T helper cell subsets specific for pseudomonas aeruginosa in healthy individuals and patients with cystic fibrosis

Background: We set out to determine the magnitude of antigen-specific memory T helper cell responses to Pseudomonas aeruginosa in healthy humans and patients with cystic fibrosis. Methods: Peripheral blood human memory CD4+ T cells were co-cultured with dendritic cells that had been infected with different strains of Pseudomonas aeruginosa. The T helper response was determined by measuring proliferation, immunoassay of cytokine output, and immunostaining of intracellular cytokines. Results: Healthy individuals and patients with cystic fibrosis had robust antigen-specific memory CD4+ T cell responses to Pseudomonas aeruginosa that not only contained a Th1 and Th17 component but also Th22 cells. In contrast to previous descriptions of human Th22 cells, these Pseudomonal-specific Th22 cells lacked the skin homing markers CCR4 or CCR10, although were CCR6+. Healthy individuals and patients with cystic fibrosis had similar levels of Th22 cells, but the patient group had significantly fewer Th17 cells in peripheral blood. Conclusions: Th22 cells specific to Pseudomonas aeruginosa are induced in both healthy individuals and patients with cystic fibrosis. Along with Th17 cells, they may play an important role in the pulmonary response to this microbe in patients with cystic fibrosis and other conditions

Experimental loophole-free violation of a Bell inequality using entangled electron spins separated by 1.3 km

For more than 80 years, the counterintuitive predictions of quantum theory have stimulated debate about the nature of reality. In his seminal work, John Bell proved that no theory of nature that obeys locality and realism can reproduce all the predictions of quantum theory. Bell showed that in any local realist theory the correlations between distant measurements satisfy an inequality and, moreover, that this inequality can be violated according to quantum theory. This provided a recipe for experimental tests of the fundamental principles underlying the laws of nature. In the past decades, numerous ingenious Bell inequality tests have been reported. However, because of experimental limitations, all experiments to date required additional assumptions to obtain a contradiction with local realism, resulting in loopholes. Here we report on a Bell experiment that is free of any such additional assumption and thus directly tests the principles underlying Bell's inequality. We employ an event-ready scheme that enables the generation of high-fidelity entanglement between distant electron spins. Efficient spin readout avoids the fair sampling assumption (detection loophole), while the use of fast random basis selection and readout combined with a spatial separation of 1.3 km ensure the required locality conditions. We perform 245 trials testing the CHSH-Bell inequality $S \leq 2$ and find $S = 2.42 \pm 0.20$. A null hypothesis test yields a probability of $p = 0.039$ that a local-realist model for space-like separated sites produces data with a violation at least as large as observed, even when allowing for memory in the devices. This result rules out large classes of local realist theories, and paves the way for implementing device-independent quantum-secure communication and randomness certification.Comment: Raw data will be made available after publicatio

Outline of a Genome Navigation System Based on the Properties of GA-Sequences and Their Flanks

Introducing a new method to visualize large stretches of genomic DNA (see Appendix S1) the article reports that most GA-sequences [1] shared chains of tetra-GA-motifs and contained upstream poly(A)-segments. Although not integral parts of them, Alu-elements were found immediately upstream of all human and chimpanzee GA-sequences with an upstream poly(A)-segment. The article hypothesizes that genome navigation uses these properties of GA-sequences in the following way. (1) Poly(A) binding proteins interact with the upstream poly(A)-segments and arrange adjacent GA-sequences side-by-side (‘GA-ribbon’), while folding the intervening DNA sequences between them into loops (‘associated DNA-loops’). (2) Genome navigation uses the GA-ribbon as a search path for specific target genes that is up to 730-fold shorter than the full-length chromosome. (3) As to the specificity of the search, each molecule of a target protein is assumed to catalyze the formation of specific oligomers from a set of transcription factors that recognize tetra-GA-motifs. Their specific combinations of tetra-GA motifs are assumed to be present in the particular GA-sequence whose associated loop contains the gene for the target protein. As long as the target protein is abundant in the cell it produces sufficient numbers of such oligomers which bind to their specific GA-sequences and, thereby, inhibit locally the transcription of the target protein in the associated loop. However, if the amount of target protein drops below a certain threshold, the resultant reduction of specific oligomers leaves the corresponding GA-sequence ‘denuded’. In response, the associated DNA-loop releases its nucleosomes and allows transcription of the target protein to proceed. (4) The Alu-transcripts may help control the general background of protein synthesis proportional to the number of transcriptionally active associated loops, especially in stressed cells. (5) The model offers a new mechanism of co-regulation of protein synthesis based on the shared segments of different GA-sequences

Unique and conserved MicroRNAs in wheat chromosome 5D revealed by next-generation sequencing

MicroRNAs are a class of short, non-coding, single-stranded RNAs that act as post-transcriptional regulators in gene expression. miRNA analysis of Triticum aestivum chromosome 5D was performed on 454 GS FLX Titanium sequences of flow sorted chromosome 5D with a total of 3,208,630 good quality reads representing 1.34x and 1.61x coverage of the short (5DS) and long (5DL) arms of the chromosome respectively. In silico and structural analyses revealed a total of 55 miRNAs; 48 and 42 miRNAs were found to be present on 5DL and 5DS respectively, of which 35 were common to both chromosome arms, while 13 miRNAs were specific to 5DL and 7 miRNAs were specific to 5DS. In total, 14 of the predicted miRNAs were identified in wheat for the first time. Representation (the copy number of each miRNA) was also found to be higher in 5DL (1,949) compared to 5DS (1,191). Targets were predicted for each miRNA, while expression analysis gave evidence of expression for 6 out of 55 miRNAs. Occurrences of the same miRNAs were also found in Brachypodium distachyon and Oryza sativa genome sequences to identify syntenic miRNA coding sequences. Based on this analysis, two other miRNAs: miR1133 and miR167 were detected in B. distachyon syntenic region of wheat 5DS. Five of the predicted miRNA coding regions (miR6220, miR5070, miR169, miR5085, miR2118) were experimentally verified to be located to the 5D chromosome and three of them : miR2118, miR169 and miR5085, were shown to be 5D specific. Furthermore miR2118 was shown to be expressed in Chinese Spring adult leaves. miRNA genes identified in this study will expand our understanding of gene regulation in bread wheat

A phase II irinotecan–cisplatin combination in advanced pancreatic cancer

We report a cisplatin and irinotecan combination in patients with biopsy-proven advanced pancreatic adenocarcinoma. Patients were selected from a specialist centre and required good performance status (KPS>70%), measurable disease on CT scan, and biochemical and haematological parameters within normal limits. Based on a two-stage phase II design, we aimed to treat 22 patients initially. The study was stopped because of the death of the 19th patient during the first treatment cycle, with neutropenic sepsis and multiorgan failure. A total of 89 treatments were administered to 17 patients. Serious grade 3/4 toxicities were haematological (neutropenia) 6%, diarrhoea 6%, nausea 7% and vomiting 6%. Using the clinical benefit response (CBR) criteria, no patients had an overall CBR. For responses confirmed by CT examination, there was one partial response (5%), three stable diseases lasting greater than 6 weeks (16%), with an overall 22% with disease control (PR+SD). The median progression-free and overall survival was 3.1 months (95% CI: 1.3-3.7) and 5.0 (95% CI: 3.9-10.1) months, respectively. Although this synergistic combination has improved the response rates and survival of other solid tumours, we recommend caution when using this combination in the palliation of advanced pancreatic cancer, because of unexpected toxicity

Ethical principles in machine learning and artificial intelligence: cases from the field and possible ways forward

Decision-making on numerous aspects of our daily lives is being outsourced to machine-learning algorithms and artificial intelligence (AI), motivated by speed and efficiency in the decision process. Machine learning (ML) approaches - one of the typologies of algorithms underpinning artificial intelligence - are typically developed as black boxes. The implication is that ML code scripts are rarely scrutinised; interpretability is usually sacrificed in favour of usability and effectiveness. Room for improvement in practices associated with programme development have also been flagged along other dimensions, including inter alia fairness, accuracy, accountability, and transparency. In this contribution, the production of guidelines and dedicated documents around these themes is discussed. The following applications of AI-driven decision making are outlined: a) Risk assessment in the criminal justice system, and b) autonomous vehicles, highlighting points of friction across ethical principles. Possible ways forward towards the implementation of governance on AI are finally examined

Dissection of the Complex Phenotype in Cuticular Mutants of Arabidopsis Reveals a Role of SERRATE as a Mediator

Mutations in LACERATA (LCR), FIDDLEHEAD (FDH), and BODYGUARD (BDG) cause a complex developmental syndrome that is consistent with an important role for these Arabidopsis genes in cuticle biogenesis. The genesis of their pleiotropic phenotypes is, however, poorly understood. We provide evidence that neither distorted depositions of cutin, nor deficiencies in the chemical composition of cuticular lipids, account for these features, instead suggesting that the mutants alleviate the functional disorder of the cuticle by reinforcing their defenses. To better understand how plants adapt to these mutations, we performed a genome-wide gene expression analysis. We found that apparent compensatory transcriptional responses in these mutants involve the induction of wax, cutin, cell wall, and defense genes. To gain greater insight into the mechanism by which cuticular mutations trigger this response in the plants, we performed an overlap meta-analysis, which is termed MASTA (MicroArray overlap Search Tool and Analysis), of differentially expressed genes. This suggested that different cell integrity pathways are recruited in cesA cellulose synthase and cuticular mutants. Using MASTA for an in silico suppressor/enhancer screen, we identified SERRATE (SE), which encodes a protein of RNA–processing multi-protein complexes, as a likely enhancer. In confirmation of this notion, the se lcr and se bdg double mutants eradicate severe leaf deformations as well as the organ fusions that are typical of lcr and bdg and other cuticular mutants. Also, lcr does not confer resistance to Botrytis cinerea in a se mutant background. We propose that there is a role for SERRATE-mediated RNA signaling in the cuticle integrity pathway

Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust

<p>Abstract</p> <p>Background</p> <p>The Australian plague locust, <it>Chortoicetes terminifera</it>, is among the most promising species to unravel the suites of genes underling the density-dependent shift from shy and cryptic solitarious behaviour to the highly active and aggregating gregarious behaviour that is characteristic of locusts. This is because it lacks many of the major phenotypic changes in colour and morphology that accompany phase change in other locust species. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method available for determining changes in gene expression. However, to accurately monitor the expression of target genes, it is essential to select an appropriate normalization strategy to control for non-specific variation between samples. Here we identify eight potential reference genes and examine their expression stability at different rearing density treatments in neural tissue of the Australian plague locust.</p> <p>Results</p> <p>Taking advantage of the new orthologous DNA sequences available in locusts, we developed primers for genes encoding 18SrRNA, ribosomal protein L32 (RpL32), armadillo (Arm), actin 5C (Actin), succinate dehydrogenase (SDHa), glyceraldehyde-3P-dehydrogenase (GAPDH), elongation factor 1 alpha (EF1a) and annexin IX (AnnIX). The relative transcription levels of these eight genes were then analyzed in three treatment groups differing in rearing density (isolated, short- and long-term crowded), each made up of five pools of four neural tissue samples from 5^thinstar nymphs. SDHa and GAPDH, which are both involved in metabolic pathways, were identified as the least stable in expression levels, challenging their usefulness in normalization. Based on calculations performed with the geNorm and NormFinder programs, the best combination of two genes for normalization of gene expression data following crowding in the Australian plague locust was EF1a and Arm. We applied their use to studying a target gene that encodes a Ca²⁺binding glycoprotein, <it>SPARC</it>, which was previously found to be up-regulated in brains of gregarious desert locusts, <it>Schistocerca gregaria</it>. Interestingly, expression of this gene did not vary with rearing density in the same way in brains of the two locust species. Unlike <it>S. gregaria</it>, there was no effect of any crowding treatment in the Australian plague locust.</p> <p>Conclusion</p> <p>Arm and EF1a is the most stably expressed combination of two reference genes of the eight examined for reliable normalization of RT-qPCR assays studying density-dependent behavioural change in the Australian plague locust. Such normalization allowed us to show that <it>C. terminifera </it>crowding did not change the neuronal expression of the <it>SPARC </it>gene, a gregarious phase-specific gene identified in brains of the desert locust, <it>S. gregaria</it>. Such comparative results on density-dependent gene regulation provide insights into the evolution of gregarious behaviour and mass migration of locusts. The eight identified genes we evaluated are also candidates as normalization genes for use in experiments involving other Oedipodinae species, but the rank order of gene stability must necessarily be determined on a case-by-case basis.</p

Structural and molecular correlates of cognitive aging in the rat

Aging is associated with cognitive decline. Herein, we studied a large cohort of old age and young adult male rats and confirmed that, as a group, old  rats display poorer spatial learning and behavioral flexibility than younger adults. Surprisingly, when animals were clustered as good and bad performers, our data revealed that while in younger animals better cognitive performance was associated with longer dendritic trees and increased levels of synaptic markers in the hippocampus and prefrontal cortex, the opposite was found in the older group, in which better performance was associated with shorter dendrites and lower levels of synaptic markers. Additionally, in old, but not young individuals, worse performance correlated with increased levels of BDNF and the autophagy substrate p62, but decreased levels of the autophagy complex protein LC3. In summary, while for younger individuals "bigger is better", "smaller is better" is a more appropriate aphorism for older subjects.Portuguese Foundation for Science and Technology (FCT) with fellowships granted to: Cristina Mota (SFRH/BD/81881/2011), Susana Monteiro (SFRH/BD/69311/2010), Sofia Pereira das Neves and Sara Monteiro-Martins (PIC/IC/83213/2007); and by the European Commission within the 7th framework program, under the grant agreement: Health-F2-2010-259772 (Switchbox). In addition, this work was co-funded by the Northern Portugal Regional Operational Programme (ON.2 SR&TD Integrated Program – NORTE-07-0124-FEDER-000021), through the European Regional Development Fund (FEDER) and by national funds granted by FCT (PEst-C/SAU/LA0026/2013), and FEDER through the COMPETE (FCOMP-01-0124-FEDER-037298)