Proteomic analysis of Artemisia annua – towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin

Background: MS-based proteomics was applied to the analysis of the medicinal plant Artemisia annua, exploiting a recently published contig sequence database (Graham et al. (2010) Science 327, 328–331) and other genomic and proteomic sequence databases for comparison. A. annua is the predominant natural source of artemisinin, the precursor for artemisinin-based combination therapies (ACTs), which are the WHO-recommended treatment for P. falciparum malaria. Results: The comparison of various databases containing A. annua sequences (NCBInr/viridiplantae, UniProt/ viridiplantae, UniProt/A. annua, an A. annua trichome Trinity contig database, the above contig database and another A. annua EST database) revealed significant differences in respect of their suitability for proteomic analysis, showing that an organism-specific database that has undergone extensive curation, leading to longer contig sequences, can greatly increase the number of true positive protein identifications, while reducing the number of false positives. Compared to previously published data an order-of-magnitude more proteins have been identified from trichome-enriched A. annua samples, including proteins which are known to be involved in the biosynthesis of artemisinin, as well as other highly abundant proteins, which suggest additional enzymatic processes occurring within the trichomes that are important for the biosynthesis of artemisinin. Conclusions: The newly gained information allows for the possibility of an enzymatic pathway, utilizing peroxidases, for the less well understood final stages of artemisinin’s biosynthesis, as an alternative to the known non-enzymatic in vitro conversion of dihydroartemisinic acid to artemisinin. Data are available via ProteomeXchange with identifier PXD000703.

When enough is not enough: information overload and metacognitive decisions to stop studying information

People are often exposed to more information than they can actually remember. Despite this frequent form of information overload, little is known about how much information people choose to remember. Using a novel “stop” paradigm, the current research examined whether and how people choose to stop receiving new—possibly overwhelming—information with the intent to maximize memory performance. Participants were presented with a long list of items and were rewarded for the number of correctly remembered words in a following free recall test. Critically, participants in a stop condition were provided with the option to stop the presentation of the remaining words at any time during the list, whereas participants in a control condition were presented with all items. Across five experiments, we found that participants tended to stop the presentation of the items to maximize the number of recalled items, but this decision ironically led to decreased memory performance relative to the control group. This pattern was consistent even after controlling for possible confounding factors (e.g., task demands). The results indicated a general, false belief that we can remember a larger number of items if we restrict the quantity of learning materials. These findings suggest people have an incomplete understanding of how we remember excessive amounts of information.

Margin squeeze: an above cost predatory pricing approach

We provide a new legal perspective for the antitrust analysis of margin squeeze conducts. Building on recent economic analysis, we explain why margin squeeze conducts should solely be evaluated under adjusted predatory pricing standards. The adjustment corresponds to an increase in the cost benchmark used in the predatory pricing test by including opportunity costs due to missed upstream sales. This can reduce both the risks of false-positives and false-negatives in margin squeeze cases. We justify this approach by explaining why classic arguments against above-cost predatory pricing typically do not hold in vertical structures where margin squeezes take place and by presenting case law evidence supporting this adjustment. Our approach can help to reconcile the divergent US and EU antitrust stances on margin squeeze.

Pragmatic abilities of high-functioning Greek-speaking children with autism

This paper reports on the findings of the pragmatic abilities of Greek-speaking children with autism spectrum disorders (ASD). Twenty high functioning children with ASD and their typically developing age and vocabulary controls were administered a pragmatics task. The task was based on the Diagnostic Evaluation of Language Variation (DELV) in the context of a larger study targeting the grammar of Greek-speaking children with autism, and assessed the children’s abilities in communicative role taking, narrative, and question asking. The children with ASD showed an uneven profile in their pragmatic abilities. The two groups did not differ in communicative role taking and question asking. However, the children with ASD had difficulties on the narrative task, and more specifically, on the items assessing reference contrast and temporal links. Yet, they performed similarly on the mental state representations and the false beliefs items. Despite their good performance on mental states and false beliefs, the ASD children’s lower performance on reference contrast can be interpreted via Theory of Mind deficits if we assume that the former involve an additional level of complexity; namely, quantifying the amount of information available to the listener. Lower performance on temporal links is in line with the ASD children’s attested difficulties in organizing events into a coherent gist. Their overall profile, and, in particular, the dissociation between the different sections of the task, does not support single deficit accounts. It rather indicates that the deficits of individuals with ASD stem from distinct deficits in core cognitive processes (Happé & Frith, 2006).

An approximate dynamic programming approach for improving accuracy of lossy data compression by Bloom filters

Bloom filters are a data structure for storing data in a compressed form. They offer excellent space and time efficiency at the cost of some loss of accuracy (so-called lossy compression). This work presents a yes-no Bloom filter, which as a data structure consisting of two parts: the yes-filter which is a standard Bloom filter and the no-filter which is another Bloom filter whose purpose is to represent those objects that were recognised incorrectly by the yes-filter (that is, to recognise the false positives of the yes-filter). By querying the no-filter after an object has been recognised by the yes-filter, we get a chance of rejecting it, which improves the accuracy of data recognition in comparison with the standard Bloom filter of the same total length. A further increase in accuracy is possible if one chooses objects to include in the no-filter so that the no-filter recognises as many as possible false positives but no true positives, thus producing the most accurate yes-no Bloom filter among all yes-no Bloom filters. This paper studies how optimization techniques can be used to maximize the number of false positives recognised by the no-filter, with the constraint being that it should recognise no true positives. To achieve this aim, an Integer Linear Program (ILP) is proposed for the optimal selection of false positives. In practice the problem size is normally large leading to intractable optimal solution. Considering the similarity of the ILP with the Multidimensional Knapsack Problem, an Approximate Dynamic Programming (ADP) model is developed making use of a reduced ILP for the value function approximation. Numerical results show the ADP model works best comparing with a number of heuristics as well as the CPLEX built-in solver (B&B), and this is what can be recommended for use in yes-no Bloom filters. In a wider context of the study of lossy compression algorithms, our researchis an example showing how the arsenal of optimization methods can be applied to improving the accuracy of compressed data.

High-throughput monitoring of wild bee diversity and abundance via mitogenomics

1. Bee populations and other pollinators face multiple, synergistically acting threats, which have led to population declines, loss of local species richness and pollination services, and extinctions. However, our understanding of the degree, distribution and causes of declines is patchy, in part due to inadequate monitoring systems, with the challenge of taxonomic identification posing a major logistical barrier. Pollinator conservation would benefit from a high-throughput identification pipeline. 2. We show that the metagenomic mining and resequencing of mitochondrial genomes (mitogenomics) can be applied successfully to bulk samples of wild bees. We assembled the mitogenomes of 48 UK bee species and then shotgun-sequenced total DNA extracted from 204 whole bees that had been collected in 10 pan-trap samples from farms in England and been identified morphologically to 33 species. Each sample data set was mapped against the 48 reference mitogenomes. 3. The morphological and mitogenomic data sets were highly congruent. Out of 63 total species detections in the morphological data set, the mitogenomic data set made 59 correct detections (93�7% detection rate) and detected six more species (putative false positives). Direct inspection and an analysis with species-specific primers suggested that these putative false positives were most likely due to incorrect morphological IDs. Read frequency significantly predicted species biomass frequency (R2 = 24�9%). Species lists, biomass frequencies, extrapolated species richness and community structure were recovered with less error than in a metabarcoding pipeline. 4. Mitogenomics automates the onerous task of taxonomic identification, even for cryptic species, allowing the tracking of changes in species richness and istributions. A mitogenomic pipeline should thus be able to contain costs, maintain consistently high-quality data over long time series, incorporate retrospective taxonomic revisions and provide an auditable evidence trail. Mitogenomic data sets also provide estimates of species counts within samples and thus have potential for tracking population trajectories.

Meta-analysis to integrate effect sizes within a paper: Possible misuse and Type-1 error inflation

In recent years an increasing number of papers have employed meta-analysis to integrate effect sizes of researchers’ own series of studies within a single paper (“internal meta-analysis”). Although this approach has the obvious advantage of obtaining narrower confidence intervals, we show that it could inadvertently inflate false-positive rates if researchers are motivated to use internal meta-analysis in order to obtain a significant overall effect. Specifically, if one decides whether to stop or continue a further replication experiment depending on the significance of the results in an internal meta-analysis, false-positive rates would increase beyond the nominal level. We conducted a set of Monte-Carlo simulations to demonstrate our argument, and provided a literature review to gauge awareness and prevalence of this issue. Furthermore, we made several recommendations when using internal meta-analysis to make a judgment on statistical significance.

The changing influence of societal culture on job satisfaction across Europe

This paper contributes to the growing multidisciplinary body of literature on subjective well-being by investigating the longitudinal stability and impact of societal cultural values (SCVs) – as opposed to the more common organizational values – on job satisfaction. It is assumed that SCVs evolve slowly; hence, their impact on job satisfaction is likely to remain stable over time. False adherence to this assumption could cause misalignment between organizational policies/practices and expectations formed by societal culture, decreasing job satisfaction and adversely affecting productivity, competiveness and prosperity. Four waves of the European Values Study are used to examine whether SCVs have evolved and their impacts on job satisfaction over a relatively short time: 1981–2008. SCVs are parameterized through reference to traditional vs secular-rational, and survival vs self-expression value continuums. Results indicate that the strength of many SCVs has declined, the impacts of traditional societal values on job satisfaction have remained fairly constant, and the impacts of survival societal values on job satisfaction have declined substantially over this sample period. These reductions in SCVs amplify the importance of accounting for such changes when designing new or adjusting existing policies/practices to enhance job satisfaction and stimulate improvements in productivity, competitiveness and prosperity.

Non-nutritive sucking scoring system for preterm newborns

Aim: To validate a non-nutritive sucking (NNS) scoring system for oral feeding in preterm newborns (PTNB). Methods: A cohort study was carried out in two phases. In phase one of the study, 22 mastered speech-language pathologists received the protocol and procedure for a NNS scoring system to evaluate the content and presentation of the form and to define the grading scale. In phase two, six speech-language pathologists evaluated 51 PTNBs weekly, using the defined scoring system. Setting: This study was carried out in the Nursery Annex to the Maternity at the Intensive and Neonatal Pediatrics Service, Instituto da Crianca, Hospital das Clinicas, School of Medicine, University of Sao Paulo (FMUSP) during the period from May 2004 to May 2006. Participants: A total of 28 speech-language pathologist experts and 51 PTNBs. Results: In the first phase of the study, 22 speech-language pathologists selected the criteria, utilized in the NNS evaluation with 80% agreement. In the second phase of the study, the NNS evaluation was carried out on 51 PTNB, and a scoring system of 50 points was proposed, which corresponds to the smallest number of false positive and negative results regarding oral feeding ability. Conclusion: An NNS evaluation system was validated that was able to indicate when oral feeding could safely begin in PTNBs with a high level of agreement among the speech-language pathologists who have participated.

Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis

The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1). B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis. (c) 2008 Elsevier Ltd. All rights reserved.

Comparison of Neck Skin Excision and Whole Carcass Rinse Sampling Methods for Microbiological Evaluation of Broiler Carcasses before and after Immersion Chilling

Sampling protocols for detecting Salmonella on poultry differ among various countries. In the United States, the U.S. Department of Agriculture Food Safety and Inspection Service dictates that whole broiler carcasses should be rinsed with 400 ml of 1% buffered peptone water, whereas in the European Union 25-g samples composed of neck skin from three carcasses are evaluated. The purpose of this study was to evaluate a whole carcass rinse (WCR) and a neck skin excision (NS) procedure for Salmonella and Escherichia coli isolation from the same broiler carcass. Carcasses were obtained from three broiler processing plants. The skin around the neck area was aseptically removed and bagged separately from the carcass, and microbiological analysis was performed. The corresponding carcass was bagged and a WCR sample was evaluated. No significant difference (alpha <= 0.05) in Salmonella prevalence was found between the samples processed by the two methods, but both procedures produced many false-negative Salmonella results. Prechill, 37% (66 carcasses), 28% (50 carcasses), and 51% (91 carcasses) of the 180 carcasses examined were positive for Salmonella by WCR, NS, and both procedures combined, respectively. Postchill, 3% (5 carcasses), 7% (12 carcasses), and 10% (17 carcasses) of the 177 carcasses examined were positive for Salmonella by the WCR, NS, and combination of both procedures, respectively. Prechill, E. coli plus coliform counts were 3.0 and 2.6 log CFU/ml by the WCR and NS methods, respectively. Postchill. E. coli plus coliform counts were 1.7 and 1.4 log CFU/ml by the WCR and NS methods, respectively.

One-step reverse transcriptase polymerase chain reaction for the diagnosis of respiratory syncytial virus in children

Human respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory tract infections in infants and children. Rapid diagnosis is required to permit appropriate care and treatment and to avoid unnecessary antibiotic use. Reverse transcriptase (RT-PCR) and indirect immunofluorescence assay (IFA) methods have been considered important tools for virus detection due to their high sensitivity and specificity. In order to maximize use-simplicity and minimize the risk of sample cross-contamination inherent in two-step techniques, a RT-PCR method using only a single tube to detect HRSV in clinical samples was developed. Nasopharyngeal aspirates from 226 patients with acute respiratory illness, ranging from infants to 5 years old, were collected at the University Hospital of the University of Sao Paulo (HU-USP), and tested using IFA, one-step RT-PCR, and semi-nested RT-PCR. One hundred and two (45.1%) samples were positive by at least one of the three methods, and 75 (33.2%) were positive by all methods: 92 (40.7%) were positive by one-step RT-PCR, 84 (37.2%) by IFA, and 96 (42.5%) by the semi-nested RT-PCR technique. One-step RT-PCR was shown to be fast, sensitive, and specific for RSV diagnosis, without the added inconvenience and risk of false positive results associated with semi-nested PCR. The combined use of these two methods enhances HRSV detection. (C) 2007 Elsevier B.V. All rights reserved.

Notes on distribution, variation and characterization of Erythrolamprus pseudocorallus Roze, 1959 (Serpentes: Colubridae) with the first records from Colombia

The Maracaibo false coral snake Erythrolamprus pseudocorallus, previously known only from Venezuela, is recorded from five departments in Colombia. These new data include the westernmost and the southernmost records presently known for the species. Two specimens previously identified as E. aesculapii, from the localities of El Valle, Distrito Federal, Venezuela, and Yarumal, Antioquia, Colombia, are now attributed to E. pseudocorallus, the first one representing the northeasternmost record of the species. Morphological characterization of E. pseudocorallus is expanded based on the new specimens.

Next Generation Sequencing of Pooled Samples Reveals New SNRNP200 Mutations Associated with Retinitis Pigmentosa

The gene SNRNP200 is composed of 45 exons and encodes a protein essential for pre-mRNA splicing, the 200 kDa helicase hBrr2. Two mutations in SNRNP200 have recently been associated with autosomal dominant retinitis pigmentosa (adRP), a retinal degenerative disease, in two families from China. In this work we analyzed the entire 35-Kb SNRNP200 genomic region in a cohort of 96 unrelated North American patients with adRP. To complete this large-scale sequencing project, we performed ultra high-throughput sequencing of pooled, untagged PCR products. We then validated the detected DNA changes by Sanger sequencing of individual samples from this cohort and from an additional one of 95 patients. One of the two previously known mutations (p.S1087L) was identified in 3 patients, while 4 new missense changes (p.R681C, p.R681H, p.V683L, p.Y689C) affecting highly conserved codons were identified in 6 unrelated individuals, indicating that the prevalence of SNRNP200-associated adRP is relatively high. We also took advantage of this research to evaluate the pool-and-sequence method, especially with respect to the generation of false positive and negative results. We conclude that, although this strategy can be adopted for rapid discovery of new disease-associated variants, it still requires extensive validation to be used in routine DNA screenings. (C) 2011 Wiley-Liss, Inc.

Separating the wheat from the chaff: unbiased filtering of background tandem mass spectra improves protein identification

Only a small fraction of spectra acquired in LC-MS/MS runs matches peptides from target proteins upon database searches. The remaining, operationally termed background, spectra originate from a variety of poorly controlled sources and affect the throughput and confidence of database searches. Here, we report an algorithm and its software implementation that rapidly removes background spectra, regardless of their precise origin. The method estimates the dissimilarity distance between screened MS/MS spectra and unannotated spectra from a partially redundant background library compiled from several control and blank runs. Filtering MS/MS queries enhanced the protein identification capacity when searches lacked spectrum to sequence matching specificity. In sequence-similarity searches it reduced by, on average, 30-fold the number of orphan hits, which were not explicitly related to background protein contaminants and required manual validation. Removing high quality background MS/MS spectra, while preserving in the data set the genuine spectra from target proteins, decreased the false positive rate of stringent database searches and improved the identification of low-abundance proteins.