements in sensitivity, throughput, and data analysis. Particular emphasis was placed on plasma proteomics and its potential for biomarker discovery across various diseases. The symposium addressed critical challenges in translating proteomic discoveries to clinical practice, including standardization, regulatory considerations, and the need for robust "business cases" to motivate adoption. Promising applications were presented in areas such as cancer diagnostics, neurodegenerative diseases, and cardiovascular health. The integration of proteomics with other omics technologies and imaging methods was explored, showcasing the power of multimodal approaches in understanding complex biological systems. Artificial intelligence emerged as a crucial tool for the acquisition of large-scale proteomic datasets, extracting meaningful insights, and enhancing clinical decision-making. By fostering dialog between academic researchers, industry leaders in proteomics technology, and clinicians, the symposium illuminated potential pathways for proteomics to transform personalized medicine, advancing the cause of more precise diagnostics and targeted therapies.

Proteomics↗

PubMed2006

Survivin regulation of vascular injury.

Survivin is a protein member of the inhibitor of apoptosis family that has attracted attention for its dual essential roles in the regulation of cell division and the control of apoptosis. Although extensively characterized as a tumor gene and exploited for new opportunities in cancer diagnostics and therapeutics, fresh experimental evidence has uncovered a critical role of survivin in vascular cell responses in vivo. Dynamically embedded in broad signaling pathways controlling smooth muscle cell adaptation, survivin not only controls vascular wall homeostasis but also acts as a critical upstream regulator of gene expression and second mediators.

Animals↗

PubMed2003

Promoter hypomethylation of a novel cancer/testis antigen gene CAGE is correlated with its aberrant expression and is seen in premalignant stage of gastric carcinoma.

Previously, we reported the identification and characterization of a novel cancer/testis antigen gene, CAGE(4), that was expressed in various histological types of tumors, but not in normal tissues, with the exception of the testis. To date, molecular mechanisms for the expression of CAGE have never been studied. In our expression analysis, we found that some cancer cell lines did not express CAGE. The expression of CAGE could be restored in these cell lines by treatment with 5(')-aza-2(')-deoxycytidine, suggesting that the expression of CAGE is mainly suppressed by hypermethylation. Bisulfite sequencing analysis of the 16 CpG sites of the CAGE promoter in various cancer cell lines and tissues revealed a close relationship between the methylation status of the CAGE promoter and the expression of CAGE. The transient transfection experiments displayed that the methylation of CpG sites inhibited the CAGE promoter activity in luciferase reporter assays. The methylation of the CpG sites inhibited the binding of transcription factors, shown by a mobility shift assay. A methylation-specific PCR analysis revealed that hypomethylation of the CAGE promoter was present at frequencies of more than 60% in breast, gastric, and lung cancers, and hepatocellular carcinomas, and at frequencies of less than 40% in prostate, uterine cervical, and laryngeal cancers. Promoter hypomethylation was found in chronic gastritis (19/55, 34.5%) and liver cirrhosis (13/22, 59%), but not in normal prostate, normal colon, or chronic hepatitis. These results suggest that the methylation status of the CpG sites of CAGE determines its expression, that the hypomethylation of CAGE precedes the development of gastric cancer and hepatocellular carcinoma, and that the high frequencies of hypomethylation of CAGE, in various cancers would be valuable as a cancer diagnostic marker.

Antigens, Nuclear↗

PubMed1981

Genital herpes simplex--concepts and treatment.

Genital herpes simplex is an increasingly common venereal disease caused by either herpes simplex virus type 1 or 2. The disease can be manifested in a primary and/or recurrent form, the clinical features and complications of which are reviewed. Potential problems include psychosocial difficulties, neonatal herpes simplex infections, and the possibility of an oncogenic role in cervical cancer. Diagnostic modalities, prophylactic measures, and management technics are discussed.

Animals↗

PubMed1998

Future trends in diagnosis using laboratory-on-a-chip technologies.

There has been an enormous growth in the development of biotechnological applications, where advances in the techniques of microelectronic fabrication and the technologies of miniaturization and integration in semiconductor industries are being applied to the production of Laboratory-on-a-Chip devices. The aim of this development is to create devices that will perform the same processes that are currently carried out in the laboratory in reduced timescales, at a lower cost, requiring less reagents, and with a greater resolution of detection and specificity. The expectations of this Laboratory-on-a-Chip revolution is that this technology will facilitate rapid advances in gene discovery, genetic mapping and gene expression with broader applications ranging from infectious diseases and cancer diagnostics to food quality and environmental testing. A review of the current state of development in this field reveals the scale of the ongoing revolution and serves to highlight the advances that can be perceived in the development of Laboratory-on-a-Chip technologies. Since miniaturization can be applied to such a wide range of laboratory processes, some of the sub-units that can be used as building blocks in these devices are described, with a brief description of some of the fabrication processes that can be used to create them.

Animals↗

PubMed2000

DNA microsatellite analysis using ion-pair reversed-phase high-performance liquid chromatography.

Genotyping based on short tandem repeat (STR) regions is used in human identification and parentage testing, gene mapping studies, cancer diagnostics, and diagnosis of hereditary diseases. Analysis of STR systems using slab gel electrophoresis requires lengthy and labor-intensive procedures. Therefore, alternative methods such as capillary electrophoresis or ion-pair reversed-phase high-performance liquid chromatography (IPRP HPLC) have been used to analyze DNA. IPRP HPLC offers an attractive substitute to gel electrophoresis for STR analysis because of the reduced analysis time, and there is no need for the waste disposal associated with radioisotopic, enzyme-linked, or fluorescence detection systems. We evaluated the use of IPRP HPLC for the sizing and typing of STR alleles from the HUMTHO1 locus. The IPRP HPLC conditions (column temperature, flow rate, percent organic modifier per minute) were optimized for the separation of PCR products. Using the optimized separation conditions, the alleles of the HUMTHO1 system were sized in their native state (double standard) with the use of internal markers. The typing results correlated 100% to accepted methods of DNA typing. The analysis time for the HUMTHO1 locus was less than 14 min, and the alleles could be peak captured for further examination following such as sequencing.

Alleles↗