Nucleotide pool imbalance and adenosine deaminase deficiency induce alterations of N-region insertions during V(D)J recombination

Template-independent nucleotide additions (N regions) generated at sites of V(D)J recombination by terminal deoxynucleotidyl transferase (TdT) increase the diversity of antigen receptors. Two inborn errors of purine metabolism, deficiencies of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), result in defective lymphoid development and aberrant pools of 2′-deoxynucleotides that are substrates for TdT in lymphoid precursors. We have asked whether selective increases in dATP or dGTP pools result in altered N regions in an extrachromosomal substrate transfected into T-cell or pre–B-cell lines. Exposure of the transfected cells to 2′-deoxyadenosine and an ADA inhibitor increased the dATP pool and resulted in a marked increase in A–T insertions at recombination junctions, with an overall decreased frequency of V(D)J recombination. Sequence analysis of VH-DH-JH junctions from the IgM locus in B-cell lines from ADA-deficient patients demonstrated an increase in A–T insertions equivalent to that found in the transfected cells. In contrast, elevation of dGTP pools, as would occur in PNP deficiency, did not alter the already rich G–C content of N regions. We conclude that the frequency of V(D)J recombination and the composition of N-insertions are influenced by increases in dATP levels, potentially leading to alterations in antigen receptors and aberrant lymphoid development. Alterations in N-region insertions may contribute to the B-cell dysfunction associated with ADA deficiency

Three microarray platforms: an analysis of their concordance in profiling gene expression

BACKGROUND: Microarrays for the analysis of gene expression are of three different types: short oligonucleotide (25–30 base), long oligonucleotide (50–80 base), and cDNA (highly variable in length). The short oligonucleotide and cDNA arrays have been the mainstay of expression analysis to date, but long oligonucleotide platforms are gaining in popularity and will probably replace cDNA arrays. As part of a validation study for the long oligonucleotide arrays, we compared and contrasted expression profiles from the three formats, testing RNA from six different cell lines against a universal reference standard. RESULTS: The three platforms had 6430 genes in common. In general, correlation of gene expression levels across the platforms was good when defined by concordance in the direction of expression difference (upregulation or downregulation), scatter plot analysis, principal component analysis, cell line correlation or quantitative RT-PCR. The overall correlations (r values) between platforms were in the range 0.7 to 0.8, as determined by analysis of scatter plots. When concordance was measured for expression ratios significant at p-values of <0.05 and at expression threshold levels of 1.5 and 2-fold, the agreement among the platforms was very high, ranging from 93% to 100%. CONCLUSION: Our results indicate that the long oligonucleotide platform is highly suitable for expression analysis and compares favorably with the cDNA and short oligonucleotide varieties. All three platforms can give similar and reproducible results if the criterion is the direction of change in gene expression and minimal emphasis is placed on the magnitude of change

Joint attention initiation with and without positive affect: risk group differences and associations with ASD symptoms.

Infants at risk for autism spectrum disorders (ASD) may have difficulty integrating smiles into initiating joint attention (IJA) bids. A specific IJA pattern, anticipatory smiling, may communicate preexisting positive affect when an infant smiles at an object and then turns the smile toward the social partner. We compared the development of anticipatory smiling at 8, 10, and 12&nbsp;months in infant siblings of children with ASD (high-risk siblings) and without ASD (low-risk siblings). High-risk siblings produced less anticipatory smiling than low-risk siblings, suggesting early differences in communicating preexisting positive affect. While early anticipatory smiling distinguished the risk groups, IJA not accompanied by smiling best predicted later severity of ASD-related behavioral characteristics among high-risk siblings. High-risk infants appear to show lower levels of motivation to share positive affect with others. However, facility with initiating joint attention in the absence of a clear index of positive affective motivation appears to be central to the prediction of ASD symptoms

A New Method for In Situ Measurements of Oxygen Isotopologues of Soil Water and Carbon Dioxide with High Time Resolution

The newly developed method allows for the first time simultaneous and continuous measurements of the oxygen isotope composition of H2O and CO2 along soil profiles. Its application in the field will contribute to reducing the uncertainties associated with soil–atmosphere CO2 oxygen isotope fluxes.The oxygen isotope composition of atmospheric CO2 (δ18Oac) can be used to disentangle ecosystem component CO2 fluxes, such as soil respiration and plant assimilation, because the δ18O composition of different water pools is transferred to CO2 during isotopic equilibration. The oxygen isotope exchange between CO2 and water in soils has been widely studied with theoretical models, but experimental data are scarce, albeit indispensable to characterization of the role of soils in determining δ18Oac. Here, we present a new methodology to monitor the δ18O of soil CO2 (δ18Osc) and of soil water (δ18Osw) in situ at varying soil water content. Infrared laser spectroscopy was combined with gas-permeable polypropylene (PP) tubing installed at different depths in a sand column. The permeable tubing did not lead to any isotopic fractionation and was suitable for combined δ18Osc and δ18Osw measurements. Soil water became gradually 18O enriched from the top of the sand over several days. Measured and δ18Osc simulated with the model MuSICA indicated incomplete CO2–H2O isotopic equilibrium. Irrigation of the sand column with tapwater resulted in a temporary reset of δ18Osw along the soil column, while δ18Osc was only influenced when the enzyme carbonic anhydrase was added to the irrigation water. Our study demonstrates that δ18Osc and δ18Osw can now be monitored in situ and online with high time resolution with minimum disturbance. With this new tool at hand, research into the oxygen isotope exchange between soil water and CO2 in natural soils has the potential to advance to a new stage and help to constrain the atmospheric CO2 budget

Lsh, an epigenetic guardian of repetitive elements

The genome is burdened with repetitive sequences that are generally embedded in silenced chromatin. We have previously demonstrated that Lsh (lymphoid-specific helicase) is crucial for the control of heterochromatin at pericentromeric regions consisting of satellite repeats. In this study, we searched for additional genomic targets of Lsh by examining the effects of Lsh deletion on repeat regions and single copy gene sequences. We found that the absence of Lsh resulted in an increased association of acetylated histones with repeat sequences and transcriptional reactivation of their silenced state. In contrast, selected single copy genes displayed no change in histone acetylation levels, and their transcriptional rate was indistinguishable compared to Lsh-deficient cells and wild-type controls. Microarray analysis of total RNA derived from brain and liver tissues revealed that <0.4% of the 15 247 examined loci were abnormally expressed in Lsh−/−embryos and almost two-thirds of these deregulated sequences contained repeats, mainly retroviral LTR (long terminal repeat) elements. Chromatin immunoprecipitation analysis demonstrated a direct interaction of Lsh with repetitive sites in the genome. These data suggest that the repetitive sites are direct targets of Lsh action and that Lsh plays an important role as ‘epigenetic guardian’ of the genome to protect against deregulation of parasitic retroviral elements

Circulating tumor DNA dynamics and response to immunotherapy in colorectal cancer.

Circulating tumor DNA (ctDNA) is increasingly being investigated as a tool to detect minimal residual disease in resected, stage I-III colorectal cancer. Recent ctDNA studies have indicated that detection of ctDNA following surgery for resectable colorectal cancer confers a significantly higher risk of recurrence than those with negative ctDNA postoperatively. In those with postoperative ctDNA positivity, clearance of minimal residual disease with adjuvant chemotherapy is a positive prognostic indicator. Lastly, ctDNA has demonstrated superior sensitivity to the conventional blood tumor marker carcinoembryonic antigen (CEA) and can offer median lead times of up to 11 months for radiographic detection of recurrence during the surveillance of resected, stage I-III colorectal cancer. In metastatic colorectal cancer (mCRC), there is growing evidence to suggest that plasma ctDNA can be used to monitor tumor response to conventional chemotherapy as well. The present case series demonstrated that plasma ctDNA is a predictor of tumor response to immunotherapy in patients with mCRC that are microsatellite stable or microsatellite instability high. Plasma ctDNA could serve as a dynamic marker of immunotherapy response even in colorectal tumors that were CEA non-producers. Overall, these findings add to ongoing efforts to establish the role of plasma ctDNA in monitoring response to immunotherapy in CRC