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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Diagnostics".

Deadline for manuscript submissions: closed (31 August 2011)

Special Issue Editors

Guest Editor
Prof. Dr. Renata Pasqualini (Website)

Department of Genitourinary Medical Oncology - Research, Division of Cancer Medicine, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 0018-1, Houston, TX 77030, USA
Guest Editor
Prof. Dr. Wadih Arap (Website)

Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 1374, Houston, TX 77030, USA

Published Papers (22 papers)

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Research

Jump to: Review

Open AccessArticle Mass Spectrometry-Based Quantitative Metabolomics Revealed a Distinct Lipid Profile in Breast Cancer Patients
Int. J. Mol. Sci. 2013, 14(4), 8047-8061; doi:10.3390/ijms14048047
Received: 1 March 2013 / Revised: 30 March 2013 / Accepted: 1 April 2013 / Published: 12 April 2013
Cited by 15 | PDF Full-text (684 KB) | HTML Full-text | XML Full-text
Abstract
Breast cancer accounts for the largest number of newly diagnosed cases in female cancer patients. Although mammography is a powerful screening tool, about 20% of breast cancer cases cannot be detected by this method. New diagnostic biomarkers for breast cancer are necessary. [...] Read more.
Breast cancer accounts for the largest number of newly diagnosed cases in female cancer patients. Although mammography is a powerful screening tool, about 20% of breast cancer cases cannot be detected by this method. New diagnostic biomarkers for breast cancer are necessary. Here, we used a mass spectrometry-based quantitative metabolomics method to analyze plasma samples from 55 breast cancer patients and 25 healthy controls. A number of 30 patients and 20 age-matched healthy controls were used as a training dataset to establish a diagnostic model and to identify potential biomarkers. The remaining samples were used as a validation dataset to evaluate the predictive accuracy for the established model. Distinct separation was obtained from an orthogonal partial least squares-discriminant analysis (OPLS-DA) model with good prediction accuracy. Based on this analysis, 39 differentiating metabolites were identified, including significantly lower levels of lysophosphatidylcholines and higher levels of sphingomyelins in the plasma samples obtained from breast cancer patients compared with healthy controls. Using logical regression, a diagnostic equation based on three metabolites (lysoPC a C16:0, PC ae C42:5 and PC aa C34:2) successfully differentiated breast cancer patients from healthy controls, with a sensitivity of 98.1% and a specificity of 96.0%. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessArticle Detection of Tumor Cell-Specific mRNA in the Peripheral Blood of Patients with Breast Cancer — Evaluation of Several Markers with Real-Time Reverse Transcription-PCR
Int. J. Mol. Sci. 2013, 14(1), 1093-1104; doi:10.3390/ijms14011093
Received: 12 November 2012 / Revised: 3 December 2012 / Accepted: 31 December 2012 / Published: 8 January 2013
Cited by 8 | PDF Full-text (716 KB) | HTML Full-text | XML Full-text
Abstract
It is widely known that cells from epithelial tumors, e.g., breast cancer, detach from their primary tissue and enter blood circulation. We show that the presence of circulating tumor cells (CTCs) in samples of patients with primary and metastatic breast cancer can [...] Read more.
It is widely known that cells from epithelial tumors, e.g., breast cancer, detach from their primary tissue and enter blood circulation. We show that the presence of circulating tumor cells (CTCs) in samples of patients with primary and metastatic breast cancer can be detected with an array of selected tumor-marker-genes by reverse transcription real-time PCR. The focus of the presented work is on detecting differences in gene expression between healthy individuals and adjuvant and metastatic breast cancer patients, not an accurate quantification of these differences. Therefore, total RNA was isolated from blood samples of healthy donors and patients with primary or metastatic breast cancer after enrichment of mononuclear cells by density gradient centrifugation. After reverse transcription real-time PCR was carried out with a set of marker genes (BCSP, CK8, Her2, MGL, CK18, CK19). B2M and GAPDH were used as reference genes. Blood samples from patients with metastatic disease revealed increased cytokine gene levels in comparison to normal blood samples. Detection of a single gene was not sufficient to detect CTCs by reverse transcription real-time PCR. Markers used here were selected based on a recent study detecting cancer cells on different protein levels. The combination of such a marker array leads to higher and more specific discovery rates, predominantly in metastatic patients. Identification of CTCs by PCR methods may lead to better diagnosis and prognosis and could help to choose an adequate therapy. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessArticle CARD15/NOD2, CD14 and Toll-like 4 Receptor Gene Polymorphisms in Saudi Patients with Crohn’s Disease
Int. J. Mol. Sci. 2012, 13(4), 4268-4280; doi:10.3390/ijms13044268
Received: 31 December 2011 / Revised: 19 March 2012 / Accepted: 20 March 2012 / Published: 2 April 2012
Cited by 7 | PDF Full-text (309 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Crohn’s disease (CD) is a multifactorial disease with a genetic component and an observed association with genes related to the innate immune response. Polymorphisms in the CARD15/NOD2 gene, in addition to functional variants of the toll-like receptor-4 (TLR4) and CD14 genes, have [...] Read more.
Crohn’s disease (CD) is a multifactorial disease with a genetic component and an observed association with genes related to the innate immune response. Polymorphisms in the CARD15/NOD2 gene, in addition to functional variants of the toll-like receptor-4 (TLR4) and CD14 genes, have been associated with the development of Crohn’s disease. There is no information about the frequency of these polymorphisms in the Saudi population. We examined the frequency of the three major CARD15/NOD2 risk alleles (Leu1007fsinsC, Arg702Trp, and Gly908Arg) and the TLR4 (Thr399Il) polymorphism as well as a functional polymorphism in the promoter of the CD14–159C/T in 46 Saudi CD patients and 50 matched controls. Genotyping was performed by allele-specific PCR or by restriction fragment length polymorphism (PCR-RFLP) analysis. The mutant genotype frequencies of the Leu1007fsinsC, Arg702Trp and Gly908Arg in the patient group were 6.5, 21.7 and 6.5%, respectively, compared with frequencies of 0, 4 and 2%, respectively, in the control group. There were 15 patients who carried the mutant alleles for all three CARD15/NOD2 variants, Leu1007fsinsC, Arg702Trp and Gly908Arg, while none of the control candidates carried the three alleles. This genetic study provides evidence that the three major CARD15/NOD2 variant alleles and the CD14 −159C/T polymorphism are associated with Crohn’s disease (CD) susceptibility in the Saudi population; however, there is no evidence that the TLR4 (Thr399Il) or CARD15/NOD2 polymorphisms can be considered risk factors for Crohn’s disease. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessArticle Use of the MLPA Assay in the Molecular Diagnosis of Gene Copy Number Alterations in Human Genetic Diseases
Int. J. Mol. Sci. 2012, 13(3), 3245-3276; doi:10.3390/ijms13033245
Received: 30 December 2011 / Revised: 28 February 2012 / Accepted: 29 February 2012 / Published: 8 March 2012
Cited by 35 | PDF Full-text (295 KB) | HTML Full-text | XML Full-text
Abstract
Multiplex Ligation-dependent Probe Amplification (MLPA) assay is a recently developed technique able to evidence variations in the copy number of several human genes. Due to this ability, MLPA can be used in the molecular diagnosis of several genetic diseases whose pathogenesis is [...] Read more.
Multiplex Ligation-dependent Probe Amplification (MLPA) assay is a recently developed technique able to evidence variations in the copy number of several human genes. Due to this ability, MLPA can be used in the molecular diagnosis of several genetic diseases whose pathogenesis is related to the presence of deletions or duplications of specific genes. Moreover, MLPA assay can also be used in the molecular diagnosis of genetic diseases characterized by the presence of abnormal DNA methylation. Due to the large number of genes that can be analyzed by a single technique, MLPA assay represents the gold standard for molecular analysis of all pathologies derived from the presence of gene copy number variation. In this review, the main applications of the MLPA technique for the molecular diagnosis of human diseases are described. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessArticle Characterization of Novel Di-, Tri-, and Tetranucleotide Microsatellite Primers Suitable for Genotyping Various Plant Pathogenic Fungi with Special Emphasis on Fusaria and Mycospherella graminicola
Int. J. Mol. Sci. 2012, 13(3), 2951-2964; doi:10.3390/ijms13032951
Received: 7 November 2011 / Revised: 9 January 2012 / Accepted: 20 February 2012 / Published: 6 March 2012
Cited by 11 | PDF Full-text (1665 KB) | HTML Full-text | XML Full-text
Abstract
The goals of this investigation were to identify and evaluate the use of polymorphic microsatellite marker (PMM) analysis for molecular typing of seventeen plant pathogenic fungi. Primers for di-, tri-, and tetranucleotide loci were designed directly from the recently published genomic sequence [...] Read more.
The goals of this investigation were to identify and evaluate the use of polymorphic microsatellite marker (PMM) analysis for molecular typing of seventeen plant pathogenic fungi. Primers for di-, tri-, and tetranucleotide loci were designed directly from the recently published genomic sequence of Mycospherlla graminicola and Fusarium graminearum. A total of 20 new microsatellite primers as easy-to-score markers were developed. Microsatellite primer PCR (MP-PCR) yielded highly reproducible and complex genomic fingerprints, with several bands ranging in size from 200 to 3000 bp. Of the 20 primers tested, only (TAGG)4, (TCC)5 and (CA)7T produced a high number of polymorphic bands from either F. graminearum or F. culmorum. (ATG)5 led to successful amplifications in M. graminicola isolates collected from Germany. Percentage of polymorphic bands among Fusarium species ranged from 9 to 100%. Cluster analysis of banding patterns of the isolates corresponded well to the established species delineations based on morphology and other methods of phylogenetic analysis. The current research demonstrates that the newly designed microsatellite primers are reliable, sensitive and technically simple tools for assaying genetic variability in plant pathogenic fungi. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessArticle Comparison of Culture and Molecular Identification of Bacteria in Chronic Wounds
Int. J. Mol. Sci. 2012, 13(3), 2535-2550; doi:10.3390/ijms13032535
Received: 1 November 2011 / Revised: 30 January 2012 / Accepted: 13 February 2012 / Published: 23 February 2012
Cited by 32 | PDF Full-text (137 KB) | HTML Full-text | XML Full-text
Abstract
Clinical diagnostics of chronic polymicrobial infections, such as those found in chronic wounds, represent a diagnostic challenge for both culture and molecular methods. In the current retrospective study, the results of aerobic bacterial cultures and culture-free bacterial identification using DNA analyses were [...] Read more.
Clinical diagnostics of chronic polymicrobial infections, such as those found in chronic wounds, represent a diagnostic challenge for both culture and molecular methods. In the current retrospective study, the results of aerobic bacterial cultures and culture-free bacterial identification using DNA analyses were compared. A total of 168 chronic wounds were studied. The majority of bacteria identified with culture testing were also identified with molecular testing, but the majority of bacteria identified with the molecular testing were not identified with culture testing. Seventeen (17) different bacterial taxa were identified with culture, and 338 different bacterial taxa were identified with molecular testing. This study demonstrates the increased sensitivity that molecular microbial identification can have over culture methodologies, and previous studies suggest that molecular bacterial identification can improve the clinical outcomes of patients with chronic wounds. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessArticle Genetic Variability and Phylogeny of High Risk HPV Type 16, 18, 31, 33 and 45 L1 Gene in Greek Women
Int. J. Mol. Sci. 2012, 13(1), 1-17; doi:10.3390/ijms13010001
Received: 5 September 2011 / Revised: 25 November 2011 / Accepted: 5 December 2011 / Published: 22 December 2011
Cited by 10 | PDF Full-text (262 KB) | HTML Full-text | XML Full-text
Abstract
The present study explores nucleotide variability, phylogeny and association with cervical neoplasia in high risk HPV types 16, 18, 31, 33 and 45 collected from Greek women. Of the 1894 women undergoing routine cervical cytology examination, 160 samples test positive for single [...] Read more.
The present study explores nucleotide variability, phylogeny and association with cervical neoplasia in high risk HPV types 16, 18, 31, 33 and 45 collected from Greek women. Of the 1894 women undergoing routine cervical cytology examination, 160 samples test positive for single infections of HPV type 16 (n = 104), HPV 31 (n = 40), HPV 33 (n = 7), HPV 18 (n = 5), and HPV 45 (n = 4) were typed by microarrays method, amplified by PCR then sequenced and phylogenetically analyzed. For HPV 16, 9 variants with nucleotide variations were included into the study. For HPV 31, 33, 18 and 45, nucleotide variations were identified in 6, 4, 2 and 3 variants, respectively. The Bayesian inference and Maximum Parsimony methods were used in order to construct the phylogenetic trees. When types were analyzed independently HPV 16 (European and non-European) and HPV 18 (African and non-African) formed distinct clades. The genomic characterization of HPV variants will be important for illuminating the geographical relatedness and biological differences and for the determination of their risk. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessArticle Molecular Diagnosis of 5α-Reductase Type II Deficiency in Brazilian Siblings with 46,XY Disorder of Sex Development
Int. J. Mol. Sci. 2011, 12(12), 9471-9480; doi:10.3390/ijms12129471
Received: 2 September 2011 / Revised: 28 November 2011 / Accepted: 13 December 2011 / Published: 19 December 2011
PDF Full-text (1297 KB) | HTML Full-text | XML Full-text
Abstract
The steroid 5α-reductase type II enzyme catalyzes the conversion of testosterone (T) to dihydrotestosterone (DHT), and its deficiency leads to undervirilization in 46,XY individuals, due to an impairment of this conversion in genital tissues. Molecular analysis in the steroid 5α-reductase type II [...] Read more.
The steroid 5α-reductase type II enzyme catalyzes the conversion of testosterone (T) to dihydrotestosterone (DHT), and its deficiency leads to undervirilization in 46,XY individuals, due to an impairment of this conversion in genital tissues. Molecular analysis in the steroid 5α-reductase type II gene (SRD5A2) was performed in two 46,XY female siblings. SRD5A2 gene sequencing revealed that the patients were homozygous for p.Gln126Arg missense mutation, which results from the CGA > CAA nucleotide substitution. The molecular result confirmed clinical diagnosis of 46,XY disorder of sex development (DSD) for the older sister and directed the investigation to other family members. Studies on SRD5A2 protein structure showed severe changes at NADPH binding region indicating that structural modeling analysis can be useful to evaluate the deleterious role of a mutation as causing 5α-reductase type II enzyme deficiency. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessArticle Loop-Mediated Amplification Accelerated by Stem Primers
Int. J. Mol. Sci. 2011, 12(12), 9108-9124; doi:10.3390/ijms12129108
Received: 31 August 2011 / Revised: 6 November 2011 / Accepted: 29 November 2011 / Published: 8 December 2011
Cited by 11 | PDF Full-text (931 KB) | HTML Full-text | XML Full-text
Abstract
Isothermal nucleic acid amplifications (iNAATs) have become an important alternative to PCR for in vitro molecular diagnostics in all fields. Amongst iNAATs Loop-mediated amplification (LAMP) has gained much attention over the last decade because of the simplicity of hardware requirements. LAMP demonstrates [...] Read more.
Isothermal nucleic acid amplifications (iNAATs) have become an important alternative to PCR for in vitro molecular diagnostics in all fields. Amongst iNAATs Loop-mediated amplification (LAMP) has gained much attention over the last decade because of the simplicity of hardware requirements. LAMP demonstrates performance equivalent to that of PCR, but its application has been limited by the challenging primer design. The design of six primers in LAMP requires a selection of eight priming sites with significant restrictions imposed on their respective positioning and orientation. In order to relieve primer design constraints we propose an alternative approach which uses Stem primers instead of Loop primers and demonstrate the application of STEM-LAMP in assaying for Clostridium difficile, Listeria monocytogenes and HIV. Stem primers used in LAMP in combination with loop-generating and displacement primers gave significant benefits in speed and sensitivity, similar to those offered by Loop primers, while offering additional options of forward and reverse orientations, multiplexing, use in conjunction with Loop primers or even omission of one or two displacement primers, where necessary. Stem primers represent a valuable alternative to Loop primers and an additional tool for IVD assay development by offering more choices for primer design at the same time increasing assay speed, sensitivity, and reproducibility. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessArticle Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer
Int. J. Mol. Sci. 2011, 12(12), 8530-8538; doi:10.3390/ijms12128530
Received: 18 September 2011 / Revised: 17 November 2011 / Accepted: 17 November 2011 / Published: 29 November 2011
Cited by 3 | PDF Full-text (239 KB) | HTML Full-text | XML Full-text
Abstract
This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC) tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and [...] Read more.
This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC) tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip). Biochip hybridization identified 17 (21%) samples to carry a KRAS mutation of which 16 (33%) were adenocarcinomas and 1 (3%) was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessArticle A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays
Int. J. Mol. Sci. 2011, 12(11), 7748-7759; doi:10.3390/ijms12117748
Received: 5 September 2011 / Revised: 31 October 2011 / Accepted: 3 November 2011 / Published: 9 November 2011
Cited by 8 | PDF Full-text (460 KB) | HTML Full-text | XML Full-text
Abstract
Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (< 30 ng/mL) determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessArticle Microsatellite Analysis in Multistage Carcinogenesis of Esophageal Squamous Cell Carcinoma from Chongqing in Southern China
Int. J. Mol. Sci. 2011, 12(11), 7401-7409; doi:10.3390/ijms12117401
Received: 15 August 2011 / Revised: 13 October 2011 / Accepted: 26 October 2011 / Published: 28 October 2011
Cited by 4 | PDF Full-text (113 KB) | HTML Full-text | XML Full-text
Abstract
In order to characterize the molecular events in the carcinogenesis of esophageal cancer and to identify biomarkers for the early detection of the disease, matched precancerous and cancerous tissues resected from 34 esophageal cancer patients in Chongqing of southern China were compared for the extent of loss of heterozygosity (LOH). Sixteen microsatellite markers on nine chromosome regions were used for the PCR-based LOH analysis. The overall frequency of LOH at the 16 microsatellite loci was significantly increased as the pathological status of the resection specimens changed from low-grade dysplasia (LGD) to high-grade dysplasia (HGD) and squamous cell carcinoma (SCC) (P < 0.001), indicating that tumorigenesis of the esophageal squamous epithelia is a progressive process involving accumulative changes of LOH. A total of eight markers showed LOH in the LGD samples, suggesting that these loci may be involved in the early-stage tumorigenesis of esophageal squamous cell carcinoma (ESCC) and that LOH analysis at these loci may help improve the early detection of this disease. In addition, heterozygosity was regained at four loci in the SCC samples of four patients compared with the HGD samples, suggesting the possibility of genetic heterogeneity in the tumorigenesis of esophageal cancer. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessCommunication Molecular Diagnosis of Analbuminemia: A New Case Caused by a Nonsense Mutation in the Albumin Gene
Int. J. Mol. Sci. 2011, 12(11), 7314-7322; doi:10.3390/ijms12117314
Received: 5 August 2011 / Revised: 15 September 2011 / Accepted: 13 October 2011 / Published: 25 October 2011
Cited by 7 | PDF Full-text (478 KB) | HTML Full-text | XML Full-text
Abstract
Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin (ALB). We report here a new case diagnosed in a 45 years old man of Southwestern Asian origin, living in Switzerland, on the basis [...] Read more.
Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin (ALB). We report here a new case diagnosed in a 45 years old man of Southwestern Asian origin, living in Switzerland, on the basis of his low ALB concentration (0.9 g/L) in the absence of renal or gastrointestinal protein loss, or liver dysfunction. The clinical diagnosis was confirmed by a mutational analysis of the albumin (ALB) gene, carried out by single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), and DNA sequencing. This screening of the ALB gene revealed that the proband is homozygous for two mutations: the insertion of a T in a stretch of eight Ts spanning positions c.1289 + 23–c.1289 + 30 of intron 10 and a c.802 G > T transversion in exon 7. Whereas the presence of an additional T in the poly-T tract has no direct deleterious effect, the latter nonsense mutation changes the codon GAA for Glu244 to the stop codon TAA, resulting in a premature termination of the polypeptide chain. The putative protein product would have a length of only 243 amino acid residues instead of the normal 585 found in the mature serum albumin, but no evidence for the presence in serum of such a truncated polypeptide chain could be obtained by two dimensional electrophoresis and western blotting analysis. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
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Open AccessArticle Screening of Molecular Virulence Markers in Staphylococcus aureus and Pseudomonas aeruginosa Strains Isolated from Clinical Infections
Int. J. Mol. Sci. 2010, 11(12), 5273-5291; doi:10.3390/ijms11125273
Received: 12 November 2010 / Revised: 2 December 2010 / Accepted: 3 December 2010 / Published: 21 December 2010
Cited by 10 | PDF Full-text (538 KB) | HTML Full-text | XML Full-text
Abstract
Staphylococcus (S.) aureus and Pseudomonas (Ps.) aeruginosa are two of the most frequently opportunistic pathogens isolated in nosocomial infections, responsible for severe infections in immunocompromised hosts. The frequent emergence of antibiotic-resistant S. aureus and Ps. aeruginosa strains has determined the development of [...] Read more.
Staphylococcus (S.) aureus and Pseudomonas (Ps.) aeruginosa are two of the most frequently opportunistic pathogens isolated in nosocomial infections, responsible for severe infections in immunocompromised hosts. The frequent emergence of antibiotic-resistant S. aureus and Ps. aeruginosa strains has determined the development of new strategies in order to elucidate the different mechanisms used by these bacteria at different stages of the infectious process, providing the scientists with new procedures for preventing, or at least improving, the control of S. aureus and Ps. aeruginosa infections. The purpose of this study was to characterize the molecular markers of virulence in S. aureus and Ps. aeruginosa strains isolated from different clinical specimens. We used multiplex and uniplex PCR assays to detect the genes encoding different cell-wall associated and extracellular virulence factors, in order to evaluate potential associations between the presence of putative virulence genes and the outcome of infections caused by these bacteria. Our results demonstrate that all the studied S. aureus and Ps. aeruginosa strains synthesize the majority of the investigated virulence determinants, probably responsible for different types of infections. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessArticle An Adsorptive Transfer Technique Coupled with Brdicka Reaction to Reveal the Importance of Metallothionein in Chemotherapy with Platinum Based Cytostatics
Int. J. Mol. Sci. 2010, 11(12), 4826-4842; doi:10.3390/ijms11124826
Received: 11 October 2010 / Revised: 10 November 2010 / Accepted: 24 November 2010 / Published: 26 November 2010
Cited by 16 | PDF Full-text (619 KB) | HTML Full-text | XML Full-text
Abstract
The drugs based on platinum metals represent one of the oldest, but also one of the most effective groups of chemotherapeutic agents. Thanks to many clinical studies it is known that resistance of tumor cells to drugs is a frequent cause of [...] Read more.
The drugs based on platinum metals represent one of the oldest, but also one of the most effective groups of chemotherapeutic agents. Thanks to many clinical studies it is known that resistance of tumor cells to drugs is a frequent cause of chemotherapy failure. With regard to platinum based drugs, multidrug resistance can also be connected with increased expression of low-molecular weight protein metallothionein (MT). This study aimed at investigating the interactions of MT with cisplatin or carboplatin, using the adsorptive transfer technique coupled with differential pulse voltammetry Brdicka reaction (AdTS DPV Brdicka reaction), and a comparison of in vitro results with results obtained in vivo. The results obtained from the in vitro study show a strong affinity between platinum based drugs and MT. Further, we analyzed extracts of neuroblastoma cell lines treated with cisplatin or carboplatin. It is clear that neuroblastoma UKF-NB-4 cisplatin-resistant and cisplatin-sensitive cell lines unlikely respond to the presence of the platinum-based cytostatics cisplatin and carboplatin. Finally, we determined the level of MT in samples from rabbits treated with carboplatin and patients with retinoblastoma treated with the same drug. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)

Review

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Open AccessReview Molecular Diagnostic and Pathogenesis of Hereditary Hemochromatosis
Int. J. Mol. Sci. 2012, 13(2), 1497-1511; doi:10.3390/ijms13021497
Received: 6 September 2011 / Revised: 12 January 2012 / Accepted: 13 January 2012 / Published: 1 February 2012
Cited by 30 | PDF Full-text (204 KB) | HTML Full-text | XML Full-text
Abstract
Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by enhanced intestinal absorption of dietary iron. Without therapeutic intervention, iron overload leads to multiple organ damage such as liver cirrhosis, cardiomyopathy, diabetes, arthritis, hypogonadism and skin pigmentation. Most HH patients carry HFE [...] Read more.
Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by enhanced intestinal absorption of dietary iron. Without therapeutic intervention, iron overload leads to multiple organ damage such as liver cirrhosis, cardiomyopathy, diabetes, arthritis, hypogonadism and skin pigmentation. Most HH patients carry HFE mutant genotypes: homozygosity for p.Cys282Tyr or p.Cys282Tyr/p.His63Asp compound heterozygosity. In addition to HFE gene, mutations in the genes that encode hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) have been associated with regulation of iron homeostasis and development of HH. The aim of this review was to identify the main gene mutations involved in the pathogenesis of type 1, 2, 3 and 4 HH and their genetic testing indication. HFE testing for the two main mutations (p.Cys282Tyr and p.His63Asp) should be performed in all patients with primary iron overload and unexplained increased transferrin saturation and/or serum ferritin values. The evaluation of the HJV p.Gly320Val mutation must be the molecular test of choice in suspected patients with juvenile hemochromatosis with less than 30 years and cardiac or endocrine manifestations. In conclusion, HH is an example that genetic testing can, in addition to performing the differential diagnostic with secondary iron overload, lead to more adequate and faster treatment. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessReview Molecular Basis of Medullary Thyroid Carcinoma: The Role of RET Polymorphisms
Int. J. Mol. Sci. 2012, 13(1), 221-239; doi:10.3390/ijms13010221
Received: 10 October 2011 / Revised: 29 November 2011 / Accepted: 20 December 2011 / Published: 27 December 2011
Cited by 16 | PDF Full-text (341 KB) | HTML Full-text | XML Full-text
Abstract
Medullary thyroid carcinoma is a rare malignant tumor originating in parafollicular C cells. It accounts for 5 to 8% of all thyroid cancers. MTC develops in either sporadic (75%) or hereditary form (25%). Genetic and molecular studies have demonstrated the involvement of [...] Read more.
Medullary thyroid carcinoma is a rare malignant tumor originating in parafollicular C cells. It accounts for 5 to 8% of all thyroid cancers. MTC develops in either sporadic (75%) or hereditary form (25%). Genetic and molecular studies have demonstrated the involvement of the RET proto-oncogene in hereditary MTC and, less often, in its sporadic form. Although a strong genotype-phenotype correlation has been described, wide clinical heterogeneity is observed among families with the same RET mutation or even in carriers of the same kindred. In recent years, several single nucleotide polymorphisms of the RET gene have been described in the general population as well as in patients with MTC. Some studies have reported associations between the presence of polymorphisms and development or progression of MTC. Nonetheless, other studies failed to demonstrate any effect of the RET variants. Differences in the genetic background of distinct populations or methodological approaches have been suggested as potential reasons for the conflicting results. Here, we review current knowledge concerning the molecular pathogenesis of sporadic and hereditary MTC. In particular, we analyze the role of RET polymorphisms in the clinical presentation and prognosis of MTC based on the current literature. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessReview Applications of Next-Generation Sequencing Technologies to Diagnostic Virology
Int. J. Mol. Sci. 2011, 12(11), 7861-7884; doi:10.3390/ijms12117861
Received: 2 September 2011 / Revised: 20 October 2011 / Accepted: 7 November 2011 / Published: 14 November 2011
Cited by 78 | PDF Full-text (207 KB) | HTML Full-text | XML Full-text
Abstract
Novel DNA sequencing techniques, referred to as “next-generation” sequencing (NGS), provide high speed and throughput that can produce an enormous volume of sequences with many possible applications in research and diagnostic settings. In this article, we provide an overview of the many [...] Read more.
Novel DNA sequencing techniques, referred to as “next-generation” sequencing (NGS), provide high speed and throughput that can produce an enormous volume of sequences with many possible applications in research and diagnostic settings. In this article, we provide an overview of the many applications of NGS in diagnostic virology. NGS techniques have been used for high-throughput whole viral genome sequencing, such as sequencing of new influenza viruses, for detection of viral genome variability and evolution within the host, such as investigation of human immunodeficiency virus and human hepatitis C virus quasispecies, and monitoring of low-abundance antiviral drug-resistance mutations. NGS techniques have been applied to metagenomics-based strategies for the detection of unexpected disease-associated viruses and for the discovery of novel human viruses, including cancer-related viruses. Finally, the human virome in healthy and disease conditions has been described by NGS-based metagenomics. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
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Open AccessReview Eighteen Years of Molecular Genotyping the Hemophilia Inversion Hotspot: From Southern Blot to Inverse Shifting-PCR
Int. J. Mol. Sci. 2011, 12(10), 7271-7285; doi:10.3390/ijms12107271
Received: 5 September 2011 / Revised: 8 October 2011 / Accepted: 19 October 2011 / Published: 24 October 2011
Cited by 6 | PDF Full-text (631 KB) | HTML Full-text | XML Full-text
Abstract
The factor VIII gene (F8) intron 22 inversion (Inv22) is a paradigmatic duplicon-mediated rearrangement, found in about one half of patients with severe hemophilia A worldwide. The identification of this prevalent cause of hemophilia was delayed for nine years after [...] Read more.
The factor VIII gene (F8) intron 22 inversion (Inv22) is a paradigmatic duplicon-mediated rearrangement, found in about one half of patients with severe hemophilia A worldwide. The identification of this prevalent cause of hemophilia was delayed for nine years after the F8 characterization in 1984. The aim of this review is to present the wide diversity of practical approaches that have been developed for genotyping the Inv22 (and related int22h rearrangements) since discovery in 1993. The sequence—Southern blot, long distance-PCR and inverse shifting-PCR—for Inv22 genotyping is an interesting example of scientific ingenuity and evolution in order to resolve challenging molecular diagnostic problems. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
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Open AccessReview Malignant Catarrhal Fever: Understanding Molecular Diagnostics in Context of Epidemiology
Int. J. Mol. Sci. 2011, 12(10), 6881-6893; doi:10.3390/ijms12106881
Received: 24 August 2011 / Revised: 22 September 2011 / Accepted: 10 October 2011 / Published: 18 October 2011
Cited by 7 | PDF Full-text (133 KB) | HTML Full-text | XML Full-text
Abstract
Malignant catarrhal fever (MCF) is a frequently fatal disease, primarily of ruminants, caused by a group of gammaherpesviruses. Due to complexities of pathogenesis and epidemiology in various species, which are either clinically-susceptible or reservoir hosts, veterinary clinicians face significant challenges in laboratory [...] Read more.
Malignant catarrhal fever (MCF) is a frequently fatal disease, primarily of ruminants, caused by a group of gammaherpesviruses. Due to complexities of pathogenesis and epidemiology in various species, which are either clinically-susceptible or reservoir hosts, veterinary clinicians face significant challenges in laboratory diagnostics. The recent development of specific assays for viral DNA and antibodies has expanded and improved the inventory of laboratory tests and opened new opportunities for use of MCF diagnostics. Issues related to understanding and implementing appropriate assays for specific diagnostic needs must be addressed in order to take advantage of molecular diagnostics in the laboratory. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessReview The Role of microRNAs in the Biology of Rare Diseases
Int. J. Mol. Sci. 2011, 12(10), 6733-6742; doi:10.3390/ijms12106733
Received: 2 September 2011 / Revised: 21 September 2011 / Accepted: 30 September 2011 / Published: 11 October 2011
Cited by 6 | PDF Full-text (183 KB) | HTML Full-text | XML Full-text
Abstract
Rare diseases (RD) are characterized by low prevalence and affect not more than five individuals per 10,000 in the European population; they are a large and heterogeneous group of disorders including more than 7,000 conditions and often involve all organs and tissues, [...] Read more.
Rare diseases (RD) are characterized by low prevalence and affect not more than five individuals per 10,000 in the European population; they are a large and heterogeneous group of disorders including more than 7,000 conditions and often involve all organs and tissues, with several clinical subtypes within the same disease. Very often information concerning either diagnosis and/or prognosis on many RD is insufficient. microRNAs are a class of small non-coding RNAs that regulate gene expression at the posttranscriptional level by either degrading or blocking translation of messenger RNA targets. Recently, microRNA expression patterns of body fluids underscored their potential as noninvasive biomarkers for various diseases. The role of microRNAs as potential biomarkers has become particularly attractive. The identification of disease-related microRNAs is essential for understanding the pathogenesis of diseases at the molecular level, and is critical for designing specific molecular tools for diagnosis, treatment and prevention. Computational analysis of microRNA-disease associations is an important complementary means for prioritizing microRNAs for further experimental examination. In this article, we explored the added value of miRs as biomarkers in a selected panel of RD hitting different tissues/systems at different life stages, but sharing the need of better biomarkers for diagnostic and prognostic purposes. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)
Open AccessReview Circulating MicroRNAs: Potential Biomarkers for Cancer
Int. J. Mol. Sci. 2011, 12(3), 2055-2063; doi:10.3390/ijms12032055
Received: 21 December 2010 / Revised: 10 February 2011 / Accepted: 10 March 2011 / Published: 22 March 2011
Cited by 35 | PDF Full-text (112 KB) | HTML Full-text | XML Full-text
Abstract
Cancer is the leading cause of death in the world. Development of minimally invasive biomarkers for early detection of cancer is urgently needed to reduce high morbidity and mortality associated with malignancy. MicroRNAs (miRNAs) are small regulatory RNAs that modulate the activity [...] Read more.
Cancer is the leading cause of death in the world. Development of minimally invasive biomarkers for early detection of cancer is urgently needed to reduce high morbidity and mortality associated with malignancy. MicroRNAs (miRNAs) are small regulatory RNAs that modulate the activity of specific mRNA targets and play important roles in a wide range of physiologic and pathologic processes. Recently, miRNAs were found to be dysregulated in a variety of diseases including cancer. Emerging evidence suggests that miRNAs are involved in tumor initiation and progression. Together, the different expression profiles of miRNAs in cancer, and the stability of circulating miRNAs, make them new potentially clinical biomarkers for cancer diagnosis, classification, therapeutic decisions, and prognosis. Full article
(This article belongs to the Special Issue Advances in Molecular Diagnostics)

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