History of Parvovirus B19 infection is associated with a DNA methylation signature in childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) likely has a multistep etiology, with initial genetic aberrations occurring early in life. An abnormal immune response to common infections has emerged as a plausible candidate for triggering the proliferation of pre-leukemic clones and the fixation of secondary genetic mutations and epigenetic alterations. We investigated whether evidence of infection with a specific common myelotropic childhood virus, parvovirus B19 (PVB19), relates to patterns of gene promoter DNA methylation in ALL patients. We serologically tested bone marrow samples at diagnosis of B-cell ALL for PVB19 infection and DNA methylation using a high-throughput bead array and found that 4.2% and 36.7% of samples were seroreactive to PVB19 IgM and IgG, respectively. Leukemia samples were grouped by DNA methylation pattern. Controlling for age and immunophenotype, unsupervised modeling confirmed that the DNA methylation pattern was associated with history of PVB19 (assessed by IgG, p = 0.02), but not recent infection (assessed by IgM). Replication assays on single genes were consistent with the association. The data indicate that a common viral illness may drive specific DNA methylation patterns in susceptible B-precursor cells, contributing to the leukemogenic potential of such cells. Infections may impact childhood leukemia by altering DNA methylation patterns and specific key genes in susceptible cells; these changes may be retained even after the clearance of infection.     

Evaluation of buccal cell collection protocols for genetic susceptibility studies

Buccal cells are increasingly used as a source of quality DNA to improve participation rates in molecular studies. Here, three buccal cell collection protocols were compared to determine factors affecting the yield of cells, total DNA per sample, and DNA yield per cell. In addition, kinetic quantitative polymerase chain reaction (PCR) (TaqMan?) was used to quantify human DNA available for PCR. The method of collection used influenced the overall DNA yield per sample. The collection buffer used influenced the number of cells but not the overall DNA yield per sample. Repeated freezing and thawing did not affect overall DNA yield per sample, DNA yield per cell, or the total number of cells collected. Mouthwashes had the highest DNA yield per sample (20.8 μg) compared with cytobrush samples (1.9 μg from three cytobrushes) and tongue depressors (0.8 μg from three tongue depressors). However, mouthwash samples may contain significant non-human DNA and other contaminants that could interfere with some molecular studies. Spectrometry grossly overestimated the total DNA recovered from mouthwash samples compared with fluorometry or quantitative PCR.     

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.     

Biochemical composition of temperate and Arctic populations of Saccharina latissima after exposure to increased pCO2 and temperature reveals ecotypic variation

Previous research suggested that the polar and temperate populations of the kelp Saccharina latissima represent different ecotypes. The ecotypic differentiation might also be reflected in their biochemical composition (BC) under changing temperatures and pCO₂. Accordingly, it was tested if the BC of Arctic (Spitsbergen) and temperate S. latissima (Helgoland) is different and if they are differently affected by changes in temperature and pCO₂. Thalli from Helgoland grown at 17 °C and 10 °C and from Spitsbergen at 10 °C and 4 °C were all tested at either 380, 800, or 1,500 µatm pCO₂, and total C-, total N-, protein, soluble carbohydrate, and lipid content, as well as C/N-ratio were measured. At 10 °C, the Arctic population had a higher content of total C, soluble carbohydrates, and lipids, whereas the N- and protein content was lower. At the lower tested temperature, the Arctic ecotype had particularly higher contents of lipids, while content of soluble carbohydrates increased in the Helgoland population only. In Helgoland-thalli, elevated pCO₂ caused a higher content of soluble carbohydrates at 17 °C but lowered the content of N and lipids and increased the C/N-ratio at 10 °C. Elevated pCO₂ alone did not affect the BC of the Spitsbergen population. Conclusively, the Arctic ecotype was more resilient to increased pCO₂ than the temperate one, and both ecotypes differed in their response pattern to temperature. This differential pattern is discussed in the context of the adaptation of the Arctic ecotype to low temperature and the polar night.     

Key epigenetic changes associated with lung cancer development: Results from dense methylation array profiling

Epigenetic alterations are a common event in lung cancer and their identification can serve to inform on the carcinogenic process and provide clinically relevant biomarkers. Using paired tumor and non-tumor lung tissues from 146 individuals from three independent populations we sought to identify common changes in DNA methylation associated with the development of non-small cell lung cancer. Pathologically normal lung tissue taken at the time of cancer resection was matched to tumorous lung tissue and together were probed for methylation using Illumina GoldenGate arrays in the discovery set (n = 47 pairs) followed by bisulfite pyrosequencing for validation sets (n = 99 pairs). For each matched pair the change in methylation at each CpG was calculated (the odds ratio), and these ratios were averaged across individuals and ranked by magnitude to identify the CpGs with the greatest change in methylation associated with tumor development. We identified the top gene-loci representing an increase in methylation (HOXA9, 10.3-fold and SOX1, 5.9-fold) and decrease in methylation (DDR1, 8.1-fold). In replication testing sets, methylation was higher in tumors for HOXA9 (p < 2.2 × 10⁻¹⁶) and SOX1 (p < 2.2 × 10⁻¹⁶) and lower for DDR1 (p < 2.2 × 10⁻¹⁶). The magnitude and strength of these changes were consistent across squamous cell and adenocarcinoma tumors. Our data indicate that the identified genes consistently have altered methylation in lung tumors. Our identified genes should be included in translational studies that aim to develop screening for early disease detection.     

UV‐radiation and elevated temperatures induce formation of reactive oxygen species in gametophytes of cold‐temperate/Arctic kelps (Laminariales,Phaeophyceae)

Enhanced UV‐radiation (UVR) through stratospheric ozone depletion and global warming are crucial stressors to marine macroalgae. Damages may arise through formation of reactive oxygen species (ROS) in gametophytes of ecologically important kelps, brown algae of the order Laminariales, Such stress‐induced damages may have a negative impact on their fitness and further impact their following life stages. In our study, gametophytes of three kelp species Alaria esculenta (L.) Grev., Laminaria digitata (Huds.) Lamour., Saccharina latissima (L.) Lane, Mayes, Druehl, Saunders from the Arctic, and of L. hyperborea (Gunnerus) Foslie from the North Sea were exposed to photosynthetically active radiation, UV‐A, and UV‐B radiation and four temperatures (2–18°C). ROS are formed predominantly in the peripheral cytoplasm and in chloroplasts especially after exposure to UVR. Superoxide (O₂*^‐) is additionally formed in small, globular cytoplasmic structures, possibly mitochondria. In the surrounding medium O₂*^‐‐concentration increased markedly at elevated temperatures and under UV stress in some cases. Ultrastructural damage was negligible pointing to a high stress tolerance of this developmental stage. Our data indicate that stress tolerant gametophytes of three Arctic kelp species should sustain their crucial function as seed bank for kelp populations even under prospective rising environmental perturbations.     

Interactive effects of radiation,temperature and salinity on different life history stages of the Arctic kelp <Emphasis Type="Italic">Alaria esculenta</Emphasis> (Phaeophyceae)

Vegetative resprouting, soil or canopy-stored seed banks, post-fire seed dispersal and germination are the major strategies by which plants regenerate after fires. Post-fire regeneration modes of plants are commonly based on the presence or absence of post-fire recruitment as well as the presence or absence of post-fire resprouting. High temperatures, smoke and ash are characteristics of fire and the post-fire environment. We hypothesized that heat, smoke, ash and pH will have differential effects on seed germination depending on species’ post-fire regeneration strategies: serotinous vs. nonserotinous (which may have soil seed banks) and resprouters vs. nonresprouters (which may be obligate seeders). Here we examined the effects of these factors on the germination of 27 common east Australian species. Most serotinous species supported our hypothesis by showing no effect or reduced germination in response to heat. However, contrary to our prediction, all nonserotinous nonresprouting species also showed no effect or reduced germination in response to heat. Smoke, contrary to our hypothesis, had a negative or no effect on all serotinous and nonresprouting species, but no clear directional effect on serotinous and resprouting species. Supporting our hypotheses, ash and high pH showed positive or nonsignificant effects on the germination of all serotinous resprouting species, and a negative or no effect on nonserotinous resprouting species. However, contrary to our prediction, it had a negative or no effect on the serotinous nonresprouting species and no clear effect on nonserotinous nonresprouting species. We also discovered large differences in germination responses between conspecific populations that varied in their degree of resprouting. Although our data confirmed several of our predictions, the overall conclusion is that the responses of seeds to heat, smoke, ash and pH are not tightly associated with post-fire regeneration functional types. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic and Epigenetic Somatic Alterations in Head and Neck Squamous Cell Carcinomas Are Globally Coordinated but Not Locally Targeted

Background

Solid tumors, including head and neck squamous cell carcinomas (HNSCC), arise as a result of genetic and epigenetic alterations in a sustained stress environment. Little work has been done that simultaneously examines the spectrum of both types of changes in human tumors on a genome-wide scale and results so far have been limited and mixed. Since it has been hypothesized that epigenetic alterations may act by providing the second carcinogenic hit in gene silencing, we sought to identify genome-wide DNA copy number alterations and CpG dinucleotide methylation events and examine the global/local relationships between these types of alterations in HNSCC.

Methodology/Principal Findings

We have extended a prior analysis of 1,413 cancer-associated loci for epigenetic changes in HNSCC by integrating DNA copy number alterations, measured at 500,000 polymorphic loci, in a case series of 19 primary HNSCC tumors. We have previously demonstrated that local copy number does not bias methylation measurements in this array platform. Importantly, we found that the global pattern of copy number alterations in these tumors was significantly associated with tumor methylation profiles (p<0.002). However at the local level, gene promoter regions did not exhibit a correlation between copy number and methylation (lowest q = 0.3), and the spectrum of genes affected by each type of alteration was unique.

Conclusion/Significance

This work, using a novel and robust statistical approach demonstrates that, although a “second hit” mechanism is not likely the predominant mode of action for epigenetic dysregulation in cancer, the patterns of methylation events are associated with the patterns of allele loss. Our work further highlights the utility of integrative genomics approaches in exploring the driving somatic alterations in solid tumors.     

Wavelength-dependent induction of UV-absorbing mycosporine-like amino acids in the red alga Chondrus crispus under natural solar radiation

Polychromatic response spectra for the induction of UV absorbing mycosporine-like amino acids (MAAs) were calculated after exposing small thalli of the red alga Chondrus crispus under various cut-off filters to natural solar radiation on the North Sea island Helgoland, Germany. The laboratory-grown specimens typically contain only traces of palythine and synthesise five different MAAs rapidly and in high concentrations after being transplanted into shallow water. The resulting qualitative and quantitative patterns of MAA induction differed markedly with respect to spectral distribution. Furthermore, the wavebands effective for MAA induction vary within the MAA. UV-B radiation had a negative effect on the accumulation of the major MAAs shinorine (λ_max=334 nm) and palythine (λ_max=320 nm), while short wavelength UV-A exhibits the highest quantum efficiency on their synthesis. In contrast, the synthesis of asterina-330 (λ_max=330 nm), palythinol (λ_max=332 nm) and palythene (λ_max=360 nm) was mainly induced by UV-B radiation. Whether the synthesis of shinorine and palythine is induced by a photoreceptor with an absorption maximum in the short wavelength UV-A and whether a second photoreceptor absorbing UV-B radiation is responsible for the induction of asterina-330, palythinol and palythene remains to be studied.Our results show that C. crispus has a high capacity to adapt flexibly the qualitative and quantitative MAA concentration to the prevailing spectral distribution of irradiance. On one hand, this is regarded as an important aspect with respect to the acclimation of algae to increasing UV-B irradiance in the context of ongoing depletion of stratospheric ozone. On the other hand, the experiment demonstrates that UV-A irradiance is more important for the induction of the major MAAs shinorine and palythine than UV-B.     

Natural succession of macroalgal-dominated epibenthic assemblages at different water depths and after transplantation from deep to shallow water on Spitsbergen

In the current study, we investigated the primary succession of seaweeds over different time periods at different water depths. Furthermore, we followed the succession of field-grown benthic communities of different successional age, developing on ceramic tiles, prior to and after transplantation from 8 to 0.5 m water depth. The transplantation simulated changes associated with the break up of sea-ice cover, e.g. light regime or wave exposure. For this purpose, we transplanted 12 and 21-month old communities, grown at 8 m water depth, together with a set of sterile tiles, onto rafts, floating in 0.5 m water depth. Our results describe for the first time the succession of macroalgal communities in the Arctic and give important insights into the effect of disturbance of differently aged communities. The primary succession at 0.5 m water depth was mainly driven by Bacillariophyta and filamentous green algae like Urospora sp. and Ulothrix implexa. Twelve-month old communities at 8 m water depth are dominated by members of the Ectocarpales (Phaeophyceae), like Pylaiella littoralis, P. varia, and Ectocarpus siliculosus and the green alga U. implexa, whereas the 21-month old community showed a higher cover of the green algal class Ulvophyceae and sessile invertebrates. After transplantation to near surface conditions, species composition of the communities changed, but this effect was differently strong between communities of different age.