International Journal of Molecular Sciences

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684 KiB

Open AccessArticle

Mass Spectrometry-Based Quantitative Metabolomics Revealed a Distinct Lipid Profile in Breast Cancer Patients

by Yunping Qiu, Bingsen Zhou, Mingming Su, Sarah Baxter, Xiaojiao Zheng, Xueqing Zhao, Yun Yen and Wei Jia

Int. J. Mol. Sci. 2013, 14(4), 8047-8061; https://doi.org/10.3390/ijms14048047 - 12 Apr 2013

Cited by 94 | Viewed by 13275

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. Here, [...] 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)

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716 KiB

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

by Ulrich Andergassen, Simone Hofmann, Alexandra C. Kölbl, Christian Schindlbeck, Julia Neugebauer, Stefan Hutter, Verena Engelstädter, Matthias Ilmer, Klaus Friese and Udo Jeschke

Int. J. Mol. Sci. 2013, 14(1), 1093-1104; https://doi.org/10.3390/ijms14011093 - 8 Jan 2013

Cited by 17 | Viewed by 7708

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 be [...] 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)

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Open AccessArticle

CARD15/NOD2, CD14 and Toll-like 4 Receptor Gene Polymorphisms in Saudi Patients with Crohn’s Disease

by Nahla Azzam, Howaida Nounou, Othman Alharbi, Abedulrahman Aljebreen and Manal Shalaby

Int. J. Mol. Sci. 2012, 13(4), 4268-4280; https://doi.org/10.3390/ijms13044268 - 2 Apr 2012

Cited by 16 | Viewed by 6659

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 been [...] 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)

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Open AccessArticle

Use of the MLPA Assay in the Molecular Diagnosis of Gene Copy Number Alterations in Human Genetic Diseases

by Liborio Stuppia, Ivana Antonucci, Giandomenico Palka and Valentina Gatta

Int. J. Mol. Sci. 2012, 13(3), 3245-3276; https://doi.org/10.3390/ijms13033245 - 8 Mar 2012

Cited by 182 | Viewed by 19333

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 related [...] 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)

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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

by Ali H. Bahkali, Kamel A. Abd-Elsalam, Jian-Rong Guo, Mohamed A. Khiyami and Joseph-Alexander Verreet

Int. J. Mol. Sci. 2012, 13(3), 2951-2964; https://doi.org/10.3390/ijms13032951 - 6 Mar 2012

Cited by 19 | Viewed by 8113

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 of [...] 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)

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Open AccessArticle

Comparison of Culture and Molecular Identification of Bacteria in Chronic Wounds

by Daniel D. Rhoads, Randall D. Wolcott, Yan Sun and Scot E. Dowd

Int. J. Mol. Sci. 2012, 13(3), 2535-2550; https://doi.org/10.3390/ijms13032535 - 23 Feb 2012

Cited by 160 | Viewed by 14323

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 compared. [...] 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)

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Open AccessArticle

Genetic Variability and Phylogeny of High Risk HPV Type 16, 18, 31, 33 and 45 L1 Gene in Greek Women

by Chara Kleio Ntova, Christine Kottaridi, Aikaterini Chranioti, Aris Spathis, Dimitrios Kassanos, Evangelos Paraskevaidis and Petros Karakitsos

Int. J. Mol. Sci. 2012, 13(1), 1-17; https://doi.org/10.3390/ijms13010001 - 22 Dec 2011

Cited by 20 | Viewed by 11770

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 infections [...] 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)

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Open AccessArticle

Molecular Diagnosis of 5α-Reductase Type II Deficiency in Brazilian Siblings with 46,XY Disorder of Sex Development

by Flávia Leme de Calais, Fernanda Caroline Soardi, Reginaldo José Petroli, Ana Letícia Gori Lusa, Roberto Benedito de Paiva e Silva, Andréa Trevas Maciel-Guerra, Gil Guerra-Júnior and Maricilda Palandi de Mello

Int. J. Mol. Sci. 2011, 12(12), 9471-9480; https://doi.org/10.3390/ijms12129471 - 19 Dec 2011

Cited by 5 | Viewed by 7551

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 gene [...] 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)

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Open AccessArticle

Loop-Mediated Amplification Accelerated by Stem Primers

by Olga Gandelman, Rebecca Jackson, Guy Kiddle and Laurence Tisi

Int. J. Mol. Sci. 2011, 12(12), 9108-9124; https://doi.org/10.3390/ijms12129108 - 8 Dec 2011

Cited by 52 | Viewed by 12368

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 performance [...] 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)

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239 KiB

Open AccessArticle

Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer

by Gernot Kriegshäuser, Gerhild Fabjani, Barbara Ziegler, Sabine Zöchbauer-Müller, Adelheid End and Robert Zeillinger

Int. J. Mol. Sci. 2011, 12(12), 8530-8538; https://doi.org/10.3390/ijms12128530 - 29 Nov 2011

Cited by 4 | Viewed by 6636

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 is [...] 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)

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460 KiB

Open AccessArticle

A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays

by Jesper Gantelius, Tarek Bass, Ronald Sjöberg, Peter Nilsson and Helene Andersson-Svahn

Int. J. Mol. Sci. 2011, 12(11), 7748-7759; https://doi.org/10.3390/ijms12117748 - 9 Nov 2011

Cited by 34 | Viewed by 9232

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 [...] Read more.

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)

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113 KiB

Open AccessArticle

Microsatellite Analysis in Multistage Carcinogenesis of Esophageal Squamous Cell Carcinoma from Chongqing in Southern China

by Ming Liu, Feng Zhang, Shen Liu, Wen Zhao, Jing Zhu and Xiaoli Zhang

Int. J. Mol. Sci. 2011, 12(11), 7401-7409; https://doi.org/10.3390/ijms12117401 - 28 Oct 2011

Cited by 5 | Viewed by 6284

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 [...] Read more.

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)

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478 KiB

Open AccessCommunication

Molecular Diagnosis of Analbuminemia: A New Case Caused by a Nonsense Mutation in the Albumin Gene

by Monica Dagnino, Gianluca Caridi, Ueli Haenni, Adrian Duss, Fabienne Aregger, Monica Campagnoli, Monica Galliano and Lorenzo Minchiotti

Int. J. Mol. Sci. 2011, 12(11), 7314-7322; https://doi.org/10.3390/ijms12117314 - 25 Oct 2011

Cited by 10 | Viewed by 7216

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 of [...] 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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Graphical abstract

538 KiB

Open AccessArticle

Screening of Molecular Virulence Markers in Staphylococcus aureus and Pseudomonas aeruginosa Strains Isolated from Clinical Infections

by Ani-Ioana Cotar, Mariana-Carmen Chifiriuc, Sorin Dinu, Marcela Bucur, Carmen Iordache, Otilia Banu, Olguta Dracea, Cristina Larion and Veronica Lazar

Int. J. Mol. Sci. 2010, 11(12), 5273-5291; https://doi.org/10.3390/ijms11125273 - 21 Dec 2010

Cited by 33 | Viewed by 10100

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 new [...] 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)

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Open AccessArticle

An Adsorptive Transfer Technique Coupled with Brdicka Reaction to Reveal the Importance of Metallothionein in Chemotherapy with Platinum Based Cytostatics

by Sona Krizkova, Ivo Fabrik, Dalibor Huska, Vojtech Adam, Petr Babula, Jan Hrabeta, Tomas Eckschlager, Pavel Pochop, Denisa Darsova, Jiri Kukacka, Richard Prusa, Libuse Trnkova and Rene Kizek

Int. J. Mol. Sci. 2010, 11(12), 4826-4842; https://doi.org/10.3390/ijms11124826 - 26 Nov 2010

Cited by 20 | Viewed by 9087

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 chemotherapy [...] 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)

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Review

Jump to: Research

204 KiB

Open AccessReview

Molecular Diagnostic and Pathogenesis of Hereditary Hemochromatosis

by Paulo C. J. L. Santos, Jose E. Krieger and Alexandre C. Pereira

Int. J. Mol. Sci. 2012, 13(2), 1497-1511; https://doi.org/10.3390/ijms13021497 - 1 Feb 2012

Cited by 67 | Viewed by 19807

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 mutant [...] 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)

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Open AccessReview

Molecular Basis of Medullary Thyroid Carcinoma: The Role of RET Polymorphisms

by Lucieli Ceolin, Débora R. Siqueira, Mírian Romitti, Carla V. Ferreira and Ana Luiza Maia

Int. J. Mol. Sci. 2012, 13(1), 221-239; https://doi.org/10.3390/ijms13010221 - 27 Dec 2011

Cited by 35 | Viewed by 9924

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 the [...] 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)

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207 KiB

Open AccessReview

Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

by Luisa Barzon, Enrico Lavezzo, Valentina Militello, Stefano Toppo and Giorgio Palù

Int. J. Mol. Sci. 2011, 12(11), 7861-7884; https://doi.org/10.3390/ijms12117861 - 14 Nov 2011

Cited by 211 | Viewed by 27137

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 applications [...] 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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Eighteen Years of Molecular Genotyping the Hemophilia Inversion Hotspot: From Southern Blot to Inverse Shifting-PCR

by Liliana C. Rossetti, Claudia P. Radic, Miguel M. Abelleyro, Irene B. Larripa and Carlos D. De Brasi

Int. J. Mol. Sci. 2011, 12(10), 7271-7285; https://doi.org/10.3390/ijms12107271 - 24 Oct 2011

Cited by 25 | Viewed by 11789

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 the [...] 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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133 KiB

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Malignant Catarrhal Fever: Understanding Molecular Diagnostics in Context of Epidemiology

by Hong Li, Cristina W. Cunha and Naomi S. Taus

Int. J. Mol. Sci. 2011, 12(10), 6881-6893; https://doi.org/10.3390/ijms12106881 - 18 Oct 2011

Cited by 36 | Viewed by 9519

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 diagnostics. [...] 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)

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The Role of microRNAs in the Biology of Rare Diseases

by Marco Salvatore, Armando Magrelli and Domenica Taruscio

Int. J. Mol. Sci. 2011, 12(10), 6733-6742; https://doi.org/10.3390/ijms12106733 - 11 Oct 2011

Cited by 14 | Viewed by 7885

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, with [...] 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)

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Circulating MicroRNAs: Potential Biomarkers for Cancer

by De-Cai Yu, Qing-Guo Li, Xi-Wei Ding and Yi-Tao Ding

Int. J. Mol. Sci. 2011, 12(3), 2055-2063; https://doi.org/10.3390/ijms12032055 - 22 Mar 2011

Cited by 87 | Viewed by 11627

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 of [...] 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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