Integrity Of Genomic DNA Research Articles

Hepatoprotective effect of dandelion (Taraxacum officinale) against induced chronic liver cirrhosis

Recently, it was found that dandelion flower extract may fight free radicals (chemical by-products known to damage DNA). Health care providers clinically use dandelion root to promote liver detoxification. Dandelion leaves water extract was investigated for hepatoprotective activity against carbon tetrachloride induced liver damage. The aim of the study was to evaluate the efficacy of the hepatoprotective activity of dandelion leaves water extract against carbon tetrachloride (CCl4) intoxication in liver albino rats. Carbon tetrachloride was used as hepatotoxic agent and dandelion leaves water extract was used as a probable hepato-protective agent. Various biochemical parameters were studied to evaluate the hepatoprotective activity of dandelion leaves water extract. The study was also supported by histopathology of liver sections and DNA extraction of the studied animals to investigate the genomic DNA integrity. The results revealed that the serum markers in rats treated with CCl4 recorded elevated concentration, indicating severe hepatic damage by CCl4, while the blood samples from dandelion treated animals showed significant reduction in the serum markers, indicating the effect of the plant extract in restoring the normal functional ability of the hepatocytes. The present study revealed that dandelion leaf water extract could afford a significant protection against CCl4 induced hepatocellular injury. Key words: Taraxacum officinale, dandelion leaf water extract (DLWE), hepatoprotective activity, carbon tetrachloride (CCl4) damage.

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage.

Integrity of bacterial genomic DNA after autoclaving: possible implications for horizontal gene transfer and clinical waste management

Current autoclaving practices are designed to kill bacteria. Little is known about the effect of autoclaving on the integrity of bacterial genomic DNA. An experiment was performed to examine the effect of standard autoclaving on the integrity of bacterial DNA, employing polymerase chain reaction (PCR) as an indicator of DNA integrity. Amplifiable PCR signal was observed at t = 10, 20 and 30 min autoclaving time for Pseudomonas aeruginosa NCTC 10662; at t = 10, 20, 30 and 40 min for Salmonella Nottingham NCTC 7832; and at t = 10 and 20 min for Escherichia coli NCTC 9001. Careful consideration should therefore be given to residual molecular artefacts in future risk and environmental impact assessments, where the legacy of residual genomic DNA from dead bacterial and higher organisms may act as a potential reservoir, thereby feeding horizontal gene transfer scenarios in viable cells with potential hazardous genes of virulence, persistence or antibiotic resistance characteristics.

Luminescent oligonucleotide-based detection of enzymes involved with DNA repair

DNA repair processes are responsible for upholding the integrity and stability of genomic DNA. Consequently, the development of analytical assays to monitor enzymes involved with DNA repair pathways is of great interest in a variety of disciplines, including biochemistry, cell biology and biotechnology. In this perspective, we highlight examples of the development of luminescent assays for the detection of enzymes involved with DNA repair that have been reported in the last several years.

Abstract 5542: Gene expression and genomic DNA stabilization in whole blood stored at room temperature

Abstract Genomic and mitochondrial DNA and gene expression profiles from blood samples are increasingly being used to diagnose specific diseases, including cancer, monitor disease progression and assess patient responses to medical treatment. However, such applications require reliable preservation of total RNA and DNA in patient samples during collection, transport and storage. Numerous agents threaten DNA and RNA profiles in collected blood samples, including nucleolytic attack, oxidative damage and hydrolysis. Such damage can severely disrupt genotyping and DNA sequencing efforts. Transcription profiles are highly dynamic and can change rapidly during and after blood collection using current methods, potentially affecting interpretation of the expression analysis, and ultimately lead to inadequate treatment options. In this study, we compare freezer storage with room temperature stabilization formulations, DNAgard Blood and PAXgene Blood DNA, in their capacities to preserve genomic DNA integrity in human whole blood for 8 months. In a separate analysis, we compare freezer storage with room temperature stabilization formulations, PAXgene Blood RNA and Biomatrica formulation DRG, in their capacities to stabilize RNA in whole blood samples. We assess changes in the relative expression of a panel of 34000 genes in whole blood specimens over the course of 7 days using RT-qPCR and the Human HT-12 Bead Array (Illumina). Our results demonstrate that room temperature blood storage is a valid alternative to cold-storage for preserving gDNA for more than a year, and RNA for at least 7 days. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5542. doi:1538-7445.AM2012-5542

Polymorphisms in the XPG gene and risk of gastric cancer in Chinese populations

DNA repair genes play an important role in maintaining stability and integrity of genomic DNA. Polymorphisms in nucleotide excision repair genes may cause variations in DNA repair capacity phenotype and thus contribute to cancer risk. In this case-control study of 1,125 gastric cancer cases and 1,196 cancer-free controls, we investigated the association between three functional single nucleotide polymorphisms (SNPs, rs2296147T > C, rs2094258C > T and rs873601G > A) in the xeroderma pigmentosum group G (XPG) gene and gastric cancer risk. We used the Taqman assays to genotype these three SNPs and logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). We found that only the rs873601A variant genotypes were associated with a significant higher risk for gastric adenocarcinoma (adjusted OR = 1.30, 95% CI = 1.03-1.64 for AA vs. GG and adjusted OR = 1.23, 95% CI = 1.01-1.49 for AA vs. GG/AG). Stratification analysis indicated that this risk was more pronounced in subgroups of older age (>59 years), males, ever-smokers, and patients with NGCA. All these were not found for the other two SNPs (rs2296147T > C and rs2094258C > T). We then performed expression analysis using gastric cancer adjacent normal tissues from 141 patients and found that the A variant allele was associated with non-significantly reduced expression of XPG mRNA (P(trend) = 0.107). Further analysis using mRNA expression data from the HapMap suggested that the A allele was associated with significantly reduced expression of XPG mRNA in normal cell lines for 45 Chinese (P(trend) = 0.003) as well as for 261 subjects with different ethnicities (P(trend) = 0.001). These support the hypothesis that functional XPG variants may contribute to the risk of gastric cancer. Larger studies with different ethnic populations are warranted to validate our findings.

Room temperature stabilization of gene transcripts and genomic DNA in whole blood

Genomic and mitochondrial DNA and gene expression profiles from blood samples are increasingly being used to diagnose specific diseases, monitor disease progression and assess patient responses to medical treatment. However, such applications require reliable preservation of total RNA and DNA in patient samples during collection, transport and storage. Numerous agents threaten DNA and RNA profiles in collected blood samples, including nucleolytic attack, oxidative damage and hydrolysis. Such damage can severely disrupt genotyping and DNA sequencing efforts. Transcription profiles are highly dynamic and can change rapidly during and after blood collection using current methods, potentially affecting interpretation of the expression analysis. In this study, we compare DNAgard Blood, PAXgene Blood DNA and freezer storage in their capacities to preserve genomic DNA integrity in human whole blood for 6 months. In a separate analysis, we compare RNA stabilization properties of PAXgene Blood RNA, freezer storage and a novel stabilization formulation by assessing changes in the relative expression of a panel of genes in whole blood specimens over the course of 7 days using qRT‐PCR and the Human Common Cytokines PCR Array (SABiosciences). Our results demonstrate the importance of selecting an appropriate blood storage method to obtain reliable gene expression and genomic data.

Damage Escape and Repair in DriedChroococcidiopsisspp. from Hot and Cold Deserts Exposed to Simulated Space and Martian Conditions

The cyanobacterium Chroococcidiopsis, overlain by 3 mm of Antarctic sandstone, was exposed as dried multilayers to simulated space and martian conditions. Ground-based experiments were conducted in the context of Lichens and Fungi Experiments (EXPOSE-E mission, European Space Agency), which were performed to evaluate, after 1.5 years on the International Space Station, the survival of cyanobacteria (Chroococcidiopsis), lichens, and fungi colonized on Antarctic rock. The survival potential and the role played by protection and repair mechanisms in the response of dried Chroococcidiopsis cells to ground-based experiments were both investigated. Different methods were employed, including evaluation of the colony-forming ability, single-cell analysis of subcellular integrities based on membrane integrity molecular and redox probes, evaluation of the photosynthetic pigment autofluorescence, and assessment of the genomic DNA integrity with a PCR-based assay. Desiccation survivors of strain CCMEE 123 (coastal desert, Chile) were better suited than CCMEE 134 (Beacon Valley, Antarctica) to withstand cellular damage imposed by simulated space and martian conditions. Exposed dried cells of strain CCMEE 123 formed colonies, maintained subcellular integrities, and, depending on the exposure conditions, also escaped DNA damage or repaired the induced damage upon rewetting.

Povidone–iodine application induces corneal cell death through fixation

Background/aimsPovidone–iodine (PI) is commonly used as a preoperative disinfectant; however, it has been shown to be cytotoxic. The present study was performed to investigate the mechanism by which PI causes...

A functional analysis of Kluyveromyces lactis glutathione reductase

Glutathione reductase (GLR) null mutants of the yeast Kluyveromyces lactis, a model eukaryotic respiratory cell, were created and phenotypically analysed. We found that cells lacking GLR show decreased resistance to oxidative stress and higher levels of reactive oxygen species and catalase activity than the wild-type strain. However, glutathione redox levels (GSH : GSSG ratio) were similar in the DeltaKlglr1 mutant and wild-type strains. The thioredoxin-thioredoxin reductase system is proposed as an alternative system for maintaining the GSH : GSSG ratio in the DeltaKlglr1 mutant. The involvement of GLR in glucose metabolism in K. lactis is suggested by the improved growth on glucose caused by the DeltaKlglr1 mutation and by the increased GLR activity in the DeltaKlgcr1 strain, KlGcr1 being a transcriptional activator of glycolytic genes. We also studied the subcellular location of GLR in K. lactis, showing that it is present in mitochondria; however, the DeltaKlglr1 mutation does not affect mitochondrial morphology. Genomic DNA integrity and life span are also unaffected by the DeltaKlglr1 mutation, at least under the conditions tested in this study.

Alkylation DNA damage in combination with PARP inhibition results in formation of S-phase-dependent double-strand breaks

The combination of poly(ADP-ribose)polymerase (PARP) inhibitors and alkylating agents is currently being investigated in cancer therapy clinical trials. However, the DNA lesions producing the synergistic cell killing effect in tumors are not fully understood. Treatment of human and mouse fibroblasts with the monofunctional DNA methylating agent methyl methanesulfonate (MMS) in the presence of a PARP inhibitor has been shown to trigger a cell cycle checkpoint response. Among other changes, this DNA damage response to combination treatment includes activation of ATM/Chk2 and phosphorylation of histone H2A.X. These changes are consistent with DNA double-strand break (DSB) formation during the response, but the measurement of DSBs has not been addressed. Such DSB evaluation is important in understanding this DNA damage response because events other than DSB formation are known to lead to ATM/Chk2 activation and H2A.X phosphorylation. Here, we examined the structural integrity of genomic DNA after the combined treatment of cells with MMS and a PARP inhibitor, i.e., exposure to a sub-lethal dose of MMS in the presence of the PARP inhibitor 4-amino-1,8-napthalimide (4-AN). We used pulsed field gel electrophoresis (PFGE) for measurement of DSBs in both human and mouse embryonic fibroblasts, and flow cytometry to follow the phosphorylated form of H2A.X (γ-H2A.X). The results indicate that DSBs are formed with the combination treatment, but not following treatment with either agent alone. Our data also show that formation of γ-H2A.X correlates with PARP-1-expressing cells in S-phase of the cell cycle. The observations support the model that persistence of PARP-1 at base excision repair intermediates, as cells move into S-phase, leads to DSBs and the attendant checkpoint responses.

Innovative technology to stabilize DNA at room temperature in tissue and cells

Current methods for storing and shipping human and animal tissue or cells for clinical, forensic and biomedical research needs are costly and can be insufficient for reliable molecular diagnostics requiring preservation of high‐quality genomic DNA. Animal tissues are commonly shipped on dry‐ice or in liquid nitrogen which are costly and often not practical for the collection of samples in the field. Another common method of tissue preservation, formalin fixation followed by paraffin embedding (FFPE), is also impractical for field collection and often results in damage to nucleic acids. We have combined synthetic chemistry with the natural principles of anhydrobiosis (the capacity of some organisms to protect their molecular integrity and survive extreme desiccation for hundreds of years) to stabilize genomic DNA in animal tissue and mammalian tissue culture cells at room temperature. DNAgard™, a novel liquid formulation preserves the integrity of genomic DNA in tissue and cells stored for at least 60 days at room temperature. DNA yield is comparable to frozen controls. The data demonstrates that genomic DNA integrity is preserved in samples stabilized with DNAgard during extended exposure to extreme heat (50°C), with DNA remaining intact for 1 month in tissue samples and for two months in cultured cell samples. Tests were designed to explore the capacity of this product as a shipping application, and results demonstrate that DNAgard™ preserves the integrity of genomic DNA in animal tissue and cell samples when exposed to extreme fluctuations in temperature.

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (beta-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (β-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart

The integrity of genomic DNA is continuously challenged by the presence of DNA base lesions or DNA strand breaks. Here we report the identification of a new DNA damage response protein, SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), which is a member of the SNF2 family and is mutated in Schimke immunoosseous dysplasia (SIOD). We demonstrate that SMARCAL1 directly interacts with Replication protein A (RPA) and is recruited to sites of DNA damage in an RPA-dependent manner. SMARCAL1-depleted cells display sensitivity to DNA-damaging agents that induce replication fork collapse, and exhibit slower fork recovery and delayed entry into mitosis following S-phase arrest. Furthermore, SIOD patient fibroblasts reconstituted with SMARCAL1 exhibit faster cell cycle progression after S-phase arrest. Thus, the symptoms of SIOD may be caused, at least in part, by defects in the cellular response to DNA replication stress.

NDRG4 Is Required for Cell Cycle Progression and Survival in Glioblastoma Cells

NDRG4 is a largely unstudied member of the predominantly tumor suppressive N-Myc downstream-regulated gene (NDRG) family. Unlike its family members NDRG1-3, which are ubiquitously expressed, NDRG4 is expressed almost exclusively in the heart and brain. Given this tissue-specific expression pattern and the established tumor suppressive roles of the NDRG family in regulating cellular proliferation, we investigated the cellular and biochemical functions of NDRG4 in the context of astrocytes and glioblastoma multiforme (GBM) cells. We show that, in contrast to NDRG2, NDRG4 expression is elevated in GBM and NDRG4 is required for the viability of primary astrocytes, established GBM cell lines, and both CD133(+) (cancer stem cell (CSC)-enriched) and CD133(-) primary GBM xenograft cells. While NDRG4 overexpression has no effect on cell viability, NDRG4 knockdown causes G(1) cell cycle arrest followed by apoptosis. The initial G(1) arrest is associated with a decrease in cyclin D1 expression and an increase in p27(Kip1) expression, and the subsequent apoptosis is associated with a decrease in the expression of XIAP and survivin. As a result of these effects on cell cycle progression and survival, NDRG4 knockdown decreases the tumorigenic capacity of established GBM cell lines and GBM CSC-enriched cells that have been implanted intracranially into immunocompromised mice. Collectively, these data indicate that NDRG4 is required for cell cycle progression and survival, thereby diverging in function from its tumor suppressive family member NDRG2 in astrocytes and GBM cells.

Tardigrade Resistance to Space Effects: First Results of Experiments on the LIFE-TARSE Mission on FOTON-M3 (September 2007)

The Tardigrade Resistance to Space Effects (TARSE) project, part of the mission LIFE on FOTON-M3, analyzed the effects of the space environment on desiccated and active tardigrades. Four experiments were conducted in which the eutardigrade Macrobiotus richtersi was used as a model species. Desiccated (in leaf litter or on paper) and hydrated tardigrades (fed or starved) were flown on FOTON-M3 for 12 days in September 2007, which, for the first time, allowed for a comparison of the effects of the space environment on desiccated and on active animals. In this paper, we report the experimental design of the TARSE project and data on tardigrade survival. In addition, data on survival, genomic DNA integrity, Hsp70 and Hsp90 expressions, antioxidant enzyme contents and activities, and life history traits were compared between hydrated starved tardigrades flown in space and those maintained on Earth as a control. Microgravity and radiation had no effect on survival or DNA integrity of active tardigrades. Hsp expressions between the animals in space and the control animals on Earth were similar. Spaceflight induced an increase of glutathione content and its related enzymatic activities. Catalase and superoxide dismutase decreased with spaceflight, and thiobarbituric acid reactive substances did not change. During the flight mission, tardigrades molted, and females laid eggs. Several eggs hatched, and the newborns exhibited normal morphology and behavior.

The Schizosaccharomyces pombe Checkpoint Kinases Chk1 and Cds1 Are Important for Cell Survival in Response to Cisplatin

BackgroundDNA damage checkpoints insure that the integrity of genomic DNA is faithfully maintained throughout the eukaryotic cell cycle. In the presence of damaged DNA, checkpoints are triggered to delay cell cycle progression to allow for DNA repair. In fission yeast, the kinases Chk1 and Cds1 are major components of these DNA damage checkpoint pathways. Both Chk1 and Cds1 are important for viability in the presence of several DNA damaging agents. In this study we hypothesized that Chk1 and Cds1 play a vital role in fission yeast cells ability to survive exposure to the DNA damaging agent cisplatin. Cisplatin is a potent chemotherapeutic drug that interacts with DNA and causes both inter- and intra-strand DNA cross-links.Methodology/Principal FindingsHere, we demonstrated that treatment with cisplatin in fission yeast causes a Chk1-dependent DNA damage signal. chk1 − cells were sensitive to cisplatin and Chk1 was phosphorylated in response to cisplatin treatment. We also showed that a Chk1-dependent DNA damage checkpoint pathway is activated in a dose-dependent fashion in cells challenged with cisplatin. Furthermore the Cds1 checkpoint kinase was also important for viability in cisplatin challenged cells. In cds1 − cells, cisplatin treatment reduced cell viability and this phenotype was exacerbated in a chk1 −/cds1 − background.Conclusions/SignificanceThus, we conclude that the concerted effort of both major checkpoint kinases in fission yeast, Chk1 and Cds1, protect cells from cisplatin induced DNA damage. These observations are significant because they suggest that various classes of inter-strand crosslinking agents may generate slightly different lesions as work by others did not observe loss of viability in cds1 − cells treated with other crosslinking agents like nitrogen mustard.

FL-DNA approach for noninvasive early diagnosis of colorectal cancer in FOBT-screened patients

11062 Background: A promising approach for the early diagnosis of colorectal cancer (CRC) is the evaluation of genomic DNA integrity (FL-DNA) extracted from stool. Pilot and confirmatory studies carried out by our group have shown that, thanks to its diagnostic accuracy, this molecular assay could be a useful tool for the non-invasive, early diagnosis of CRC. The aim of the present study was to verify whether the FL-DNA method could represent a valid alternative to the fecal occult blood test (FOBT) or whether it could be used alongside FOBT in screening programs to unmask false FOBT positives and spare patients from unnecessary colonoscopy. For this purpose, stool samples were collected from all individuals participating in the FOBT (OC-Sensor, Alpha Wassermann) Regional Screening Program run by the Cancer Prevention Unit of Morgagni-Pierantoni Hospital in Forli. Methods: The program recruited subjects of either sex aged 50 to 69 years. Of the 560 individuals with FOBT-positive stool subjected to colonoscopy, 26 were diagnosed with adenocarcinoma, 264 with high-grade adenoma and 54 with low-grade adenoma. More than one third (216) of the group had only benign disease (hemorrhoids, diverticulitis, inflammation, etc), hyperplastic polyps or nothing. The integrity of fecal DNA was analyzed blindly by the FL-DNA test on the same specimen used for the occult blood determination. Results: Using a cut-off of 10 ng, the molecular analysis detected over 90% of the colorectal cancers and about 50% of the high- and low-grade adenomas. In particular, diagnostic accuracy was similar for lesions of any localization, level of dysplasia and histopathological characteristic, and higher for lesions > 2 cm and multiple lesions. The test also confirmed its capacity to identify colorectal cancer in asymptomatic individuals. Conclusions: FL-DNA represents a valid alternative to or support for approaches currently used in screening programs. A more in depth DNA stool evaluation in negative FOBT individuals could reveal the test's usefulness in unmasking colorectal tumors and adenomas missed by FOBT. No significant financial relationships to disclose.

Development and optimization of an internally controlled dried blood spot assay for surveillance of human immunodeficiency virus type-1 drug resistance

We present the evaluation of a methodology for the genotypic assessment of human immunodeficiency virus type-1 (HIV-1) drug resistance, optimized for use with dried blood spots (DBS). The ability to generate HIV-1 protease (PR) and reverse transcriptase (RT) contiguous amplicons and nucleotide sequences from DBS was evaluated. Different collection matrices and extraction methodologies were compared. The relative subtype sensitivity of the amplification strategy was assessed using a comprehensive panel of plasmids representing A-H subtypes. A panel of DBS and plasma specimens was subjected to HIV genotyping. Sequences generated from each sample type were compared. Extensive replicate testing revealed most sensitivity with the use of 903 filter paper and silica/guanidine extraction, which had an estimated 95% inclusivity endpoint of 1542 proviral copies/mL, as compared with 21 573 proviral copies/mL for the FTA system. All HIV-1 group M subtypes analysed-with the exception of subtypes A2, AE, AG, F and H-had a relative sensitivity of </=10 plasmid copies/PCR reaction. The PCR was multiplexed to include amplification of a human housekeeping gene to monitor the integrity of the human genomic DNA. Using a panel of clinical samples, we demonstrated the ability to amplify and sequence from 83% (n = 10) in the PR region and 100% (n = 12) in the RT region, of samples with detectable viral load. All specimens with an HIV-1 RNA load >/=1000 copies/mL were successfully amplified and sequenced. Twelve specimens had pol genotyping from both plasma and DBS samples. Sequence analysis and drug resistance interpretation revealed that 10 (83%) provided concordant drug resistance interpretation. Our results demonstrate that the technique is appropriate for surveillance of drug resistance in untreated individuals and those with virological failure on therapy.