Repair of a minimal DNA double-strand break by NHEJ requires DNA-PKcs and is controlled by the ATM/ATR checkpoint

Mammalian cells primarily rejoin DNA double-strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono-specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single-chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA-PK catalytic subunit (DNA-PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3′-hydroxyl and 5′-phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA-PKcs, suggesting an absolute requirement for DNA-PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.     

Adjusting HIV Prevalence for Survey Non-Response Using Mortality Rates: An Application of the Method Using Surveillance Data from Rural South Africa

Background

The main source of HIV prevalence estimates are household and population-based surveys; however, high refusal rates may hinder the interpretation of such estimates. The study objective was to evaluate whether population HIV prevalence estimates can be adjusted for survey non-response using mortality rates.

Methodology/Principal Findings

Data come from the longitudinal Africa Centre Demographic Information System (ACDIS), in rural South Africa. Mortality rates for persons tested and not tested in the 2005 HIV surveillance were available from routine household surveillance. Assuming HIV status among individuals contacted but who refused to test (non-response) is missing at random and mortality among non-testers can be related to mortality of those tested a mathematical model was developed. Non-parametric bootstrapping was used to estimate the 95% confidence intervals around the estimates. Mortality rates were higher among untested (16.9 per thousand person-years) than tested population (11.6 per thousand person-years), suggesting higher HIV prevalence in the former. Adjusted HIV prevalence for females (15–49 years) was 31.6% (95% CI 26.1–37.1) compared to observed 25.2% (95% CI 24.0–26.4). For males (15–49 years) adjusted HIV prevalence was 19.8% (95% CI 14.8–24.8), compared to observed 13.2% (95% CI 12.1–14.3). For both sexes (15–49 years) combined, adjusted prevalence was 27.5% (95% CI 23.6–31.3), and observed prevalence was 19.7% (95% CI 19.6–21.3). Overall, observed prevalence underestimates the adjusted prevalence by around 7 percentage points (37% relative difference).

Conclusions/Significance

We developed a simple approach to adjust HIV prevalence estimates for survey non-response. The approach has three features that make it easy to implement and effective in adjusting for selection bias than other approaches. Further research is needed to assess this approach in populations with widely available HIV treatment (ART).     

Phylogenetic distribution of intron positions in alpha-amylase genes of bilateria suggests numerous gains and losses

Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that "resets" of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

Use of digital webcam images to track spring green-up in a deciduous broadleaf forest

Understanding relationships between canopy structure and the seasonal dynamics of photosynthetic uptake of CO₂ by forest canopies requires improved knowledge of canopy phenology at eddy covariance flux tower sites. We investigated whether digital webcam images could be used to monitor the trajectory of spring green-up in a deciduous northern hardwood forest. A standard, commercially available webcam was mounted at the top of the eddy covariance tower at the Bartlett AmeriFlux site. Images were collected each day around midday. Red, green, and blue color channel brightness data for a 640 × 100-pixel region-of-interest were extracted from each image. We evaluated the green-up signal extracted from webcam images against changes in the fraction of incident photosynthetically active radiation that is absorbed by the canopy (f _APAR), a broadband normalized difference vegetation index (NDVI), and the light-saturated rate of canopy photosynthesis (A _max), inferred from eddy flux measurements. The relative brightness of the green channel (green %) was relatively stable through the winter months. A steady rising trend in green % began around day 120 and continued through day 160, at which point a stable plateau was reached. The relative brightness of the blue channel (blue %) also responded to spring green-up, although there was more day-to-day variation in the signal because blue % was more sensitive to changes in the quality (spectral distribution) of incident radiation. Seasonal changes in blue % were most similar to those in f _APAR and broadband NDVI, whereas changes in green % proceeded more slowly, and were drawn out over a longer period of time. Changes in A _max lagged green-up by at least a week. We conclude that webcams offer an inexpensive means by which phenological changes in the canopy state can be quantified. A network of cameras could offer a novel opportunity to implement a regional or national phenology monitoring program.     

A new non-equilibrium enzyme linked immunosorbent assay for a glycogen-derived urinary tetrasaccharide

The tetrasaccharide, Glc1-6Glc1-4Glc1-4Glc, denoted (Glc)₄, is a limit dextrin formed by amylolytic degradation of glycogen. In order to evaluate the possible clinical importance of (Glc)₄ excretion as an indicator of certain physiological and pathological conditions, we have developed a new rapid and inexpensive immunoassay using a monoclonal antibody raised against (Glc)₄ glycosidically-linked to a carrier protein. As the antibody is highly specific, it can be used to measure native (Glc)₄ directly, without the chemical reduction step required in previous methods. A new type of non-equilibrium ELISA inhibition test was developed based on the capacity of free (Glc)₄ to decrease initial rates of antibody binding to (Glc)₄-coated microtiter wells. The method is highly reproducible and is as sensitive and accurate as the gas chromatography method or radioimmunoassay used previously.Abbreviations (Glc)₄ Glc1-6Glc1-4Glc1-4Glc - KLH keyhole limpet hemacyanin - BSA bovine serum albumin - PEG polyethylene glycol     

Dynamic light scattering and fluorescence study of the interaction between double-stranded DNA and poly(amido amine) dendrimers

The interaction between a cationic poly(amido amine) (PAMAM) dendrimer of generation 4 and double-stranded salmon sperm DNA in 10 mM NaBr solution has been investigated using dynamic light scattering (DLS) and steady-state fluorescence spectroscopy. The structural parameters of the formed aggregates as well as the complex formation process were studied in dilute solutions. When DNA is mixed with PAMAM dendrimers, it undergoes a transition from a semiflexible coil to a more compact conformation due to the electrostatic interaction present between the cationic dendrimer and the anionic polyelectrolyte. The DLS results reveal that one salmon sperm DNA molecule forms a discrete aggregate in dilute solution with several PAMAM dendrimers with a mean apparent hydrodynamic radius of 50 nm. These discrete complexes coexist with free DNA at low molar ratios of dendrimer to DNA, which shows that cooperativity is present in the complex formation. The formation of the complexes was confirmed by agarose gel electrophoresis measurements. DNA in the complexes was also found to be significantly more protected against DNase catalyzed digestion compared to free DNA. The number of dendrimers per DNA chain in the complexes was found to be approximately 35 as determined by steady-state fluorescence spectroscopy.     

Phase I trial of intravenous infusion of ex-vivo-activated autologous blood-derived macrophages in patients with non-small-cell lung cancer: Toxicity and immunomodulatory effects

Summary The purpose of this phase I study was to evaluate the toxicity and biological activity of autologous blood-derived macrophages activated ex-vivo with recombinant human interferon (rhuIFN) [monokine-activated killer (MAK) cells] and administered intravenously to 11 lung cancer patients once a week for 6 consecutive weeks. Peripheral blood monocytes were collected by leukapheresis and then purified by counterflow elutriation. The MAK cells were generated by culturing the purified monocytes in Teflon bags for 7 days and adding rhuIFN to the cultured cells for the last 18 h. These MAK cells expressed differentiation-associated surface antigen MAX1, and were cytotoxic in vitro against tumour cell line U937. The MAK cells were infused at dose levels from 1 × 10⁷ to 5 × 10⁸ on an intrapatient dose-escalating schedule. No severe adverse side-effects occurred. Toxicity was mild to moderate [primarly fever (75%) and chills (32%)], non-dose-dependent, and non-cumulative. No consistent change in haemostatic function, or liver or renal function was observed. Dose-limiting toxicity was not reached at 5 × 10⁸ cells (optimal dose reproduced for each patient). The maximum tolerated dose was not determined. The immunomodulatory activity of i.v. infused MAK cells was demonstrated both in vivo by significant increases in granulocyte count and neopterin level in the patients' peripheral blood postinfusion and in vitro by secretory products (IL-1. TNF, neopterin, and thromboplastin-like substance) in the culture supernatants. The in vivo traffic patterns of autologous MAK cells labelled ex-vivo with¹¹¹In oxine were studied in 7 patients. Gamma imaging showed an immediate but transient lung uptake (<24 h), and a progressive uptake of radioactivity in the liver and spleen was seen from 6 h to 72 h post-infusion. Our results indicate that the preparation of high numbers of autologous, blood-derived MAK cells is a feasible procedure, and their transfusion is safe for patients. This immunotherapeutic approach seems to be encouraging from the point of view of establishing an adjuvant therapeutic modality in cancer patients with minimal residual disease.This work was supported in part by a grant 6911 from the Association pour la Recherche contre le Cancer (ARC), grants from the Ligue Nationale contre le cancer and the Ligues Regionales (Bas-Rhin, Haut-Rhin) contre le cancer, and contract 891013 from the Institut National pour la Santé et la Recherche Médicale (INSERM), France