Redundancy of mammalian Y family DNA polymerases in cellular responses to genomic DNA lesions induced by ultraviolet light

Short-wave ultraviolet light induces both mildly helix-distorting cyclobutane pyrimidine dimers (CPDs) and severely distorting (6–4) pyrimidine pyrimidone photoproducts ((6–4)PPs). The only DNA polymerase (Pol) that is known to replicate efficiently across CPDs is Polη, a member of the Y family of translesion synthesis (TLS) DNA polymerases. Phenotypes of Polη deficiency are transient, suggesting redundancy with other DNA damage tolerance pathways. Here we performed a comprehensive analysis of the temporal requirements of Y-family Pols ι and κ as backups for Polη in (i) bypassing genomic CPD and (6–4)PP lesions in vivo, (ii) suppressing DNA damage signaling, (iii) maintaining cell cycle progression and (iv) promoting cell survival, by using mouse embryonic fibroblast lines with single and combined disruptions in these Pols. The contribution of Polι is restricted to TLS at a subset of the photolesions. Polκ plays a dominant role in rescuing stalled replication forks in Polη-deficient mouse embryonic fibroblasts, both at CPDs and (6–4)PPs. This dampens DNA damage signaling and cell cycle arrest, and results in increased survival. The role of relatively error-prone Pols ι and κ as backups for Polη contributes to the understanding of the mutator phenotype of xeroderma pigmentosum variant, a syndrome caused by Polη defects.     

Mammalian keratin associated proteins (KRTAPs) subgenomes: disentangling hair diversity and adaptation to terrestrial and aquatic environments

Background

Adaptation of mammals to terrestrial life was facilitated by the unique vertebrate trait of body hair, which occurs in a range of morphological patterns. Keratin associated proteins (KRTAPs), the major structural hair shaft proteins, are largely responsible for hair variation.

Results

We exhaustively characterized the KRTAP gene family in 22 mammalian genomes, confirming the existence of 30 KRTAP subfamilies evolving at different rates with varying degrees of diversification and homogenization. Within the two major classes of KRTAPs, the high cysteine (HS) subfamily experienced strong concerted evolution, high rates of gene conversion/recombination and high GC content. In contrast, high glycine-tyrosine (HGT) KRTAPs showed evidence of positive selection and low rates of gene conversion/recombination. Species with more hair and of higher complexity tended to have more KRATP genes (gene expansion). The sloth, with long and coarse hair, had the most KRTAP genes (175 with 141 being intact). By contrast, the “hairless” dolphin had 35 KRTAPs and the highest pseudogenization rate (74% relative to the 19% mammalian average). Unique hair-related phenotypes, such as scales (armadillo) and spines (hedgehog), were correlated with changes in KRTAPs. Gene expression variation probably also influences hair diversification patterns, for example human have an identical KRTAP repertoire as apes, but much less hair.

Conclusions

We hypothesize that differences in KRTAP gene repertoire and gene expression, together with distinct rates of gene conversion/recombination, pseudogenization and positive selection, are likely responsible for micro and macro-phenotypic hair diversification among mammals in response to adaptations to ecological pressures.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-779) contains supplementary material, which is available to authorized users.     

Function of leaf hamamelitol as a compatible solute during water stress treatment of Hedera helix L.

Hamamelitol is an unusual branched-chain sugar alcohol previously suggested to function as a leaf compatible solute. In this study, we have examined the leaf metabolism and intracelluiar compartmentalization of hamamelitol and other soluble sugars during long-term water stress treatment of Hedera helix (English ivy). Total leaf hamamelitol content was relatively low in greenhouse control plants, but increased 2-fold during water stress treatment to levels approaching those observed in field-grown plants (6–7 μmol g^?1 fresh weight). Using density gradient fractionation with non-aqueous solvents, we showed that hamamelitol occurs primarily in the cytoplasm and vacuoles of leaf mesophyll cells. During water stress treatment most of the increase in leaf hamamelitol occurred in the mesophyll cytoplasm, compensating osmotically for a decrease in cytoplasmic sucrose concentration. The maximum concentration of cytoplasmic hamamelitol was 155 mol m^?3 and occurred in field-grown plants. Labelling experiments showed that hamamelitol is slowly synthesized from ¹⁴CO₂ in leaves of H. helix, but is very long-lived (estimated t_1/2 of 4 years). Together, these data indicate that hamamelitol probably functions during long-term stress conditions as an osmotically active, compatible solute in plant leaves. We suggest that the signal for enhanced accumulation of hamamelitol during the water stress treatment was initiated by decreased plant growth and increased leaf sucrose hydrolysis.     

Enzymes and proteins from extremophiles as hyperstable probes in nanotechnology: the use of D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus litoralis</Emphasis> for sugars monitoring

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose (Mal) uptake system. In the hyperthermophilic archaeon Thermococcus litoralis, this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds sugars, such as maltose, trehalose and glucose, with high affinity. In this work, we have immobilized TMBP on a porous silicon wafer. The immobilization of TMBP to the chip was monitored by reflectivity and Fourier Transformed Infrared spectroscopy. Furthermore, we have tested the optical response of the protein-Chip complex to glucose binding. The obtained data suggest the use of this protein for the design of advanced optical non-consuming analyte biosensors for glucose detection. The authors wish to dedicate this work to Prof. Ignacy Gryczynski, University of North Texas, TX, USA, for his outstanding contribution to the development of new sensing methodologies.     

Geographic variations in underwater male Weddell seal Trills suggest breeding area fidelity

Adult Weddell seals (Leptonychotes weddellii) exhibit site fidelity to where they first breed but juveniles, and perhaps transient adult males, may disperse from their natal location. If there is mixing between adjacent breeding groups, we would expect that common vocalizations would exhibit clinal patterns. Underwater Trill vocalizations of male Weddell seals at Mawson, Davis, Casey, McMurdo Sound, Neumayer and Drescher Inlet separated by ca. 500 to >9,000 km, were examined for evidence of clinal variation. Trills are only emitted by males and have a known territorial defense function. Trills from Davis and Mawson, ca. 630 km apart, were distinct from each other and exhibited the greatest number of unique frequency contour patterns. The acoustic features (duration, waveform, frequency contour) of Trills from Neumayer and Drescher Inlet, ca. 500 km apart, were more distinct from each other than they were from the other four locations. General Discriminant Analysis and Classification Tree Analysis correctly classified 65.8 and 76.9% of the Trills to the correct location. The classification errors assigned more locations to sites >630 km away than to nearest neighbours. Weddell seal Trills exhibit geographic variation but there is no evidence of a clinal pattern. This suggests that males remain close to single breeding areas throughout their lifetime.     

Phase-I study of Innacell γδ™, an autologous cell-therapy product highly enriched in γ9δ2 T lymphocytes, in combination with IL-2, in patients with metastatic renal cell carcinoma

PURPOSE: gamma9delta2 T lymphocytes have been shown to be directly cytotoxic against renal carcinoma cells. Lymphocytes T gammadelta can be selectively expanded in vivo with BrHPP (IPH1101, Phosphostim) and interleukin 2 (IL-2). A phase I Study was conducted in patients with metastatic renal cell carcinoma (mRCC) to determine the maximum-tolerated dose and safety of Innacell gammadeltatrade mark, an autologous cell-therapy product based on gamma9delta2 T lymphocytes, in patients with mRCC. EXPERIMENTAL DESIGN: A 1-h intravenous infusion of gamma9delta2 T lymphocytes was administered alone during treatment cycle 1 and combined with a low dose of subcutaneous interleukin-2 (IL-2, 2 MIU/m(2) from Day 1 to Day 7) in the two subsequent cycles (at 3-week intervals). The dose of gamma9delta2 T lymphocytes was escalated from 1 up to 8 x 10(9) cells. RESULTS: Ten patients underwent a total of 27 treatment cycles. Immunomonitoring data demonstrate that gamma9delta2 T lymphocytes are initially cleared from the blood to reappear at the end of IL-2 administration. Dose-limiting toxicity occurred in one patient at the dose of 8 x 10(9) cells (disseminated intravascular coagulation). Other treatment-related adverse events (AEs) included mainly gastrointestinal disorders and flu-like symptoms (fatigue, pyrexia, rigors). Hypotension and tachycardia also occurred, especially with co-administered IL-2. Six patients showed stabilized disease. Time to progression was 25.7 weeks. CONCLUSION: The data collected in ten patients with mRCC indicate that repeated infusions of Innacell gammadeltatrade mark at different dose levels (up to 8 x 10(9) total cells), either alone or with IL-2 is well tolerated. These results are in favor of the therapeutic value of cell therapy with Innacell gammadeltatrade mark for the treatment of cancers.     

Analysis of FGF-Dependent and FGF-Independent Pathways in Otic Placode Induction

The inner ear develops from a patch of thickened cranial ectoderm adjacent to the hindbrain called the otic placode. Studies in a number of vertebrate species suggest that the initial steps in induction of the otic placode are regulated by members of the Fibroblast Growth Factor (FGF) family, and that inhibition of FGF signaling can prevent otic placode formation. To better understand the genetic pathways activated by FGF signaling during otic placode induction, we performed microarray experiments to estimate the proportion of chicken otic placode genes that can be up-regulated by the FGF pathway in a simple culture model of otic placode induction. Surprisingly, we find that FGF is only sufficient to induce about 15% of chick otic placode-specific genes in our experimental system. However, pharmacological blockade of the FGF pathway in cultured chick embryos showed that although FGF signaling was not sufficient to induce the majority of otic placode-specific genes, it was still necessary for their expression in vivo. These inhibitor experiments further suggest that the early steps in otic placode induction regulated by FGF signaling occur through the MAP kinase pathway. Although our work suggests that FGF signaling is necessary for otic placode induction, it demonstrates that other unidentified signaling pathways are required to co-operate with FGF signaling to induce the full otic placode program.     

The Rescue of miR-148a Expression in Pancreatic Cancer: An Inappropriate Therapeutic Tool

MicroRNAs are small non-coding RNAs that physiologically modulate proteins expression, and regulate numerous cellular mechanisms. Alteration of microRNA expression has been described in cancer and is associated to tumor initiation and progression. The microRNA 148a (miR-148a) is frequently down-regulated in cancer. We previously demonstrated that its down-regulation by DNA hypermethylation is an early event in pancreatic ductal adenocarcinoma (PDAC) carcinogenesis, suggesting a tumor suppressive function. Here, we investigate the potential role of miR-148a over-expression in PDAC as a therapeutic tool. We first report the consequences of miR-148a over-expression in PDAC cell lines. We demonstrate that miR-148a over-expression has no dramatic effect on cell proliferation and cell chemo-sensitivity in four well described PDAC cell lines. We also investigate the modulation of protein expression by a global proteomic approach (2D-DIGE). We show that despite its massive over-expression, miR-148a weakly modulates protein expression, thus preventing the identification of protein targets in PDAC cell lines. More importantly, in vivo data demonstrate that modulating miR-148a expression either in the epithelia tumor cells and/or in the tumor microenvironment does not impede tumor growth. Taken together, we demonstrate herein that miR-148a does not impact PDAC proliferation both in vitro and in vivo thus suggesting a weak potential as a therapeutic tool.     

Improved Detection of Common Variants Associated with Schizophrenia by Leveraging Pleiotropy with Cardiovascular-Disease Risk Factors

Several lines of evidence suggest that genome-wide association studies (GWASs) have the potential to explain more of the “missing heritability” of common complex phenotypes. However, reliable methods for identifying a larger proportion of SNPs are currently lacking. Here, we present a genetic-pleiotropy-informed method for improving gene discovery with the use of GWAS summary-statistics data. We applied this methodology to identify additional loci associated with schizophrenia (SCZ), a highly heritable disorder with significant missing heritability. Epidemiological and clinical studies suggest comorbidity between SCZ and cardiovascular-disease (CVD) risk factors, including systolic blood pressure, triglycerides, low- and high-density lipoprotein, body mass index, waist-to-hip ratio, and type 2 diabetes. Using stratified quantile-quantile plots, we show enrichment of SNPs associated with SCZ as a function of the association with several CVD risk factors and a corresponding reduction in false discovery rate (FDR). We validate this “pleiotropic enrichment” by demonstrating increased replication rate across independent SCZ substudies. Applying the stratified FDR method, we identified 25 loci associated with SCZ at a conditional FDR level of 0.01. Of these, ten loci are associated with both SCZ and CVD risk factors, mainly triglycerides and low- and high-density lipoproteins but also waist-to-hip ratio, systolic blood pressure, and body mass index. Together, these findings suggest the feasibility of using genetic-pleiotropy-informed methods for improving gene discovery in SCZ and identifying potential mechanistic relationships with various CVD risk factors.