CpG-based immunotherapy impairs antitumor activity of BRAF inhibitors in a B-cell-dependent manner.

Combining immunotherapy with targeted therapy has increasingly become an appealing therapeutic paradigm for cancer treatment due to its great potential for generating durable and synergistic antitumor response. In this study, however, we unexpectedly found that two types of CpG-based tumor peptide vaccine treatments consistently negated the antitumor activity of a selective BRAF inhibitor in tumors with BRAF mutation rather than showing a synergistic antitumor effect. Our further studies demonstrated that CpG alone was sufficient to dampen BRAF inhibitor-induced antitumor responses, suggesting that the impaired antitumor activity of the BRAF inhibitor observed in mice receiving CpG-based peptide vaccine is mainly dependent upon the use of CpG. Mechanistically, CpG increased the number of circulating B cells, which produced elevated amounts of tumor necrosis factor-α (TNFα) that contributed to the increased tumor resistance to BRAF inhibitors. More importantly, B-cell depletion or TNFα neutralization can restore the antitumor effect of BRAF inhibition in mice receiving CpG treatment, indicating that TNFα-secreting B cells play an indispensable role in BRAF inhibitor resistance induced by CpG. Taken together, our results strongly suggest that precautions must be implemented when designing combinatorial approaches for cancer treatment, because distinct regimens, despite their respective therapeutic benefit as monotherapy, may together provide antagonistic clinical outcomes.

Methanogenesis produces strong 13C enrichment in stromatolites of Lagoa Salgada, Brazil: a modern analogue for Palaeo-/Neoproterozoic stromatolites?

Holocene stromatolites characterized by unusually positive inorganic δ(13) CPDB values (i.e. up to +16‰) are present in Lagoa Salgada, a seasonally brackish to hypersaline lagoon near Rio de Janeiro (Brazil). Such positive values cannot be explained by phototrophic fixation of CO2 alone, and they suggest that methanogenesis was a dominating process during the growth of the stromatolites. Indeed, up to 5 mm methane was measured in the porewater. The archaeal membrane lipid archaeol showing δ(13) C values between -15 and 0‰ suggests that archaea are present and producing methane in the modern lagoon sediment. Moreover, (13) C-depleted hopanoids diplopterol and 3ß-methylated C32 17ß(H),21ß(H)-hopanoic acid (both -40‰) are preserved in lagoon sediments and are most likely derived from aerobic methanotrophic bacteria thriving in the methane-enriched water column. Loss of isotopically light methane through the water column would explain the residual (13) C-enriched pool of dissolved inorganic carbon from where the carbonate constituting the stromatolites precipitated. The predominance of methanogenic archaea in the lagoon is most likely a result of sulphate limitation, suppressing the activity of sulphate-reducing bacteria under brackish conditions in a seasonally humid tropical environment. Indeed, sulphate-reduction activity is very low in the modern sediments. In absence of an efficient carbonate-inducing metabolic process, we propose that stromatolite formation in Lagoa Salgada was abiotically induced, while the (13) C-enriched organic and inorganic carbon pools are due to methanogenesis. Unusually, (13) C-enriched stromatolitic deposits also appear in the geological record of prolonged periods in the Palaeo- and Neoproterozoic. Lagoa Salgada represents a possible modern analogue to conditions that may have been widespread in the Proterozoic, at times when low sulphate concentrations in sea water allowed methanogens to prevail over sulphate-reducing bacteria.

Models of intellectual disability: towards a perspective of (poss)ability.

BACKGROUND: The social and medical models of disability configure the relationship between disability and impairment differently. Neither of these models has provided a comprehensive theoretical or practical basis for talking about intellectual disability (ID). Models that emphasise the interactive nature of disability appear to be more promising. This study explores the ways in which models of disability are reflected in disability discourse in an empirical discourse analysis conducted in the Eastern Cape Province of South Africa. METHODS: Q methodology was used in this study as a discourse analysis tool. Adults with ID, parents of children with ID and professionals who work with people with ID completed a sorting task where they stated the degree to which they agreed or disagreed with statements that are made about people with ID. This exercise resulted in a pattern of responses for each participant, termed a Q sort and these were used as data in a factor analysis using dedicated Q method software. A second order factor analysis was then performed on the resulting factors. RESULTS: Four discourses were identified: the Social Model/Human Rights Discourse, the Medical Model/Professional Religious Discourse, the Community Model/Community Religious Discourse and the Interactive Discourse. Except for the last one, each of these discourses adopts a model of disability with a static view of impairment as fixed. The Interactive Discourse appears to be related to dynamic, environmental conceptions of disability where competence is built through social interaction. CONCLUSIONS: A theory of (poss)ability is proposed and some of its concerns are suggested. This perspective views impairment as an interaction between individuals and their environment and postulates that competence is a function of context, rather than a property of the individual.

Obstructive sleep apnoea and perioperative complications in bariatric patients.

BACKGROUND: The objective of this study was to determine the relationship between perioperative complications and the severity of obstructive sleep apnoea (OSA) in patients undergoing bariatric surgery who had undergone preoperative polysomnography (PSG). METHODS: The records of 797 patients, age >18 yr, who underwent bariatric operations (442 open and 355 laparoscopic procedures) at Mayo Clinic and were assessed before operation by PSG, were reviewed retrospectively. OSA was quantified using the apnoea-hypopnoea index (AHI) as none (≤ 4), mild (5-15), moderate (16-30), and severe (≥ 31). Pulmonary, surgical, and 'other' complications within the first 30 postoperative days were analysed according to OSA severity. Logistic regression was used to assess the multivariable association of OSA, age, sex, BMI, and surgical approach with postoperative complications. RESULTS: Most patients with OSA (93%) received perioperative positive airway pressure therapy, and all patients were closely monitored after operation with pulse oximetry on either regular nursing floors or in intensive or intermediate care units. At least one postoperative complication occurred in 259 patients (33%). In a multivariable model, the overall complication rate was increased with open procedures compared with laparoscopic. In addition, increased BMI and age were associated with increased likelihood of pulmonary and other complications. Complication rates were not associated with OSA severity. CONCLUSIONS: In obese patients evaluated before operation by PSG before bariatric surgery and managed accordingly, the severity of OSA, as assessed by the AHI, was not associated with the rate of perioperative complications. These results cannot determine whether unrecognized and untreated OSA increases risk.

Positional cloning of a cyromazine resistance gene in Drosophila melanogaster.

Cyromazine is an effective insecticide used to control dipteran insects. Its precise mode of action is yet to be determined, although it has been suggested that it interferes with the hormone system, sclerotization of the cuticle, or nucleic acid metabolism. To understand the way in which cyromazine acts, we have positionally cloned a cyromazine resistance gene from Drosophila melanogaster. Six cyromazine resistance alleles had previously been generated by ethyl methanasulphonate treatment. Two of these failed to complement each other and here we identify them as having independent non-sense mutations in CG32743, which is an ortholog of Smg1 of worms and mammals and encodes a phosphatidylinositol kinase-like kinase (PIKK). RNAi experiments confirm that cyromazine resistance can be achieved by knocking down CG32743. These are the first cyromazine resistant mutations identified at the nucleotide level. In mammals Smg1 phosphorylates P53 in response to DNA damage. This finding supports the hypothesis that cyromazine interferes with nucleic acid metabolism.

Molecular systematics and biogeography of the bent-wing bat complex Miniopterus schreibersii (Kuhl, 1817) (Chiroptera: Vespertilionidae).

The complete mitochondrial ND2 gene (1037 bp) was sequenced to examine relationships within the bent-wing bat complex, Miniopterus schreibersii (Family Vespertilionidae). It was found that M. schreibersii is a paraphyletic assemblage comprising several species. Two major lineages were identified, one of which was restricted to the Palearctic-Ethiopian regions and the other to the Oriental-Australasian regions. This pattern of differentiation was mirrored by the genus as a whole. Speciation and differentiation within the genus Miniopterus appears to have a hierarchical geographical pattern. The earliest divergence corresponds to the Ethiopian-Palearctic and the Oriental-Australasian biogeographical zones. This early divergence is then followed by radiations within each of the Ethiopian, Oriental and Australasian regions. The study also revealed that the number of species currently recognized (11 or 13) is a gross underestimate of the number of actual species. The emerging picture is one of a relatively speciose genus with most species having relatively restricted distributions; few, if any, occur in more than one biogeographical region.

A method for the extraction of ammonium from freshwaters for nitrogen isotope analysis.

The measurement of delta15N values of inorganic nitrogen species is an important analytical tool to trace nitrogen species in order to understand nitrogen cycling in aquatic systems. Nitrogen isotope analysis of freshwater ammonium has, however, been hindered by the lack of a simple and reliable technique to measure delta15N values at natural abundance levels. We present a simple and rapid method to concentrate ammonium from freshwater samples for on-line N-isotope ratio determination. Ammonium is collected by adsorption on N-free cation exchange resins. The dried N-loaded exchange resin is then directly combusted to produce N2 gas for subsequent delta15N analysis. The method was evaluated with simulated freshwater solutions containing varying amounts of standard NH4+-N (delta15N = 2.1 per thousand) and potentially interfering inorganic and organic compounds. In general, the cation exchange resin method gives accurate and reproducible delta15N values (sigma1 < 0.3 per thousand; n = 10). Because of adsorption interference, high concentrations of cations in solution may cause ammonium loss but do not result in measurable isotope fractionation. Replicate extractions of the ammonium standard added to water collected from four Swiss lakes demonstrate the good performance of this method when applied to low ionic strength natural water samples with modest concentrations of dissolved organic nitrogen.

Oligonucleotides with bistranded abasic sites interfere with substrate binding and catalysis by human apurinic/apyrimidinic endonuclease.

Apurinic/apyrimidinic endonuclease (AP endo) is a key enzyme in oxidative damage DNA repair. The enzyme, which repairs abasic sites, makes a single nick 5' to the phosphodeoxyribose, leaving a free 3'-hydroxyl. We recently described single turnover kinetics for human recombinant AP endo acting on an oligonucleotide with a single abasic site. We hypothesized that the structural changes induced by the presence of a second abasic site might provide insight into how AP endo recognizes the first abasic site. Here we performed steady state and single turnover experiments using bistranded abasic site substrates, with the second site located on the complementary strand to the one being followed and either opposite to the first or displaced in the 5' direction. All sites on the complementary strand were within half a helical turn of the first. The catalytic efficiency was reduced 80 to 96% and the Kd for substrate binding and dissociation was elevated 40- to 125-fold. The smaller changes occurred when the second site was opposite the first site or displaced by four nucleotides. In addition, if the second abasic site was directly across the helix or displaced by 1 or 3 nucleotides from the first abasic site, cleavage of the first abasic site was subject to apparent substrate inhibition, which did not occur if the second abasic site was displaced by four nucleotides from the first. While a substrate containing a nick without a phosphodeoxyribose on the contralateral strand abasic site did not inhibit nicking of the first strand, a substrate with a nicked abasic site on the contralateral strand was an even stronger inhibitor of enzyme action than an oligonucleotide containing the corresponding abasic site on each strand. Consequently, the inhibitory effect of the second abasic site is probably the result of prior cleavage of the abasic site on the contralateral strand with resulting distortions to the DNA helix that interfere with enzyme binding and/or cleavage.

Evaluation of the cleaning and disinfection efficacy of the DEKO-190; award-based automated washer/disinfector.

The DEKO-190 Washer-Disinfector combines both automatic washing and thermal disinfection functions and is designed for the decontamination of ward utensils (such as bedpans and urine bottles) and instruments used in minor surgery prior to sterilization. We undertook a microbiological evaluation of the disinfection efficacy of the machine, using its short wash plus disinfection programme and sealed suspensions of test organisms placed at various points within the instrument. Suspensions of Enterococcus faecalis and Poliovirus were totally inactivated, the counts of aerobic organisms within a stool specimen being reduced by a factor of 10(4)whilst spores of Clostridium perfringens were unaffected. The cleaning efficacy of the DEKO-190 was also evaluated under clinical conditions by visual inspection and was found to be satisfactory. Ward-based combined washer-disinfection machines, such as the DEKO-190, have the potential to improve the efficacy of cleaning protocols within healthcare institutions.

Wings and bristles: character specificity of the asymmetry phenotype in insecticide-resistant strains of Lucilia cuprina.

We investigated the hypothesis that observed higher levels of asymmetry displayed by insecticide-resistance genotypes of Lucilia cuprina are restricted to bristle characters, due to the action of resistance genes in bristle cell development, rather than through the disruption of genomic coadaptation. We compared the level of asymmetry of three bristle characters and three wing characters in non-modified and modified-resistance genotypes. Consistent with previous studies, resistance genotypes displayed greater levels of bristle asymmetry than either susceptible or modified genotypes. However, there were no differences among genotypes for any of the wing characters. To confirm that this result is attributable to the action of the resistance and modifier genes themselves, we also examined the responses of both bristle and wing characters to the more general developmental stress of extreme temperature. Sub-optimal temperature was shown to increase both bristle and wing asymmetry, suggesting that there are no underlying differences between the two character types which could, of themselves, explain the differential response observed in the resistance genotypes.

The character or the variation: the genetic analysis of the insecticide-resistance phenotype.

In this critique it is argued that the genetic basis of the evolution of resistance is dependent on how the phenotypic, and underlying genotypic, variation is channelled during a selective response. A polygenic response is preferentially favoured if selection acts within the phenotypic distribution of susceptibles; a monogenic response is predicted if selection screens rare mutations with phenotypes outside that susceptible distribution. The relevance of this model to the method of genetic analysis, the prediction of resistance mechanisms to novel insecticides, the generation of resistant beneficial insects and the development of the most effective resistance and integrated pest management programmes is discussed.

A genetic analysis of cyromazine resistance in Drosophila melanogaster (Diptera: Drosophilidae).

Flies resistant to the insect growth regulator cyromazine were selected in the F1 generation from a cyromazine-susceptible strain of Drosophila melanogaster (Meigen) treated with ethyl methanesulfonate. Four resistant strains were isolated by screening with cyromazine at a concentration > LC100 of susceptibles. In each strain, resistance is conferred by a single gene mutation. Cyromazine resistance in two of the mutants (rst(1a)cyr1 and rst(1a)cyr2) localizes to map position 17 of the X chromosome. Evidence is presented that these mutations are alleles of the gene rst(1a)cyr. Cyromazine resistance in another of the mutants (Rst(1b)Cyr) is also X-linked, and localizes to map position 49 of the X chromosome. The location of the gene conferring cyromazine resistance in the other mutant (Rst(2b)Cyr) is map position 66 of chromosome II. This is possibly an allele of a previously characterized cyromazine resistance gene, Rst(2)Cyr. Dosage-mortality analyses demonstrate a low level of cyromazine resistance is conferred in all strains.

Dieldrin resistance in Lucilia cuprina (the australian sheep blowfly): chance, selection and response.

Discrete-generation population cages of Lucilia cuprina were initiated with dieldrin-resistant allele (Rdl ) frequencies of 1 or 5% and maintained for 17 generations on media with concentrations of dieldrin in the range 0-0.006% (w/v). The probability of the initial establishment of the Rdl allele in a population was consistently greater at the 5% frequency and dependent on the concentration of dieldrin in the medium for both starting frequencies. Once the resistant allele was established responses to selection were concentration-dependent. It was concluded that in the absence of dieldrin the susceptible allele was selectively favoured, at 0. 00005% (w/v) concentration selection and random genetic drift influenced changes in allele frequency and at concentrations above this the Rdl allele was at a selective advantage. Fixation of Rdl occurred at the higher concentrations. The influence of random genetic drift and selection on the genetic response during the evolution of insecticide resistance is discussed.

Fluctuating asymmetry for specific bristle characters in notch mutants of Drosophila melanogaster.

Asymmetry has been used as a measure of developmental stability for bilaterally symmetrical organisms. Most studies have failed to partition the genetic and environmental contributions to the asymmetry phenotype due to the limitations of the systems used or the shortcomings in experimental design. The Notch mutants of Drosophila melanogaster were used to study the genetic contribution to asymmetry for six different bristle characters. Asymmetry response was character specific for the mutants examined. For Nspl, N(Co), N264-47, Ax71d, Ax9B2, AxE2, 1(1)N(B) and nd2 significant asymmetry responses, relative to wildtype Canton-S, were observed for some characters. N60g11 and nd1 did not exhibit significant asymmetry for any of the characters examined. All of the mutants except N60g11 and nd1 showed thoracic bristle asymmetry. However, when asymmetry scores were pooled over the five bristle characters which individually exhibited fluctuating asymmetry, no significant differences were found between any genotypes. Therefore pooling asymmetry values across characters obscures the significant character specific asymmetry values observed. Thus caution is necessary when using the asymmetry phenotype of specific characters to draw organism wide conclusions about developmental stability.

Biogeochemistry. Sulfate reducers--dominant players in a low-oxygen world?

Sulfate reducing bacteria can adapt to extreme physical and chemical conditions and play an important role in global geochemical cycles, but their role in the formation of ore deposits has remained controversial. Strong support for such a role is provided by Labrenz et al., who have discovered sulfate-reducing bacteria that can tolerate low levels of oxygen and can precipitate zinc sulfide minerals. The results may have implications for bioremediation and may provide clues to processes that may have been more widespread in the geologic past.

Risk-assessment scores, prevention strategies, and the incidence of pressure ulcers among the elderly in four Canadian health-care facilities.

The accurate prediction of pressure ulcer (PU) development among hospitalized elderly patients is a complex endeavour. A prospective, longitudinal, cohort study of 330 patients over age 65 in 2 Canadian tertiary-care teaching hospitals and 2 long-term-care facilities examined the association between risk-assessment scores, prevention strategies, and PU incidence. The overall PU incidence rate was 9.7%, with half of the subjects who developed a PU doing so in the first week of hospitalization. The incidence rate for "at risk" patients (10.1%) was similar to the rate for "not at risk" patients (9.3%). The number of prevention strategies used was related to risk-assessment scores and to PU development. Paradoxically, the incidence rate increased with the number of prevention strategies employed. The total risk-assessment score that appeared to have the best balance of sensitivity (69%) and specificity (55%) was 19. Four of the 6 risk-assessment subscales were associated with PU development. Logistic regression modelling confirmed the univariate results that the number of prevention strategies used was the best single predictor of PU development. The data confirm that predicting PU development for individual patients is difficult at best. Results suggest that use of a risk-assessment scale alone is not sufficient to accurately predict PU development. The clinical judgement and experience of nurses are required in providing supplementary information to standard measurement instruments.

Molecular characterization of the Notch homologue from the Australian sheep blowfly, Lucilia cuprina.

The Drosophila melanogaster Notch gene product as a receptor of intercellular signals and is central to cell fate specification. The Scalloped wings (Scl) gene is the homologue of Notch in the Australian sheep blowfly, Lucilia cuprina. An allele of Scl is thought to be involved in the modification of Darwinian fitness and bristle asymmetry in flies resistant to organophosphorous chemicals (OPs). As a first step towards the testing of this hypothesis we cloned and sequenced Scl. A full-length cDNA segment representing the mRNA of Scl is 8503 bp and encodes a protein of 2653 amino acids, which shares 73.6% identity with Notch. All functional motifs including EGF-like repeats, LNR repeats, cdc 10/ankyrin repeats, opa and PEST elements are present in the same order as in Notch and the sequence identities peak in these motifs. With respect to genomic structure, intron/exon boundaries are conserved but, in most cases, the Scl introns are larger. Sequence analysis of the upstream genomic region reveals that the gene has a TATA-less promoter. Consistent with a central role in embryogenesis and imaginal development, high levels of Scl expression were detected in the early embryonic and pupal stages.

Genotypically dependent effects of cyromazine on reproduction and offspring development in the Australian Lucilia cuprina (Diptera: Calliphoridae).

The effects of cyromazine on egg production and subsequent egg-to-adult survival were examined on susceptible, heterozygous, and homozygous cyromazine-resistant genotypes of the Australian sheep blowfly Lucilia cuprina (Wiedemann) by administering to adults 10 ppm of cyromazine in drinking water. Cyromazine reduced egg production, hatch, and subsequent larval survival in susceptible genotypes by acting at the embryonic stage of development. Resistance negated these effects dominantly for egg production and egg hatch and in a partially dominant manner for egg-to-adult survivorship.

Predicting insecticide resistance: mutagenesis, selection and response.

Strategies to manage resistance to a particular insecticide have usually been devised after resistance has evolved. If it were possible to predict likely resistance mechanisms to novel insecticides before they evolved in the field, it might be feasible to have programmes that manage susceptibility. With this approach in mind, single-gene variants of the Australian sheep blowfly, Lucilia cuprina, resistant to dieldrin, diazinon and malathion, were selected in the laboratory after mutagenesis of susceptible strains. The genetic and molecular bases of resistance in these variants were identical to those that had previously evolved in natural populations. Given this predictive capacity for known resistances, the approach was extended to anticipate possible mechanisms of resistance to cyromazine, an insecticide to which L. cuprina populations remain susceptible after almost 20 years of exposure. Analysis of the laboratory-generated resistant variants provides an explanation for this observation. The variants show low levels of resistance and a selective advantage over susceptibles for only a limited concentration range. These results are discussed in the context of the choice of insecticides for control purposes and of delivery strategies to minimize the evolution of resistance.

Stress and asymmetry during arrested development of the Australian sheep blowfly.

The dieldrin and diazinon resistance systems of the Australian sheep blowfly (Lucilia cuprina) have been used previously to relate stress, departures from bilateral symmetry, developmental stability and relative fitness. These systems are now used to consider stress and asymmetry in a developmental context. Larval to adult development is shown to be significantly impaired after arrested development at 8 degrees C, however the asymmetry score of adults of a given genotype is similar after arrested or continuous development. Selection against dieldrin-resistant and unmodified diazinon-resistant genotypes occurs during arrested development because greater proportions of these genotypes pupae at 8 degrees C than do susceptible or modified diazinon-resistant genotypes. Pre-pupae of all genotypes complete development equally successfully when transferred from 8 degrees C to 27 degrees C. Adults fail to emerge when pupae formed at 8 degrees C undergo this temperature transition. Temperature-shift experiments show the asymmetry score is determined between pre-pupal and pupal stages of the life cycle. This stage occurs at 27 degrees C in arrested and continuously developing cultures providing an explanation for the independence of stress, selective mortality during developmental arrest and asymmetry score. The results emphasize the need for genetic, environmental and developmental data before an asymmetry phenotype can be directly related to developmental stability and relative fitness.