G-spots cause incorrect expression measurement in Affymetrix microarrays

Abstract Background High Density Oligonucleotide arrays (HDONAs), such as the Affymetrix HG-U133A GeneChip, use sets of probes chosen to match specified genes, with the expectation that if a particular gene is highly expressed then all the probes in that gene's probe set will provide a consistent message signifying the gene's presence. However, probes that contain a G-spot (a sequence of four or more guanines) behave abnormally and it has been suggested that these probes are responding to some biochemical effect such as the formation of G-quadruplexes. Results We have tested this expectation by examining the correlation coefficients between pairs of probes using the data on thousands of arrays that are available in the NCBI Gene Expression Omnibus (GEO) repository. We confirm the finding that G-spot probes are poorly correlated with others in their probesets and reveal that, by contrast, they are highly correlated with one another. We demonstrate that the correlation is most marked when the G-spot is at the 5' end of the probe. Conclusion Since these G-spot probes generally show little correlation with the other members of their probesets they are not fit for purpose and their values should be excluded when calculating gene expression values. This has serious implications, since more than 40% of the probesets in the HG-U133A GeneChip contain at least one such probe. Future array designs should avoid these untrustworthy probes. </jats:sec

A Comparative Study of the Impact of G-Stack Probes on Various Affymetrix GeneChips of Mammalia

We have previously discovered that probes containing runs of four or more contiguous guanines are not reliable for measuring gene expression in the Human HG_U133A Affymetrix GeneChip data. These probes are not correlated with other members of their probe set, but they are correlated with each other. We now extend our analysis to different3′GeneChip designs of mouse, rat, and human. We find that, in all these chip designs, the G-stack probes (probes with a run of exactly four consecutive guanines) are correlated highly with each other, indicating that such probes are not reliable measures of gene expression in mammalian studies. Furthermore, there is no specific position of G-stack where the correlation is highest in all the chips. We also find that the latest designs of rat and mouse chips have significantly fewer G-stack probes compared to their predecessors, whereas there has not been a similar reduction in G-stack density across the changes in human chips. Moreover, we find significant changes in RMA values (after removing G-stack probes) as the number of G-stack probes increases.</jats:p

SHRIMP ion probe zircon geochronology and Sr and Nd isotope geochemistry for southern Longwood Range and Bluff Peninsula intrusive rocks of Southland, New Zealand

Permian–Jurassic ultramafic to felsic intrusive complexes at Bluff Peninsula and in the southern Longwood Range along the Southland coast represent a series of intraoceanic magmatic arcs with ages spanning a time interval of 110 m.y. New SHRIMP U-Pb zircon data for a quartz diorite from the Flat Hill complex, Bluff Peninsula, yield an age of 259 ± 4 Ma, consistent with other geochronological and paleontological evidence confirming a Late Permian age. The new data are consistent with an age of c. 260 Ma for the intrusive rocks of the Brook Street Terrane. SHRIMP U-Pb zircon ages for the southern Longwood Range confirm that intrusions become progressively younger from east to west across the complex. A gabbro at Oraka Point (eastern end of coastal section) has an age of 245 ± 4 Ma and shows virtually no evidence of zircon inheritance. The age is significantly different from that of the Brook Street Terrane intrusives. Zircon ages from the western parts of the section are younger and more varied (203–227 Ma), indicating more complex magmatic histories. A leucogabbro dike from Pahia Point gives the youngest emplacement age of 142 Ma, which is similar to published U-Pb zircon ages for the Anglem Complex and Paterson Group on Stewart Island

Influence of oxidative stress, diaphragm fatigue, and inspiratory muscle training on the plasma cytokine response to maximum sustainable voluntary ventilation

The influence of oxidative stress, diaphragm fatigue, and inspiratory muscle training (IMT) on the cytokine response to maximum sustainable voluntary ventilation (MSVV) is unknown. Twelve healthy males were divided equally into an IMT or placebo (PLA) group, and before and after a 6-wk intervention they undertook, on separate days, 1h of (1) passive rest and (2) MSVV, whereby participants undertook volitional hyperpnea at rest that mimicked the breathing and respiratory muscle recruitment patterns commensurate with heavy cycling exercise. Plasma cytokines remained unchanged during passive rest. There was a main effect of time (P < 0.01) for plasma interleukin-1 (IL-1) and interleukin-6 (IL-6) concentrations and a strong trend (P = 0.067) for plasma interleukin-1 receptor antagonist concentration during MSVV. Plasma IL-6 concentration was reduced after IMT by 27 + 18% (main effect of intervention, P = 0.029), whereas there was no change after PLA (P = 0.753). There was no increase in a systemic marker of oxidative stress [DNA damage in peripheral blood mononuclear cells (PBMC)], and diaphragm fatigue was not related to the increases in plasma IL-1 and IL-6 concentrations. A dose-response relationship was observed between respiratory muscle work and minute ventilation and increases in plasma IL-6 concentration. In conclusion, increases in plasma IL-1 and IL-6 concentrations during MSVV were not due to diaphragm fatigue or DNA damage in PBMC. Increases in plasma IL-6 concentration during MSVV are attenuated following IMT, and the plasma IL-6 response is dependent upon the level of respiratory muscle work and minute ventilation

Natural selection on individual variation in tolerance of gastrointestinal nematode infection

This is the final version of the article. Available from the publisher via the DOI in this record.Hosts may mitigate the impact of parasites by two broad strategies: resistance, which limits parasite burden, and tolerance, which limits the fitness or health cost of increasing parasite burden. The degree and causes of variation in both resistance and tolerance are expected to influence host-parasite evolutionary and epidemiological dynamics and inform disease management, yet very little empirical work has addressed tolerance in wild vertebrates. Here, we applied random regression models to longitudinal data from an unmanaged population of Soay sheep to estimate individual tolerance, defined as the rate of decline in body weight with increasing burden of highly prevalent gastrointestinal nematode parasites. On average, individuals lost weight as parasite burden increased, but whereas some lost weight slowly as burden increased (exhibiting high tolerance), other individuals lost weight significantly more rapidly (exhibiting low tolerance). We then investigated associations between tolerance and fitness using selection gradients that accounted for selection on correlated traits, including body weight. We found evidence for positive phenotypic selection on tolerance: on average, individuals who lost weight more slowly with increasing parasite burden had higher lifetime breeding success. This variation did not have an additive genetic basis. These results reveal that selection on tolerance operates under natural conditions. They also support theoretical predictions for the erosion of additive genetic variance of traits under strong directional selection and fixation of genes conferring tolerance. Our findings provide the first evidence of selection on individual tolerance of infection in animals and suggest practical applications in animal and human disease management in the face of highly prevalent parasites.This study was funded by Natural Environment Research Council (http://www.nerc.ac.uk/) Standard Grant no. NE/G004854/1 and European Research Council (http://erc.europa.eu/) Large Grant no. 250098 to JMP. ADH is supported by European Research Council grant no. R/120448-11-1 to V. Lummaa; DHN is supported by Biotechnology and Biological Sciences Research Council (http://www.bbsrc.ac.uk/) David Phillips Fellowship no. BB/H021686/1; AJW is supported by BBSRC David Phillips Fellowship no. BB/G022976/1; ALG is supported by Princeton University and the Research and Policy for Infectious Disease Dynamics (RAPIDD) program of the Science and Technology Directorate, U.S. Department of Homeland Security, and the Fogarty International Center of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Extremely high He isotope ratios in MORB-source mantle from the proto-Iceland plume

The high &lt;sup&gt;3&lt;/sup&gt;He/&lt;sup&gt;4&lt;/sup&gt;He ratio of volcanic rocks thought to be derived from mantle plumes is taken as evidence for the existence of a mantle reservoir that has remained largely undegassed since the Earth's accretion. The helium isotope composition of this reservoir places constraints on the origin of volatiles within the Earth and on the evolution and structure of the Earth's mantle. Here we show that olivine phenocrysts in picritic basalts presumably derived from the proto-Iceland plume at Baffin Island, Canada, have the highest magmatic &lt;sup&gt;3&lt;/sup&gt;He/&lt;sup&gt;4&lt;/sup&gt;He ratios yet recorded. A strong correlation between &lt;sup&gt;3&lt;/sup&gt;He/&lt;sup&gt;4&lt;/sup&gt;He and &lt;sup&gt;87&lt;/sup&gt;Sr/&lt;sup&gt;86&lt;/sup&gt;Sr, &lt;sup&gt;143&lt;/sup&gt;Nd/&lt;sup&gt;144&lt;/sup&gt;Nd and trace element ratios demonstrate that the &lt;sup&gt;3&lt;/sup&gt;He-rich end-member is present in basalts that are derived from large-volume melts of depleted upper-mantle rocks. This reservoir is consistent with the recharging of depleted upper-mantle rocks by small volumes of primordial volatile-rich lower-mantle material at a thermal boundary layer between convectively isolated reservoirs. The highest &lt;sup&gt;3&lt;/sup&gt;He/&lt;sup&gt;4&lt;/sup&gt;He basalts from Hawaii and Iceland plot on the observed mixing trend. This indicates that a &lt;sup&gt;3&lt;/sup&gt;He-recharged depleted mantle (HRDM) reservoir may be the principal source of high &lt;sup&gt;3&lt;/sup&gt;He/&lt;sup&gt;4&lt;/sup&gt;He in mantle plumes, and may explain why the helium concentration of the 'plume' component in ocean island basalts is lower than that predicted for a two-layer, steady-state model of mantle structure

Identification of novel subgroup a variants with enhanced receptor binding and replicative capacity in primary isolates of anaemogenic strains of feline leukaemia virus

&lt;b&gt;BACKGROUND:&lt;/b&gt; The development of anaemia in feline leukaemia virus (FeLV)-infected cats is associated with the emergence of a novel viral subgroup, FeLV-C. FeLV-C arises from the subgroup that is transmitted, FeLV-A, through alterations in the amino acid sequence of the receptor binding domain (RBD) of the envelope glycoprotein that result in a shift in the receptor usage and the cell tropism of the virus. The factors that influence the transition from subgroup A to subgroup C remain unclear, one possibility is that a selective pressure in the host drives the acquisition of mutations in the RBD, creating A/C intermediates with enhanced abilities to interact with the FeLV-C receptor, FLVCR. In order to understand further the emergence of FeLV-C in the infected cat, we examined primary isolates of FeLV-C for evidence of FeLV-A variants that bore mutations consistent with a gradual evolution from FeLV-A to FeLV-C.&lt;p&gt;&lt;/p&gt; &lt;b&gt;RESULTS:&lt;/b&gt; Within each isolate of FeLV-C, we identified variants that were ostensibly subgroup A by nucleic acid sequence comparisons, but which bore mutations in the RBD. One such mutation, N91D, was present in multiple isolates and when engineered into a molecular clone of the prototypic FeLV-A (Glasgow-1), enhanced replication was noted in feline cells. Expression of the N91D Env on murine leukaemia virus (MLV) pseudotypes enhanced viral entry mediated by the FeLV-A receptor THTR1 while soluble FeLV-A Env bearing the N91D mutation bound more efficiently to mouse or guinea pig cells bearing the FeLV-A and -C receptors. Long-term in vitro culture of variants bearing the N91D substitution in the presence of anti-FeLV gp70 antibodies did not result in the emergence of FeLV-C variants, suggesting that additional selective pressures in the infected cat may drive the subsequent evolution from subgroup A to subgroup C.&lt;p&gt;&lt;/p&gt; &lt;b&gt;CONCLUSIONS:&lt;/b&gt; Our data support a model in which variants of FeLV-A, bearing subtle differences in the RBD of Env, may be predisposed towards enhanced replication in vivo and subsequent conversion to FeLV-C. The selection pressures in vivo that drive the emergence of FeLV-C in a proportion of infected cats remain to be established

Mice lacking NF-κB1 exhibit marked DNA damage responses and more severe gastric pathology in response to intraperitoneal tamoxifen administration

Tamoxifen (TAM) has recently been shown to cause acute gastric atrophy and metaplasia in mice. We have previously demonstrated that the outcome of Helicobacter felis infection, which induces similar gastric lesions in mice, is altered by deletion of specific NF-κB subunits. Nfkb1-/- mice developed more severe gastric atrophy than wild-type (WT) mice 6 weeks after H. felis infection. In contrast, Nfkb2-/- mice were protected from this pathology. We therefore hypothesized that gastric lesions induced by TAM may be similarly regulated by signaling via NF-κB subunits. Groups of five female C57BL/6 (WT), Nfkb1-/-, Nfkb2-/- and c-Rel-/- mice were administered 150 mg/kg TAM by IP injection. Seventy-two hours later, gastric corpus tissues were taken for quantitative histological assessment. In addition, groups of six female WT and Nfkb1-/- mice were exposed to 12 Gy γ-irradiation. Gastric epithelial apoptosis was quantified 6 and 48 h after irradiation. TAM induced gastric epithelial lesions in all strains of mice, but this was more severe in Nfkb1-/- mice than in WT mice. Nfkb1-/- mice exhibited more severe parietal cell loss than WT mice, had increased gastric epithelial expression of Ki67 and had an exaggerated gastric epithelial DNA damage response as quantified by γH2AX. To investigate whether the difference in gastric epithelial DNA damage response of Nfkb1-/- mice was unique to TAM-induced DNA damage or a generic consequence of DNA damage, we also assessed gastric epithelial apoptosis following γ-irradiation. Six hours after γ-irradiation, gastric epithelial apoptosis was increased in the gastric corpus and antrum of Nfkb1-/- mice. NF-κB1-mediated signaling regulates the development of gastric mucosal pathology following TAM administration. This is associated with an exaggerated gastric epithelial DNA damage response. This aberrant response appears to reflect a more generic sensitization of the gastric mucosa of Nfkb1-/- mice to DNA damage

The systematic guideline review: method, rationale, and test on chronic heart failure

Background: Evidence-based guidelines have the potential to improve healthcare. However, their de-novo-development requires substantial resources-especially for complex conditions, and adaptation may be biased by contextually influenced recommendations in source guidelines. In this paper we describe a new approach to guideline development-the systematic guideline review method (SGR), and its application in the development of an evidence-based guideline for family physicians on chronic heart failure (CHF). Methods: A systematic search for guidelines was carried out. Evidence-based guidelines on CHF management in adults in ambulatory care published in English or German between the years 2000 and 2004 were included. Guidelines on acute or right heart failure were excluded. Eligibility was assessed by two reviewers, methodological quality of selected guidelines was appraised using the AGREE instrument, and a framework of relevant clinical questions for diagnostics and treatment was derived. Data were extracted into evidence tables, systematically compared by means of a consistency analysis and synthesized in a preliminary draft. Most relevant primary sources were re-assessed to verify the cited evidence. Evidence and recommendations were summarized in a draft guideline. Results: Of 16 included guidelines five were of good quality. A total of 35 recommendations were systematically compared: 25/35 were consistent, 9/35 inconsistent, and 1/35 un-rateable (derived from a single guideline). Of the 25 consistencies, 14 were based on consensus, seven on evidence and four differed in grading. Major inconsistencies were found in 3/9 of the inconsistent recommendations. We re-evaluated the evidence for 17 recommendations (evidence-based, differing evidence levels and minor inconsistencies) - the majority was congruent. Incongruity was found where the stated evidence could not be verified in the cited primary sources, or where the evaluation in the source guidelines focused on treatment benefits and underestimated the risks. The draft guideline was completed in 8.5 man-months. The main limitation to this study was the lack of a second reviewer. Conclusion: The systematic guideline review including framework development, consistency analysis and validation is an effective, valid, and resource saving-approach to the development of evidence-based guidelines

A New Era in the Quest for Dark Matter

There is a growing sense of `crisis' in the dark matter community, due to the absence of evidence for the most popular candidates such as weakly interacting massive particles, axions, and sterile neutrinos, despite the enormous effort that has gone into searching for these particles. Here, we discuss what we have learned about the nature of dark matter from past experiments, and the implications for planned dark matter searches in the next decade. We argue that diversifying the experimental effort, incorporating astronomical surveys and gravitational wave observations, is our best hope to make progress on the dark matter problem.Comment: Published in Nature, online on 04 Oct 2018. 13 pages, 1 figur