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Younger and older adults were tested for their ability to process and retrieve information from texts. The authors focused on the construction and retrieval of situation models relative to other types of text representations. The results showed that during memory retrieval, younger adults showed superior memory for surface form and textbase knowledge (what the text was), whereas older adults had equivalent or superior memory for situation model information (what the text was about). The results also showed that during reading, older and younger adults were similar in their sensitivity to various aspects of the texts. Overall, these findings suggest that although there are age-related declines in the processing and memory for text-based information, for higher level representations, these abilities appear to be preserved. Several possibilities for why this is the case are discussed, including an in-depth consideration of one possibility that involves W. Kintsch's (1988) construction-integration model. [ABSTRACT FROM AUTHOR]

Tremendous advances in mass spectrometric and bioinformatic approaches have expanded proteomics into the field of microbial ecology. The commonly used spectral annotation method for metaproteomics data relies on database searching, which requires sample-specific databases obtained from whole metagenome sequencing experiments. However, creating these databases is complex, time-consuming, and prone to errors, potentially biasing experimental outcomes and conclusions. This asks for alternative approaches that can provide rapid and orthogonal insights into metaproteomics data. Here, we present NovoLign, a de novo metaproteomics pipeline that performs sequence alignment of de novo sequences from complete metaproteomics experiments. The pipeline enables rapid taxonomic profiling of complex communities and evaluates the taxonomic coverage of metaproteomics outcomes obtained from database searches. Furthermore, the NovoLign pipeline supports the creation of reference sequence databases for database searching to ensure comprehensive coverage. We assessed the NovoLign pipeline for taxonomic coverage and false positive annotations using a wide range of in silico and experimental data, including pure reference strains, laboratory enrichment cultures, synthetic communities, and environmental microbial communities. In summary, we present NovoLign, a de novo metaproteomics pipeline that employs large-scale sequence alignment to enable rapid taxonomic profiling, evaluation of database searching outcomes, and the creation of reference sequence databases. The NovoLign pipeline is publicly available via: https://github.com/hbckleikamp/NovoLign., Competing Interests: The authors declare that they have no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Mycobacterium tuberculosis complex (MTBC) whole genome sequencing (WGS) turnaround time and WGS success rates are highly influenced by DNA extraction protocols even from cultures. Efficient mycobacterial lysis is crucial for obtaining sufficient DNA from cultures to facilitate reliable genomic drug susceptibility prediction and accurate genotyping with WGS. We compared four DNA extraction protocols from BD BACTEC™ Mycobacterial Growth Indicator Tubes (MGIT) for WGS with a focus on the lysis step: protocol A) column-based protocol without mechanical lysis; protocol B) an adapted protocol including a bead beating step; protocol C) DNA extraction from primary received cultures using bead beating: and protocol D) DNA extraction from pre MGIT-positive (enriched) cultures. Protocol B increased DNA yield approximately 60-fold, and significantly improved the sequencing success rate. The increased yield also allowed DNA extraction from primary cultures with high success rates (protocol C). Additionally, by using pre-positive enriched MGIT cultures, we demonstrated that bead beating opens the possibility of reliable WGS up to five days before a MGIT tube would be flagged positive (protocol D). The most optimal bead beating-based DNA extraction was also evaluated for Nanopore sequencing. Shortening bead beating duration to 15 s resulted in longer read lengths (N50 from 1.4 kb to 2.6 kb) while still providing efficient lysis. Furthermore, AmpureXP bead beating-based DNA capture / purification proved to be as efficient as Qiagen column-based DNA extraction, further simplifying and shortening the DNA extraction protocol. Adding a mechanical lysis step to our routine MTBC DNA extraction protocol has allowed us to reduce the turnaround time while maintaining DNA quality sequencing success rates., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kristin Kremer, Noud Hermans, Richard Anthony reports financial support was provided by Netherlands National Postcode Lottery. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The tremendous progress in sequencing technologies has made DNA sequencing routine for microbiome studies. Additionally, advances in mass spectrometric techniques have extended conventional proteomics into the field of microbial ecology. However, systematic studies that provide a better understanding of the complementary nature of these 'omics' approaches, particularly for complex environments such as wastewater treatment sludge, are urgently needed. Here, we describe a comparative metaomics study on aerobic granular sludge from three different wastewater treatment plants. For this, we employed metaproteomics, whole metagenome, and 16S rRNA amplicon sequencing to study the same granule material with uniform size. We furthermore compare the taxonomic profiles using the Genome Taxonomy Database (GTDB) to enhance the comparability between the different approaches. Though the major taxonomies were consistently identified in the different aerobic granular sludge samples, the taxonomic composition obtained by the different omics techniques varied significantly at the lower taxonomic levels, which impacts the interpretation of the nutrient removal processes. Nevertheless, as demonstrated by metaproteomics, the genera that were consistently identified in all techniques cover the majority of the protein biomass. The established metaomics data and the contig classification pipeline are publicly available, which provides a valuable resource for further studies on metabolic processes in aerobic granular sludge., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Salt stress is one of the unfavorable environmental factors to affect plants. Salinity represses root growth, resulting in reduced biomass of agricultural plants. Little is known about how plants maintain root growth to counteract salt stress. The AP2-domain transcription factors PLETHORA1/2 (PLT1/2) act as master regulators in root meristem maintenance in Arabidopsis. In this study, we report that the salt overly sensitive (SOS) pathway component SOS2 regulates PLT1/2 at the post-transcriptional level. Salt-activated SOS2 interacts and phosphorylates PLT1/2 through their conserved C-terminal motifs to stabilize PLT1/2, critical for root apical meristem maintenance under salt stress. The phospho-mimetic version of PLT1/2 restored meristem and primary root length reduction of sos2-2 and plt1-4 plt2-2 mutants on salt treatment. Moreover, SOS2-mediated PLT1/2 phosphorylation improves root growth recovery after salt stress alleviation. We identify a SOS2-PLT1/2 core protein module that is required for protecting primary root growth and meristem maintenance from salt stress., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The routine use of whole genome sequencing (WGS) as a reference typing technique for Mycobacterium tuberculosis epidemiology combined with the catalogued and extensive knowledge base of resistance-associated mutations means an initial susceptibility prediction can be derived from all cultured isolates in our laboratories based on WGS data alone. Preliminary work has confirmed, in our low-burden settings, these predictions are for first-line drugs, reproducible, robust with an accuracy similar to phenotypic drug susceptibility testing (pDST) and in many cases able to also predict the level of resistance (MIC). Routine screening for drug resistance by WGS results in approximately 80% of the isolates received being predicted as fully susceptible to the first-line drugs. Parallel testing with both WGS and pDST has demonstrated that routine pDST of genotypically fully susceptible isolates yields minimal additional information. Thus, rather than re-confirming all fully sensitive WGS-based predictions, we suggest that a more efficient use of available mycobacterial culture capacity in our setting is the development of a more extensive and detailed pDST targeted at any mono or multi-drug-resistant isolates identified by WGS screening. Phenotypic susceptibility retains a key role in the determination of an extended susceptibility profile for mono/multi-drugresistant isolates identified by WGS screening. The pDST information collected is also needed to support the development of future catalogues of resistance-associated mutations.

Plants can express different phenotypic responses following polyploidization, but ploidy-dependent phenotypic variation has so far not been assigned to specific genetic factors. To map such effects, segregating populations at different ploidy levels are required. The availability of an efficient haploid inducer line in Arabidopsis thaliana allows for the rapid development of large populations of segregating haploid offspring. Because Arabidopsis haploids can be self-fertilised to give rise to homozygous doubled haploids, the same genotypes can be phenotyped at both the haploid and diploid ploidy level. Here, we compared the phenotypes of recombinant haploid and diploid offspring derived from a cross between two late flowering accessions to map genotype × ploidy (G × P) interactions. Ploidy-specific quantitative trait loci (QTLs) were detected at both ploidy levels. This implies that mapping power will increase when phenotypic measurements of monoploids are included in QTL analyses. A multi-trait analysis further revealed pleiotropic effects for a number of the ploidy-specific QTLs as well as opposite effects at different ploidy levels for general QTLs. Taken together, we provide evidence of genetic variation between different Arabidopsis accessions being causal for dissimilarities in phenotypic responses to altered ploidy levels, revealing a G × P effect. Additionally, by investigating a population derived from late flowering accessions, we revealed a major vernalisation-specific QTL for variation in flowering time, countering the historical bias of research in early flowering accessions.

Here, we report the development and key features of the first external quality assessment (EQA) scheme for Mycobacterium tuberculosis whole-genome sequencing (WGS). The results of four rounds (2017 to 2020) of implementation within the European tuberculosis reference laboratories network (ERLTB-Net-2) are presented and discussed. EQA panels comprising 10 genomic DNAs were distributed to ERLTB-Net 2 laboratories volunteering to participate in this exercise. Since 2018, five FASTQ files were added to better assess the dry WGS processes, and in 2020, three of the five files were replaced by synthetic files (providing additional flexibility for the mutations included in the panels). Ten National tuberculosis reference laboratories participated in all four EQA rounds, and seven participated in at least one. High-confidence resistance mutations were correctly identified by all laboratories, but challenges remained with respect to the identification of mixed loci and interpretation of rare mutations. M. tuberculosis genotyping and clustering analysis was >90% accurate for pure samples with the main challenges being related to the analysis of mixed genotypes and DNA FASTQ files. The development and implementation of this WGS EQA scheme has contributed to the continuous improvement in performance of participating laboratories in M. tuberculosis WGS and data analysis. This scheme can serve as a model of comprehensive quality assessment for M. tuberculosis WGS that can be replicated in different settings worldwide. IMPORTANCE The wider availability of whole-genome sequencing (WGS) coupled to new developments in bioinformatic tools and databases to interpret Mycobacterium tuberculosis complex WGS data has accelerated the adoption of this method for the routine prediction of antimycobacterial drug resistance and genotyping, thus necessitating the establishment of a comprehensive external quality control system. Here, we report 4 years of development and results from such a panel.

In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.

Plant breeding applications exploiting meiotic mutant phenotypes (like the increase or decrease of crossover (CO) recombination) have been proposed over the last years. As recessive meiotic mutations in breeding lines may affect fertility or have other pleiotropic effects, transient silencing techniques may be preferred. Reverse breeding is a breeding technique that would benefit from the transient downregulation of CO formation. The technique is essentially the opposite of plant hybridization: a method to extract parental lines from a hybrid. The method can also be used to efficiently generate chromosome substitution lines (CSLs). For successful reverse breeding, the two homologous chromosome sets of a heterozygous plant must be divided over two haploid complements, which can be achieved by the suppression of meiotic CO recombination and the subsequent production of doubled haploid plants. Here we show the feasibility of transiently reducing CO formation using virus-induced gene silencing (VIGS) by targeting the meiotic gene MSH5 in a wild-type heterozygote of Arabidopsis thaliana. The application of VIGS (rather than using lengthy stable transformation) generates transgene-free offspring with the desired genetic composition: we obtained parental lines from a wild-type heterozygous F 1 in two generations. In addition, we obtained 20 (of the 32 possible) CSLs in one experiment. Our results demonstrate that meiosis can be modulated at will in A. thaliana to generate CSLs and parental lines rapidly for hybrid breeding. Furthermore, we illustrate how the modification of meiosis using VIGS can open routes to develop efficient plant breeding strategies., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

A wide variety of multicellular organisms across the kingdoms display remarkable ability to restore their tissues or organs when they suffer damage. However, the ability to repair damage is not uniformly distributed throughout body parts. Here, we unravel the elusive mechanistic basis of boundaries on organ regeneration potential using root tip resection as a model and show that the dosage of gradient-expressed PLT2 transcription factor is the underlying cause. While transient downregulation of PLT2 in distinct set of plt mutant backgrounds renders meristematic cells incapable of regeneration, forced expression of PLT2 acts through auto-activation to confer regeneration potential to the cells undergoing differentiation. Surprisingly, sustained exposure to nuclear PLT2, beyond a threshold, leads to reduction of regeneration potential despite giving rise to longer meristem. Our studies reveal dosage-dependent role of gradient-expressed PLT2 in root tip regeneration and uncouple the size of an organ from its regeneration potential., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Drug-susceptibility testing (DST) of Mycobacterium tuberculosis complex (MTBC) isolates by the Mycobacteria Growth Indicator Tube (MGIT) approach is the most widely applied reference standard. However, the use of WGS is increasing in many developed countries to detect resistance and predict susceptibility. We investigated the reliability of WGS in predicting drug susceptibility, and analysed the discrepancies between WGS and MGIT against the first-line drugs rifampicin, isoniazid, ethambutol and pyrazinamide., Methods: DST by MGIT and WGS was performed on MTBC isolates received in 2016/2017. Nine genes and/or their promotor regions were investigated for resistance-associated mutations: rpoB, katG, fabG1, ahpC, inhA, embA, embB, pncA and rpsA. Isolates that were discrepant in their MGIT/WGS results and a control group with concordant results were retested in the MGIT, at the critical concentration and a lower concentration, and incubated for up to 45 days after the control tube became positive in the MGIT., Results: In total, 1136 isolates were included, of which 1121 were routine MTBC isolates from the Netherlands. The negative predictive value of WGS was ≥99.3% for all four first-line antibiotics. The majority of discrepancies for isoniazid and ethambutol were explained by growth at the lower concentrations, and for rifampicin by prolonged incubation in the MGIT, both indicating low-level resistance., Conclusions: Applying WGS in a country like the Netherlands, with a low TB incidence and low prevalence of resistance, can reduce the need for phenotypic DST for ∼90% of isolates and accurately detect mutations associated with low-level resistance, often missed in conventional DST., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Little is known how patterns of cross-over (CO) numbers and distribution during meiosis are established. Here, we reveal that cyclin-dependent kinase A;1 (CDKA;1), the homolog of human Cdk1 and Cdk2, is a major regulator of meiotic recombination in Arabidopsis Arabidopsis plants with reduced CDKA;1 activity experienced a decrease of class I COs, especially lowering recombination rates in centromere-proximal regions. Interestingly, this reduction of type I CO did not affect CO assurance, a mechanism by which each chromosome receives at least one CO, resulting in all chromosomes exhibiting similar genetic lengths in weak loss-of-function cdka ;1 mutants. Conversely, an increase of CDKA;1 activity resulted in elevated recombination frequencies. Thus, modulation of CDKA;1 kinase activity affects the number and placement of COs along the chromosome axis in a dose-dependent manner., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Plants are sessile and have to cope with environmentally induced damage through modification of growth and defense pathways. How tissue regeneration is triggered in such responses and whether this involves stem cell activation is an open question. The stress hormone jasmonate (JA) plays well-established roles in wounding and defense responses. JA also affects growth, which is hitherto interpreted as a trade-off between growth and defense. Here, we describe a molecular network triggered by wound-induced JA that promotes stem cell activation and regeneration. JA regulates organizer cell activity in the root stem cell niche through the RBR-SCR network and stress response protein ERF115. Moreover, JA-induced ERF109 transcription stimulates CYCD6;1 expression, functions upstream of ERF115, and promotes regeneration. Soil penetration and response to nematode herbivory induce and require this JA-mediated regeneration response. Therefore, the JA tissue damage response pathway induces stem cell activation and regeneration and activates growth after environmental stress., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Previous studies on factors influencing the place of death have focused on cancer patients dying at home. However, home is not always the preferred place., Aim: To provide insight into the extent to which patients with various diseases die in their preferred place and into factors associated with dying in the preferred place., Design: A secondary analysis of the data set of the evaluation study of the National Quality Improvement Programme for Palliative Care was undertaken to explore factors related to 'dying in the preferred place'., Setting/participants: A total of 797 bereaved relatives filled in the Consumer Quality Index Palliative Care, a validated and reliable questionnaire., Results: Two-thirds of the patients died in the preferred place. A preference for dying at home and having had a stroke decreased the likelihood of dying in the preferred place, while having a partner, dementia, contact with the general practitioner in the last week before death, and continuity of care between professionals increased the likelihood of dying in the preferred place. Furthermore, people who wanted to die at home and also had dementia were more likely to die elsewhere than people without dementia who wanted to die at home., Conclusion: Positive associations were found between continuity of care between healthcare professionals and contact with the general practitioner and the chance of people dying in their preferred place. Moreover, special attention for people who have had a stroke and for people with dementia who want to die at home seems indicated as their diagnosis is negatively associated with dying in their preferred place.

Background: The ongoing epidemic of multidrug-resistant tuberculosis (MDR-TB) in Georgia highlights the need for more effective control strategies. A new regimen to treat MDR-TB that includes pyrazinamide (PZA) is currently being evaluated and PZA resistance status will largely influence the success of current and future treatment strategies. PZA susceptibility testing was not routinely performed at the National Reference Laboratory (NRL) in Tbilisi between 2010 and September 2015. We here provide a first insight into the prevalence of PZA resistant TB in this region., Methods: Phenotypic susceptibility to PZA was determined in a convenience collection of well-characterised TB patient isolates collected at the NRL in Tbilisi between 2012 and 2013. In addition, the pncA gene was sequenced and whole genome sequencing was performed on two isolates., Results: Out of 57 isolates tested 33 (57.9%) showed phenotypic drug resistance to PZA and had a single pncA mutation. All of these 33 isolates were MDR-TB strains. pncA mutations were absent in all but one of the 24 PZA susceptible isolate. In total we found 18 polymorphisms in the pncA gene. From the two major MDR-TB clusters represented (94-32 and 100-32), 10 of 15, 67.0% and 13 of 14, 93.0% strains, respectively were PZA resistant. We also identified a member of the potentially highly transmissive clade A strain carrying the characteristic I6L substitution in PncA. Another strain with the same MLVA type as the clade A strain acquired a different mutation in pncA and was genetically more distantly related suggesting that different branches of this particular lineage have been introduced into this region., Conclusion: In this high MDR-TB setting more than half of the tested MDR-TB isolates were resistant to PZA. As PZA is part of current and planned MDR-TB treatment regimens this is alarming and deserves the attention of health authorities. Based on our typing and sequence analysis results we conclude that PZA resistance is the result of primary transmission as well as acquisition within the patient and recommend prospective genotyping and PZA resistance testing in high MDR-TB settings. This is of utmost importance in order to preserve bacterial susceptibility to PZA to help protect (new) second line drugs in PZA containing regimens.

Strong selection on a beneficial mutation can cause a selective sweep, which fixes the mutation in the population and reduces the genetic variation in the region flanking the mutation [1-3]. These flanking regions have increased in frequency due to their physical association with the selected loci, a phenomenon called "genetic hitchhiking" [4]. Theoretically, selection could extend the hitchhiking to unlinked parts of the genome, to the point that selection on organelles affects nuclear genome diversity. Such indirect selective sweeps have never been observed in nature. Here we show that strong selection on a chloroplast gene in the wild plant species Arabidopsis thaliana has caused widespread and lasting hitchhiking of the whole nuclear genome. The selected allele spread more than 400 km along the British railway network, reshaping the genetic composition of local populations. This demonstrates that selection on organelle genomes can significantly reduce nuclear genetic diversity in natural populations. We expect that organelle-mediated genetic draft is a more common occurrence than previously realized and needs to be considered when studying genome evolution., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We consider the Vector Scheduling problem, a natural generalization of the classical makespan minimization problem to multiple resources. Here, we are given n jobs, represented as d -dimensional vectors in [ 0 , 1 ] d , and m identical machines, and the goal is to assign the jobs to machines such that the maximum load of each machine over all the coordinates is at most 1. For fixed d , the problem admits an approximation scheme, and the best known running time is n f ( ϵ , d ) where f ( ϵ , d ) = ( 1 / ϵ ) O ~ ( d ) ( O ~ suppresses polylogarithmic terms in d ). In particular, the dependence on d is double exponential. In this paper we show that a double exponential dependence on d is necessary, and give an improved algorithm with essentially optimal running time. Specifically, we let exp ( x ) denote 2 x and show that: (1) For any ϵ < 1 , there is no ( 1 + ϵ ) -approximation with running time exp o ( ⌊ 1 / ϵ ⌋ d / 3 ) unless the Exponential Time Hypothesis fails. (2) No ( 1 + ϵ ) -approximation with running time exp ⌊ 1 / ϵ ⌋ o ( d ) exists, unless NP has subexponential time algorithms. (3) Similar lower bounds also hold even if ϵ m extra machines are allowed (i.e. with resource augmentation), for sufficiently small ϵ > 0 . (4) We complement these lower bounds with a ( 1 + ϵ ) -approximation that runs in time exp ( 1 / ϵ ) O ( d log log d ) + n d . This gives the first efficient approximation scheme (EPTAS) for the problem., (© The Author(s) 2016.)

Background: The performance of molecular drug susceptibility testing in countries with a low prevalence of drug resistance, such as the Netherlands, has not been adequately studied., Objective: To evaluate the diagnostic accuracy of the GenoType(®) MTBDRplus and MTBDRsl assays to detect resistance to first- and second-line anti-tuberculosis drugs in the context of a nationwide screening programme in the Netherlands., Results: The MTBDRplus assay had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 100%, 99%, 80% and 100% for detecting rifampicin resistance. The sensitivity, specificity, PPV and NPV of either a katG or inhA mutation for detecting isoniazid resistance were 88%, 100%, 100% and 99%. The MTBDRsl assay had a sensitivity, specificity, PPV and NPV of 100%, 99%, 83%, and 100% for detecting moxifloxacin resistance; 62%, 71%, 58% and 74%, respectively, for detecting ethambutol resistance; 86%, 99%, 86% and 99% for detecting amikacin resistance; and 50%, 96%, 71% and 91% for detecting capreomycin resistance., Conclusion: The MTBDRplus and MTBDRsl assays may aid in decision making in tuberculosis treatment in low-level drug resistance settings and should preferably be used to exclude resistance.

In the Netherlands, clinical isolation of nontuberculous mycobacteria (NTM) has increased over the past decade. Proper identification of isolates is important, as NTM species differ strongly in clinical relevance. Most of the currently applied identification methods cannot distinguish between all different Mycobacterium species and complexes within species. rpoB gene sequencing exhibits a promising level of discrimination among rapidly and slowly growing mycobacteria, including the Mycobacterium avium complex. In this study, we prospectively compared rpoB gene sequencing with our routine algorithm of reverse line blot identification combined with partial 16S rRNA gene sequencing of 455 NTM isolates. rpoB gene sequencing identified 403 isolates to species level as 45 different known species and identified 44 isolates to complex level, and eight isolates remained unidentifiable to species level. In contrast, our reference reverse line blot assay with adjunctive 16S rRNA gene sequencing identified 390 isolates to species level (30 distinct species) and identified 56 isolates to complex level, and nine isolates remained unidentified. The higher discriminatory power of rpoB gene sequencing results largely from the distinction of separate species within complexes and subspecies. Also, Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium interjectum were separated into multiple groupings with relatively low sequence similarity (98 to 94%), suggesting that these are complexes of closely related species. We conclude that rpoB gene sequencing is a more discriminative identification technique than the combination of reverse line blot and 16S rRNA gene sequencing and could introduce a major improvement in clinical care of NTM disease and the research on the epidemiology and clinical relevance of NTM., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Hybrid crop varieties are traditionally produced by selecting and crossing parental lines to evaluate hybrid performance. Reverse breeding allows doing the opposite: selecting uncharacterized heterozygotes and generating parental lines from them. With these, the selected heterozygotes can be recreated as F1 hybrids, greatly increasing the number of hybrids that can be screened in breeding programs. Key to reverse breeding is the suppression of meiotic crossovers in a hybrid plant to ensure the transmission of nonrecombinant chromosomes to haploid gametes. These gametes are subsequently regenerated as doubled-haploid (DH) offspring. Each DH carries combinations of its parental chromosomes, and complementing pairs can be crossed to reconstitute the initial hybrid. Achiasmatic meiosis and haploid generation result in uncommon phenotypes among offspring owing to chromosome number variation. We describe how these features can be dealt with during a reverse-breeding experiment, which can be completed in six generations (∼1 year).

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.

The mycobacterium growth indicator tube (MGIT960) automated liquid medium testing method is becoming the international gold standard for second-line drug susceptibility testing of multidrug- and extensively drug-resistant Mycobacterium tuberculosis complex isolates. We performed a comparative study of the current gold standard in the Netherlands, the Middlebrook 7H10 agar dilution method, the MGIT960 system, and the GenoType MTBDRsl genotypic method for rapid screening of aminoglycoside and fluoroquinolone resistance. We selected 28 clinical multidrug- and extensively drug-resistant M. tuberculosis complex strains and M. tuberculosis H37Rv. We included amikacin, capreomycin, moxifloxacin, prothionamide, clofazimine, linezolid, and rifabutin in the phenotypic test panels. For prothionamide and moxifloxacin, the various proposed breakpoint concentrations were tested by using the MGIT960 method. The MGIT960 method yielded results 10 days faster than the agar dilution method. For amikacin, capreomycin, linezolid, and rifabutin, results obtained by all methods were fully concordant. Applying a breakpoint of 0.5 microg/ml for moxifloxacin led to results concordant with those of both the agar dilution method and the genotypic method. For prothionamide, concordance was noted only at the lowest and highest MICs. The phenotypic methods yielded largely identical results, except for those for prothionamide. Our study supports the following breakpoints for the MGIT960 method: 1 microg/ml for amikacin, linezolid, and clofazimine, 0.5 microg/ml for moxifloxacin and rifabutin, and 2.5 microg/ml for capreomycin. No breakpoint was previously proposed for clofazimine. For prothionamide, a division into susceptible, intermediate, and resistant seems warranted, although the boundaries require additional study. The genotypic assay proved a reliable and rapid method for predicting aminoglycoside and fluoroquinolone resistance.

Objectives: To determine the clinical relevance of Mycobacterium chelonae-abscessus group isolation from clinical samples., Methods: We retrospectively reviewed medical files of all patients from whom these mycobacteria were isolated between January 1999 and January 2005 and re-identified the isolates by rpoB sequencing. We applied the American Thoracic Society (ATS) diagnostic criteria to establish clinical relevance., Results: Ninety-five patients were traced (56 M. chelonae, 25 Mycobacterium abscessus, 8 Mycobacterium massiliense, 6 Mycobacterium bolletii). Most isolates were cultured from pulmonary samples in patients with pre-existing pulmonary disease. Among patients with pulmonary isolates, 27% (20/74) meets ATS criteria; M. abscessus is most relevant (50%; 9/18), followed by M. massiliense (29%; 2/7), M. bolletii (20%; 1/5) and M. chelonae (18%; 8/44). Extrapulmonary disease presented as disseminated skin disease, eye disease specific for M. chelonae and otomastoiditis for M. abscessus. Treatment, especially for pulmonary M. abscessus disease, yielded limited results., Conclusions: One-fourth of the patients with pulmonary M. chelonae-abscessus group isolates met the ATS criteria; this percentage differs by species. Species distribution and clinical relevance differ from other regions. M. abscessus isolation in cystic fibrosis patients warrants special attention. Current ATS criteria might be too lenient to diagnose M. chelonae-abscessus group disease.

The esat-6 and cfp-10 genes are essential for virulence in Mycobacterium tuberculosis. Among nontuberculous mycobacteria, we found these genes only in M. kansasii, M. szulgai, M. marinum, and M. riyadhense, with unique sequences. This adds a phylogenetic and taxonomical characteristic and may represent a virulence factor for nontuberculous mycobacteria.

The effect of smoking urges on cognitive performance is relatively short-lived (R. A. Zwaan, R. A. Stanfield, & C. M. Madden, 2000). The authors examined whether this results from the short-lived nature of the elicited urge itself or from practice effects on the cognitive task. Smokers listened to 1 of 2 imagery scripts (urge vs. neutral) and subsequently performed 2 cognitive tasks (math and language comprehension). Exposure to the urge script produced significantly less accurate performance at the onset of the 1st task than exposure to the neutral script, but there was no difference at the onset of the 2nd task. Thus, the quick disappearance of the urge effect seems to be due to the transient nature of the elicited urge itself rather than to practice effects.

Perceptual symbol systems assume an analogue relationship between a symbol and its referent, whereas amodal symbol systems assume an arbitrary relationship between a symbol and its referent. According to perceptual symbol theories, the complete representation of an object, called a simulation, should reflect physical characteristics of the object. Amodal theories, in contrast, do not make this prediction. We tested the hypothesis, derived from perceptual symbol theories, that people mentally represent the orientation of an object implied by a verbal description. Orientation (vertical-horizontal) was manipulated by having participants read a sentence that implicitly suggested a particular orientation for an object. Then recognition latencies to pictures of the object in each of the two orientations were measured. Pictures matching the orientation of the object implied by the sentence were responded to faster than pictures that did not match the orientation. This finding is interpreted as offering support for theories positing perceptual symbol systems.

Several studies have provided empirical support for S. T. Tiffany's (1990) hypothesis that drug urges interfere with cognitive performance. The authors examined the persistence of this effect. Results from an experiment involving 48 smokers and 46 nonsmokers, using a paradigm developed by R. A. Zwaan and T. P. Truitt (1998), suggest that the effect of smoking urges in cognitive performance dissipates over time. The implications of this finding for cognitive theories of drug urges and for future research on the effects of smoking urges are discussed.

Narrative descriptions of events often depart from how these events would have occurred in "real time." For example, narratives often contain time shifts in which events that are irrelevant to the plot are omitted. Zwaan (1996) has shown that these time shifts may affect on-line comprehension. Specifically, they are associated with increases in processing load and a deactivation of previous information. The experiments in the present article show that the situation is more complex. Specifically, there is only a deactivation of previous events if they are not assumed to be ongoing after a time shift. Furthermore, explicit discontinuations of events, as in he stopped walking also lead to deactivations when compared with explicit continuations and resumptions.

Twenty-four smokers and 24 nonsmokers performed a modified version of M. A. Gernsbacher, K. R. Varner, and M. E. Faust's (1990) suppression task, involving presentations of sentences on a computer screen. Each sentence was followed by a word that either was or was not related to the meaning of the sentence. Participants judged whether the word was related to the sentence by pressing either a "yes" or "no" key on a button box. In the experimental sentences, the test word was related to one meaning of the final word of the sentence, but this was not necessarily the meaning intended in the sentence. In half of the experimental sentences, the last word was a smoking-related word (e.g., tar or ashes). Smokers had relatively longer response latencies and lower accuracy scores than nonsmokers when the final word was smoking related, whereas both groups performed similarly on items unrelated to smoking, suggesting that smokers had more difficulty than nonsmokers inhibiting task-irrelevant, smoking-related information but that they did not have a general inability to inhibit irrelevant information.

Time is an important part of establishing situations in the world. As such, temporal information should be reflected in the organization of information into situation models. This article reports 3 experiments that explore whether people will integrate sets of related facts into situation models in a time-based fashion. People memorized lists of facts and then took a speeded recognition test. A retrieval interference methodology was used to assess whether they had integrated the facts into situation models. The presence of interference indicated a lack of integration. In contrast, a marked reduction or an absence in interference indicated integration. In 2 experiments, time-based integration was observed when common time periods were referred to by either events (e.g., "when the camera flashed") or verb tense (i.e., past, present, and future). A 3rd experiment demonstrated that common time periods alone are not sufficient; the information must be allowed to occur potentially within the same situation.

We examined the hypothesis that cigarette smoking urges take up working memory resources and interfere with language comprehension. Thirty-six smokers and 36 nonsmokers participated in the experiment. Eighteen members of each group were exposed to an imagery script that was intended to elicit a smoking urge in smokers, and the other 18 were exposed to a neutral script. The participants then performed a sentence comprehension task. The results showed that the urge script had a detrimental effect on the smokers' comprehension accuracy. As expected, there was no urge effect for the nonsmokers. Furthermore, smokers read the sentences significantly faster than nonsmokers. The results are discussed with reference to cognitive theories of urges and a capacity model of language processing.

This article reviews research on the use of situation models in language comprehension and memory retrieval over the past 15 years. Situation models are integrated mental representations of a described state of affairs. Significant progress has been made in the scientific understanding of how situation models are involved in language comprehension and memory retrieval. Much of this research focuses on establishing the existence of situation models, often by using tasks that assess one dimension of a situation model. However, the authors argue that the time has now come for researchers to begin to take the multidimensionality of situation models seriously. The authors offer a theoretical framework and some methodological observations that may help researchers to tackle this issue.

The field of discourse processing has dissected many of the levels of representation that are constructed when individuals read or listen to connected discourse. These levels include the surface code, the propositional textbase, the referential situation model, the communication context, and the discourse genre. Discourse psychologists have developed models that specify how these levels are mentally represented and how they are dynamically built during comprehension. This chapter focuses on the meaning representations that are constructed when adults read written text, such as literary stories, technical expository text, and experimenter-generated "textoids." Recent psychological models have attempted to account for the identification of referents of referring expressions (e.g. which person in the text does she refer to), the connection of explicit text segments, the establishment of local and global coherence, and the encoding of knowledge-based inferences.