The processive kinetics of gene conversion in bacteria.

Gene conversion, non-reciprocal transfer from one homologous sequence to another, is a major force in evolutionary dynamics, promoting co-evolution in gene families and maintaining similarities between repeated genes. However, the properties of the transfer - where it initiates, how far it proceeds and how the resulting conversion tracts are affected by mismatch repair - are not well understood. Here, we use the duplicate tuf genes in Salmonella as a quantitatively tractable model system for gene conversion. We selected for conversion in multiple different positions of tuf, and examined the resulting distributions of conversion tracts in mismatch repair-deficient and mismatch repair-proficient strains. A simple stochastic model accounting for the essential steps of conversion showed excellent agreement with the data for all selection points using the same value of the conversion processivity, which is the only kinetic parameter of the model. The analysis suggests that gene conversion effectively initiates uniformly at any position within a tuf gene, and proceeds with an effectively uniform conversion processivity in either direction limited by the bounds of the gene.

Uneven distribution of human papillomavirus 16 in cervical carcinoma in situ and squamous cell carcinoma in older females: A retrospective database study.

Human papillomavirus (HPV) 16 is the dominant cofactor in cervical cancer development. The present report investigated the age-specific prevalence of HPV16 in cervical carcinoma in situ (CIS) in females attending organised cervical cancer screening. A retrospective observational study was performed based on individual data from two databases. A total of 162 females aged between 20 and 65 years from Uppsala County, Sweden with CIS and an HPV test conducted between 2010 and 2011, preceding or concomitant to CIS diagnosis, were included. Females with cervical squamous cell carcinoma (SCC; n=35) were used for comparison. In total, 96% (n=156) of females with CIS were positive for high-risk HPV; HPV16 was the most prevalent (44.5%), followed by HPV33/52/58 (19.5%), HPV31 (13.1%) and HPV18/45 (9.5%). HPV16 was most frequently detected in females with CIS aged between 20 and 29 years (73.6%) and least frequently detected in those aged between 50 and 65 years (33.3%), with a statistically significant age-specific difference (P=0.001). Among the HPV16-positive females, multiple infections were most frequent in the younger age groups. The prevalence of HPV16 in females with CIS decreased with age, whereas a high prevalence of HPV16 remained in females with SCC. These results may indicate that HPV16 has increased oncogenic potential in older females.

Primary high-risk HPV screening for cervical cancer in post-menopausal women.

OBJECTIVE: The present study was conducted to examine the value of screening for high-risk HPV in post-menopausal women. METHODS: A cohort of post-menopausal women (n=2113), age range 55-76 years, from Uppsala County, Sweden, were offered testing for both high-risk HPV and a Pap smear in the gynaecological screening during 2008-2010. For the HPV test the cervical smear sample was applied to a filter paper matrix, an indicating FTA elute card and HPV typing performed using a real-time PCR assay. Histological verified CIN2+ lesion was used as an end-point measurement. RESULTS: High-risk HPV were found in 6.2% (95% CI 5.2-7.3%) of the women (n=130) and 22% (95% CI 14-32%) (n=17) of these had CIN2+ lesions based on histology. The Pap smear taken in conjunction with the HPV test was abnormal in 9.7% (95% CI 5.7-16.3%) (n=12) of HPV positive women. Among HPV positive women with an abnormal Pap smear, the frequency of histology verified CIN2+ lesions was 67% (95% CI 38-86%) (n=8), as compared to 14% (95% CI 7-24%) (n=9) in HPV positive women with a normal smear. The prevalence of HPV16 in CIN2+ lesions (29%, 95% CI 22-37%) in post-menopausal women was less than half of previous estimates in pre-menopausal women from this population. CONCLUSIONS: Most histological CIN2+ lesions in post-menopausal women are not recognized by a single Pap smear. A large fraction of pre-invasive cervical cancer cases in post-menopausal women result from infections by HPV types not included in the present vaccine formulas.

Type-specific detection of high-risk human papillomavirus (HPV) in self-sampled cervicovaginal cells applied to FTA elute cartridge.

BACKGROUND: Most procedures for self-sampling of cervical cells are based on liquid-based media for transportation and storage. An alternative is to use a solid support, such as dry filter paper media. OBJECTIVES: To evaluate if self-sampling of cervicovaginal fluid using a cytobrush (Viba-brush; Rovers Medical Devices B.V., Oss, The Netherlands) and a solid support such as the Whatman Indicating FTA Elute cartridge (GE Healthcare, United Kingdom) can be used for reliable typing of human papillomavirus (HPV), as compared to cervical samples obtained by a physician using a cytobrush and the indicating FTA Elute Micro card and biopsy analysis. STUDY DESIGN: A total of 50 women with a previous high-risk (HR) HPV positive test were invited to perform self-sampling using the Viba-brush and the FTA cartridge and thereafter a physician obtained a cervical sample using the cytobrush and a FTA card, together with a cervical biopsy for histology and HPV typing. Detection of HR-HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 was performed using three multiplex real-time polymerase chain reaction (PCR) assays. RESULT: All samples contained sufficient amounts of genomic DNA and the self-samples yielded on average 3.5 times more DNA than those obtained by the physician. All women that were positive for HR-HPV in the biopsy sample also typed positive both by self-sampling and physician-obtained sampling. For women with a histological diagnosis of cervical intraepithelial neoplasia grades 2-3 (CIN 2-3) all three HPV samples showed 100% concordance. A higher number of women were HPV positive by self-sampling than by physician-obtained sampling or by biopsy analysis. CONCLUSION: The Viba-brush and the FTA cartridge are suitable for self-sampling of vaginal cells and subsequent HR-HPV typing.

Quantitative evaluation of p16(INK4a) promoter methylation using pyrosequencing in de novo diffuse large B-cell lymphoma.

The p16(INK4a) tumor suppressor gene can be inactivated by a variety of events including promoter hypermethylation. In diffuse large B-cell lymphoma (DLBCL), p16(INK4a) methylation has been associated with advanced disease stage and higher IPI. The prognostic impact of p16(INK4a) methylation in DLBCL remains unclear; however, it has been suggested to correlate with inferior outcome. To further investigate the clinical impact of p16(INK4a) methylation in DLBCL, promoter methylation of this gene was assessed quantitatively by pyrosequencing. Forty-two of 113 (37%) DLBCL patients with methylation level above 5% were categorized as methylated and subsequently divided into low, intermediate and high methylation categories. Overall, no association was shown between the extent of p16(INK4a) methylation and patients' clinical characteristics, except disease stage (P=0.049). Moreover, we could not reveal any impact of p16(INK4a) methylation on lymphoma-specific survival. Although >25% of p16(INK4a) methylation correlated with a better progression-free survival (P=0.048) in patients <65 years old, the significance of this finding, if any, needs to be further investigated. In conclusion, our finding questions the role of p16(INK4a) promoter methylation as a negative prognostic factor in DLBCL.

KRAS analysis in colorectal carcinoma: analytical aspects of Pyrosequencing and allele-specific PCR in clinical practice.

BACKGROUND: Epidermal growth factor receptor inhibitor therapy is now approved for treatment of metastatic colorectal carcinomas (CRC) in patients with tumors lacking KRAS mutations. Several procedures to detect KRAS mutations have been developed. However, the analytical sensitivity and specificity of these assays on routine clinical samples are not yet fully characterised. METHODS: The practical aspects and clinical applicability of a KRAS-assay based on Pyrosequencing were evaluated in a series of 314 consecutive CRC cases submitted for diagnostic KRAS analysis. The performance of Pyrosequencing compared to allele-specific, real-time PCR was then explored by a direct comparison of CE-IVD-marked versions of Pyrosequencing and TheraScreen (DxS) KRAS assays for a consecutive subset (n = 100) of the 314 clinical CRC samples. RESULTS: Using Pyrosequencing, 39% of the 314 CRC samples were found KRAS-mutated and several of the mutations (8%) were located in codon 61. To explore the analytical sensitivity of the Pyrosequencing assay, mutated patient DNA was serially diluted with wild-type patient DNA. Dilutions corresponding to 1.25-2.5% tumor cells still revealed detectable mutation signals. In clinical practice, our algorithm for KRAS analysis includes a reanalysis of samples with low tumor cell content (< 10%, n = 56) using an independent assay (allele-specific PCR, DxS). All mutations identified by Pyrosequencing were then confirmed and, in addition, one more mutated sample was identified in this subset of 56 samples. Finally, a direct comparison of the two technologies was done by re-analysis of a subset (n = 100) of the clinical samples using CE-IVD-marked versions of Pyrosequencing and TheraScreen KRAS assays in a single blinded fashion. The number of samples for which the KRAS codon 12/13 mutation status could be defined using the Pyrosequencing or the TheraScreen assay was 94 and 91, respectively, and both assays detected the same number of codon 12 and 13 mutations. CONCLUSIONS: KRAS mutation detection using Pyrosequencing was evaluated on a consecutive set of clinical CRC samples. Pyrosequencing provided sufficient analytical sensitivity and specificity to assess the mutation status in routine formalin-fixed CRC samples, even in tissues with a low tumor cell content.

Use of FTA card for dry collection, transportation and storage of cervical cell specimen to detect high-risk HPV.

BACKGROUND: The FTA elute micro card, which enable the collection, transport, and archiving of DNA could be an attractive alternative to a liquid based collection system for detection of human papillomavirus (HPV). OBJECTIVES: To develop a method based on the FTA elute micro card for dry collection of cervical epithelial cell samples, suitable for subsequent PCR-based HPV testing. STUDY DESIGN: The method was evaluated by a comparison of the DNA collected by cytobrush and the regular FTA elute micro card from 50 cervical cell samples. The method was then used to estimate the DNA amount in 1040 samples applied to the indicating FTA elute micro card. RESULT: The agreement in HPV positivity between the cytobrush and FTA samples (94%) was excellent (kappa=0.88, 95% CI 0.748-1). All the 1040 samples on the indicating FTA card had sufficient amounts of genomic DNA (>10 copies of a single copy gene) to be suitable for HPV typing. In 53 of the 1040 women the day in the menstrual cycle was noted, and the copy number during follicular phase day 9-13 was found to be statistically significantly lower than for the other three stages in the menstrual cycle (day 4-8, 14, >14) and during menopause. CONCLUSION: The indicating FTA elute micro card represents a suitable medium for collection of cervical cell samples, although follow-up studies are needed to verify the detection of low frequency HPV types.

TP53 mutations predict for poor survival in de novo diffuse large B-cell lymphoma of germinal center subtype.

Presence of TP53 mutations has been associated with poor prognosis in diffuse large B-cell lymphoma (DLBCL), although this has remained controversial. The TP53 codon 72 polymorphism has shown negative impact on cancer survival, but this has not been analyzed in DLBCL. Furthermore, the MDM2 SNP309 has been associated with earlier age of onset in DLBCL. Here, we investigated the clinical impact of TP53 mutations, MDM2 SNP309 and TP53 codon 72 polymorphisms on survival in DLBCL of germinal center (GC) and non-GC subtypes. Thirteen of the 102 (12.7%) patients displayed TP53 mutations. Overall, TP53 mutations had a significant effect on lymphoma-specific survival (LSS, P=0.009) and progression-free survival (PFS, P=0.028). In particular, inferior survival was observed in TP53-mutated DLBCLs of GC subtype (LSS, P=0.002 and PFS, P=0.006). Neither MDM2 SNP309 nor the TP53 codon 72 polymorphism had an impact on age of onset or survival. Altogether, our data suggests that TP53 mutations are associated with poor outcome in GC-DLBCL patients.

Adipose tissue inflammation and increased ceramide content characterize subjects with high liver fat content independent of obesity.

OBJECTIVE: We sought to determine whether adipose tissue is inflamed in individuals with increased liver fat (LFAT) independently of obesity. RESEARCH DESIGN AND METHODS: A total of 20 nondiabetic, healthy, obese women were divided into normal and high LFAT groups based on their median LFAT level (2.3 +/- 0.3 vs. 14.4 +/- 2.9%). Surgical subcutaneous adipose tissue biopsies were studied using quantitative PCR, immunohistochemistry, and a lipidomics approach to search for putative mediators of insulin resistance and inflammation. The groups were matched for age and BMI. The high LFAT group had increased insulin (P = 0.0025) and lower HDL cholesterol (P = 0.02) concentrations. RESULTS: Expression levels of the macrophage marker CD68, the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1alpha, and plasminogen activator inhibitor-1 were significantly increased, and those of peroxisome proliferator-activated receptor-gamma and adiponectin decreased in the high LFAT group. CD68 expression correlated with the number of macrophages and crown-like structures (multiple macrophages fused around dead adipocytes). Concentrations of 154 lipid species in adipose tissue revealed several differences between the groups, with the most striking being increased concentrations of triacylglycerols, particularly long chain, and ceramides, specifically Cer(d18:1/24:1) (P = 0.01), in the high LFAT group. Expression of sphingomyelinases SMPD1 and SMPD3 were also significantly increased in the high compared with normal LFAT group. CONCLUSIONS: Adipose tissue is infiltrated with macrophages, and its content of long-chain triacylglycerols and ceramides is increased in subjects with increased LFAT compared with equally obese subjects with normal LFAT content. Ceramides or their metabolites could contribute to adverse effects of long-chain fatty acids on insulin resistance and inflammation.

Expression of genes encoding for drug metabolising cytochrome P450 enzymes and P-glycoprotein in the rat small intestine; comparison to the liver.

The level of expression of genes encoding for nine major xenobiotic metabolising Cytochrome P450s (CYPs) and the P-glycoprotein (Pgp) was determined in three different regions of the small intestine of male and female Sprague Dawley rats and the expression was compared with that in the liver. A semi-quantitative RT-PCR method, using the total RNA from the tissues, was established for the determination of the level of gene expression. Four of the CYP genes: the CYP2B1, CYP2C6, CYP2C11 and CYP2D1 and the Pgp were expressed at as high levels in the small intestine as in the liver. The expression of the other CYP genes was remarkably different in the two organs. The CYP1A2, CYP2A3, CYP2E1 and CYP3A1 showed a strong expression in the liver but only a comparatively weak or no expression in the small intestine. The CYP1A1 on the other hand exhibited a stronger expression in the small intestine than in the liver. With the exception of the CYP2A3, none of the genes showed a clear regional distribution in their small intestinal expression. Furthermore, no obvious sex difference in the expression of the CYP and Pgp genes could be observed. Our results indicate that several of the enzymes, central for drug metabolism are differently expressed in the liver and in the small intestine of the rat which should be taken into account when using rat as a model for the bioavailability and organ specific toxicity studies of orally administered xenobiotics. The apparently strong small intestinal expression of the CYP2C genes suggests that these enzymes could play a key role in the intestinal drug metabolism in rats and therefore affect the bioavailability of those orally used drugs which are substrates of the CYP2Cs. This possibility should be investigated in more detail both in rats and humans.