Development of a gLCR-based KRAS mutation detection approach and its comparison with other screening methods.

A gapped ligase chain reaction (gLCR)-based technique was developed and tested on clinical formalin-fixed, paraffin-embedded (FFPE) tissues from colorectal cancer patients. The technique was designed to detect low-level KRAS codon 12 or 13 mutations or confirming doubtful results gained by less sensitive KRAS screening techniques. The gLCR approach was compared with mutation screening techniques commonly used in routine diagnostics regarding sensitivity and specificity. The herein described monoplex gLCR technique is a useful and powerful tool for detecting low-level KRAS codon 12 and 13 mutations in a vast majority of wild type DNA. The gLCR has the capacity to detect one mutated allele in an excess of at least one million wild type alleles (0.0001 %) and is therefore an ideal technique for confirming doubtful KRAS mutation screening results obtained by other techniques. The variance of the gLCRs signal amplitude was very low and is showing a high reproducibility with constant sensitivity even at higher dilutions. The financial effort and the handling time for this technique are low and comparable to a standard cycle sequencing reaction. Additionally, the gLCR technique is easy extendable for the detection of many other clinical relevant mutation hotspots.

Development of a DUSP9 methylation screening assay.

A methylation screening assay for DUSP9 (dual-specificity phosphatase 9) has been developed and applied on 79 FFPE samples from patients with colorectal cancer (CRC) and 22 corresponding tumor free colon samples in this study. Quantitative pyrosequencing was used for the determination of the methylation in the promoter CpG island, including 83 CpG motifs. In this way, the methylation pattern of the 11 tumor samples with the weakest and the strongest methylation could be identified and were compared to their corresponding tumor free colon samples. Forty six percent of the weakly methylated samples showed no significant difference to their tumor free counterparts, whereas in 27% of the cases an increased or reduced methylation was detectable. For the strongly methylated tumor samples only 18% showed no significant difference to their tumor free counterparts, whereas 82% were significantly stronger methylated. In CRC, the aberrant promoter methylation of tumor suppressor genes is one aspect that defines the CpG island methylator phenoptype (CIMP) and is frequently observed in a subpopulation of cases. Patients harboring a CIMP phenotype often show additional clinicopathological characteristics, the so called CIMP features. Interestingly, no CIMP features were found for the weakly methylated samples analyzed in this study but could be seen in 82% of the strongly methylated cases, indicating a possible use for DUSP9 as CIMP marker.

Faster rates of post-puberty kidney deterioration in males is correlated with elevated oxidative stress in males vs females at early puberty.

BACKGROUND: Post-puberty deterioration of kidneys is more rapid in males than in females. To reveal the underlying molecular mechanisms for this difference, we analyzed gender-dependent gene expression in kidneys of three groups of 36 day-old rats. RESULTS: The number of genes exhibiting gender-dependent expression was highly influenced by the genetic background of the rat group examined. 373, 288 and 79 genes showed differential gene expression between males and females (p = 0.001) in US, Mhm and Mhm*BN rats, respectively. Of all gender dependently expressed genes, only 39 genes were differentially expressed in all tested groups and the direction of expression change was the same for those genes for all groups. The gene expression profile suggests higher metabolic and transport activities, enhanced cell proliferation, elevated oxidative stress, and altered vascular biology in males. Furthermore, elevated levels of superoxide anion (two- to three-fold) in males compared to females were detected at early puberty, but neither at pre-puberty nor at late puberty/early adulthood. CONCLUSION: Our data suggest that early puberty, with gender-related elevation in oxidative stress in males, is a key compromising factor on kidneys in males.

Differential expression of glucose-regulated (grp78) and heat-shock-inducible (hsp70) genes during asexual development of Neurospora crassa.

The expression of a glucose-regulated gene (grp78) changes significantly during the vegetative life cycle of Neurospora crassa: the amounts of grp78 mRNA are low in dormant conidia, increase during germination and exponential growth, decline in young aerial hyphae and reach a maximum in late (15-18 h) aerial hyphae. Heat shock (30 min at 45 degrees C) elevated the mRNA level of this gene especially in early aerial hyphae, whereas no increase above the high constitutive amount was found after heat treatment of late aerial hyphae. The expression of the inducible hsp70 gene after heat shock also varied with the state of development and showed the highest inducibility in late aerial hyphae. Surface mycelium, from which aerial hyphae emerge, showed a similar increase in the amounts of both mRNA species. A developmental mutant (acon-2), which is defective in minor constriction budding of aerial hyphae, showed lower levels of con-2 mRNA as well as of grp78 and hsp70 mRNA (after heat shock) in late aerial hyphae. The acon-2 mutant did not form conidia at this stage. It is concluded that the high constitutive and inducible expression of stress genes in late aerial hyphae is due to a developmental activation of their transcription or, alternatively, to a lower degradation rate of their mRNA during this stage.