Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.

Real-time quantitative polymerase-chain-reaction (qPCR) is a standard technique in most laboratories used for various applications in basic research. Analysis of qPCR data is a crucial part of the entire experiment, which has led to the development of a plethora of methods. The released tools either cover specific parts of the workflow or provide complete analysis solutions. Here, we surveyed 27 open-access software packages and tools for the analysis of qPCR data. The survey includes 8 Microsoft Windows, 5 web-based, 9 R-based and 5 tools from other platforms. Reviewed packages and tools support the analysis of different qPCR applications, such as RNA quantification, DNA methylation, genotyping, identification of copy number variations, and digital PCR. We report an overview of the functionality, features and specific requirements of the individual software tools, such as data exchange formats, availability of a graphical user interface, included procedures for graphical data presentation, and offered statistical methods. In addition, we provide an overview about quantification strategies, and report various applications of qPCR. Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.

Many B-lineage-specific genes are down-regulated in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). We investigated the involvement of epigenetic modifications in gene silencing in cHL cell lines and in microdissected primary HRS cells. We assessed the expression and methylation status of CD19, CD20, CD79B, SYK, PU.1, BOB.1/OBF.1, BCMA, and LCK, all of which are typically down-regulated in cHL. We could reactivate gene expression in cHL cell lines with the DNA demethylating agent 5-aza-deoxycytidine (5-aza-dC). Using methylation-specific polymerase chain reaction (MSP), bisulfite genomic sequencing, and digestion with methylation-sensitive endonuclease followed by polymerase chain reaction (PCR), we determined the methylation status of promoter regions of PU.1, BOB.1/OBF.1, CD19, SYK, and CD79B. Down-regulation of transcription typically correlated with hypermethylation. Using bisulfite genomic sequencing we found that in microdissected HRS cells of primary cHL SYK, BOB.1/OBF.1, and CD79B promoters were also hypermethylated. Ectopic expression of both Oct2 and PU.1 in a cHL cell line potentiated endogenous PU.1 and SYK expression after 5-aza-dC treatment. These observations indicate that silencing of the B-cell-specific genes in cHL may be the consequence of a compromised regulatory network where down-regulation of a few master transcription factors results in silencing of numerous genes. (Blood. 2006;107:2493-2500)

By means of calcitonin screening programs, sporadic and hereditary medullary thyroid carcinoma (MTC) can be detected at an early stage. We investigated the histopathologic findings of 16 familial (mean age 32 ± 21 years, female/male ratio 1.6:1) and 34 sporadic (mean age 58 ± 15 years; female/male ratio 2.4:1) MTCs with stage T1 comparatively. Patients with hereditary tumors were younger. Hereditary tumors were more often found multifocal (13 of 16 vs 3 of 34; p <0.001), bilateral (11 of 16 vs 3 of 34; p <0.001), displaying desmoplastic stroma (14 of 16 vs 19 of 34; p = 0.02), and accompanied by C cell hyperplasia (16 of 16 vs 24 of 34; p = 0.01), but all of these factors were present in some sporadic patients. Only tumors with desmoplastic stroma showed lymph node metastasis, which was observed in eight of the 50 patients. After surgery all patients showed permanent normalization of calcitonin levels. We conclude that 1) morphologic parameters considered to indicate familial MTC risk are of no value in the individual patient, 2) many sporadic MTCs develop on the background of CCH, 3) tumors with desmoplastic stroma are more likely to develop lymph node metastasis, and 4) early detection of MTC permits curative surgery in the majority of patients.

Routine screening of calcitonin serum levels in patients with nodular thyroid disorders has led to an increased rate of total thyroidectomies. We investigated prevalence and interrelationship of C-cell hyperplasia (CCH) and medullary thyroid carcinoma (MTC) in patients with thyroid and parathyroid disorders that showed increased calcitonin serum levels detected by routine screening. Within two years, 30 (mean age, 60 ± 14 years) of 667 patients had a pentagastrin-stimulated calcitonin level of more than 100 pg/mL. All 30 underwent total thyroidectomy and were tested for germ-line mutations of the ret protooncogene. Entire surgical specimens were blocked, and C-cell disorders were assessed using conventional histology and immunohistochemistry. C-cell hyperplasia was defined by the presence of more than 50 C cells/1 low-power field in both lobes and was classified as focal, diffuse, nodular, or neoplastic. Nineteen patients (female/male = 14/5) had MTC, and 11 males but no females had CCH only. Six of 16 patients with sporadic MTC had concomitant CCH. Three patients were index cases of new MTC families. We conclude that MTC with concomitant CCH is an unreliable marker for hereditary MTC risk and that CCH has a preneoplastic potential in the absence of germ-line mutations. In this series, CCH alone was not found in females.

Disease-specific alterations of the cell-free DNA methylation status are frequently found in serum samples and are currently considered to be suitable biomarkers. Candidate markers were identified by bisulfite conversion-based genome-wide methylation screening of lung tissue from lung cancer, fibrotic ILD, and COPD. cfDNA from 400 μl serum (n = 204) served to test the diagnostic performance of these markers. Following methylation-sensitive restriction enzyme digestion and enrichment of methylated DNA via targeted amplification (multiplexed MSRE enrichment), a total of 96 markers were addressed by highly parallel qPCR. Lung cancer was efficiently separated from non-cancer and controls with a sensitivity of 87.8%, (95%CI: 0.67-0.97) and specificity 90.2%, (95%CI: 0.65-0.98). Cancer was distinguished from ILD with a specificity of 88%, (95%CI: 0.57-1), and COPD from cancer with a specificity of 88% (95%CI: 0.64-0.97). Separation of ILD from COPD and controls was possible with a sensitivity of 63.1% (95%CI: 0.4-0.78) and a specificity of 70% (95%CI: 0.54-0.81). The results were confirmed using an independent sample set (n = 46) by use of the four top markers discovered in the study (HOXD10, PAX9, PTPRN2, and STAG3) yielding an AUC of 0.85 (95%CI: 0.72-0.95). This technique was capable of distinguishing interrelated complex pulmonary diseases suggesting that multiplexed MSRE enrichment might be useful for simple and reliable diagnosis of diverse multifactorial disease states.