Control of box C/D snoRNP assembly by N⁶-methylation of adenine

N6-methyladenine is the most widespread mRNA modification. A sub-set of human box C/D snoRNA species have target GAC sequences that lead to formation of N6-methyladenine at a key trans Hoogsteen-sugar A•G basepair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5 kDa protein and the induced folding of the RNA. Thus the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N6-methylation of adenine prevents the formation of trans Hoogsteen-sugar A•G basepairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A•G basepairs (but not Watson-Crick basepairs) are more susceptible to disruption by N6mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared basepair with guanine and mediates a key tertiary interaction

An Anti-Human ICAM-1 Antibody Inhibits Rhinovirus-Induced Exacerbations of Lung Inflammation

Human rhinoviruses (HRV) cause the majority of common colds and acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Effective therapies are urgently needed, but no licensed treatments or vaccines currently exist. Of the 100 identified serotypes, ∼90% bind domain 1 of human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, making this an attractive target for development of therapies; however, ICAM-1 domain 1 is also required for host defence and regulation of cell trafficking, principally via its major ligand LFA-1. Using a mouse anti-human ICAM-1 antibody (14C11) that specifically binds domain 1 of human ICAM-1, we show that 14C11 administered topically or systemically prevented entry of two major groups of rhinoviruses, HRV16 and HRV14, and reduced cellular inflammation, pro-inflammatory cytokine induction and virus load in vivo. 14C11 also reduced cellular inflammation and Th2 cytokine/chemokine production in a model of major group HRV-induced asthma exacerbation. Interestingly, 14C11 did not prevent cell adhesion via human ICAM-1/LFA-1 interactions in vitro, suggesting the epitope targeted by 14C11 was specific for viral entry. Thus a human ICAM-1 domain-1-specific antibody can prevent major group HRV entry and induction of airway inflammation in vivo

Identification of Giardia lamblia DHHC Proteins and the Role of Protein S-palmitoylation in the Encystation Process

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.Fil: Merino, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Zamponi, Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Vranych, Cecilia Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Touz, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Ropolo, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin

Managerial Work in a Practice-Embodying Institution - The role of calling, the virtue of constancy

What can be learned from a small scale study of managerial work in a highly marginal and under-researched working community? This paper uses the ‘goods-virtues-practices-institutions’ framework to examine the managerial work of owner-directors of traditional circuses. Inspired by MacIntyre’s arguments for the necessity of a narrative understanding of the virtues, interviews explored how British and Irish circus directors accounted for their working lives. A purposive sample was used to select subjects who had owned and managed traditional touring circuses for at least 15 years, a period in which the economic and reputational fortunes of traditional circuses have suffered badly. This sample enabled the research to examine the self-understanding of people who had, at least on the face of it, exhibited the virtue of constancy. The research contributes to our understanding of the role of the virtues in organizations by presenting evidence of an intimate relationship between the virtue of constancy and a ‘calling’ work orientation. This enhances our understanding of the virtues that are required if management is exercised as a domain-related practice

Serratamolide is a hemolytic factor produced by Serratia marcescens

Serratia marcescens is a common contaminant of contact lens cases and lenses. Hemolytic factors of S. marcescens contribute to the virulence of this opportunistic bacterial pathogen. We took advantage of an observed hyper-hemolytic phenotype of crp mutants to investigate mechanisms of hemolysis. A genetic screen revealed that swrW is necessary for the hyper-hemolysis phenotype of crp mutants. The swrW gene is required for biosynthesis of the biosurfactant serratamolide, previously shown to be a broad-spectrum antibiotic and to contribute to swarming motility. Multicopy expression of swrW or mutation of the hexS transcription factor gene, a known inhibitor of swrW expression, led to an increase in hemolysis. Surfactant zones and expression from an swrW-transcriptional reporter were elevated in a crp mutant compared to the wild type. Purified serratamolide was hemolytic to sheep and murine red blood cells and cytotoxic to human airway and corneal limbal epithelial cells in vitro. The swrW gene was found in the majority of contact lens isolates tested. Genetic and biochemical analysis implicate the biosurfactant serratamolide as a hemolysin. This novel hemolysin may contribute to irritation and infections associated with contact lens use. © 2012 Shanks et al

Protein-based signatures of functional evolution in Plasmodium falciparum.

BACKGROUND: It has been known for over a decade that Plasmodium falciparum proteins are enriched in non-globular domains of unknown function. The potential for these regions of protein sequence to undergo high levels of genetic drift provides a fundamental challenge to attempts to identify the molecular basis of adaptive change in malaria parasites. RESULTS: Evolutionary comparisons were undertaken using a set of forty P. falciparum metabolic enzyme genes, both within the hominid malaria clade (P. reichenowi) and across the genus (P. chabaudi). All genes contained coding elements highly conserved across the genus, but there were also a large number of regions of weakly or non-aligning coding sequence. These displayed remarkable levels of non-synonymous fixed differences within the hominid malaria clade indicating near complete release from purifying selection (dN/dS ratio at residues non-aligning across genus: 0.64, dN/dS ratio at residues identical across genus: 0.03). Regions of low conservation also possessed high levels of hydrophilicity, a marker of non-globularity. The propensity for such regions to act as potent sources of non-synonymous genetic drift within extant P. falciparum isolates was confirmed at chromosomal regions containing genes known to mediate drug resistance in field isolates, where 150 of 153 amino acid variants were located in poorly conserved regions. In contrast, all 22 amino acid variants associated with drug resistance were restricted to highly conserved regions. Additional mutations associated with laboratory-selected drug resistance, such as those in PfATPase4 selected by spiroindolone, were similarly restricted while mutations in another calcium ATPase (PfSERCA, a gene proposed to mediate artemisinin resistance) that reach significant frequencies in field isolates were located exclusively in poorly conserved regions consistent with genetic drift. CONCLUSION: Coding sequences of malaria parasites contain prospectively definable domains subject to neutral or nearly neutral evolution on a scale that appears unrivalled in biology. This distinct evolutionary landscape has potential to confound analytical methods developed for other genera. Against this tide of genetic drift, polymorphisms mediating functional change stand out to such an extent that evolutionary context provides a useful signal for identifying the molecular basis of drug resistance in malaria parasites, a finding that is of relevance to both genome-wide and candidate gene studies in this genus

Normative data on regional sweat-sodium concentrations of professional male team-sport athletes

Background: The purpose of this paper was to report normative data on regional sweat sweat-sodium concentrations of various professional male team-sport athletes, and to compare sweat-sodium concentrations among sports. Data to this effect would inform our understanding of athlete sodium requirements, thus allowing for the individualisation of sodium replacement strategies. Accordingly, data from 696 athletes (Soccer, n = 270; Rugby, n = 181; Baseball, n = 133; American Football, n = 60; Basketball, n = 52) were compiled for a retrospective analysis. Regional sweat-sodium concentrations were collected using the pilocarpine iontophoresis method, and compared to self-reported measures collected via questionnaire. Results: Sweat-sodium concentrations were significantly higher (p < 0.05) in American football (50.4 ± 15.3 mmol·L-1), baseball (54.0 ± 14.0 mmol·L-1), and basketball (48.3 ± 14.0 mmol·L-1) than either soccer (43.2 ± 12.0 mmol·L-1) or rugby (44.0 ± 12.1 mmol·L-1), but with no differences among the N.American or British sports. There were strong positive correlations between sweat-sodium concentrations and self-reported sodium losses in American football (rs = 0.962, p < 0.001), basketball (rs = 0.953, p < 0.001), rugby (rs = 0.813, p < 0.001), and soccer (rs = 0.748, p < 0.001). Conclusions: The normative data provided on sweat-sodium concentrations might assist sports science/medicine practitioners in generating bespoke hydration and electrolyte-replacement strategies to meet the sodium demands of professional team-sport athletes. Moreover, these novel data suggest that self-reported measures of sodium loss might serve as an effective surrogate in the absence of direct measures; i.e., those which are more expensive or non-readily available

Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing

Impact of a workplace 'sit less, move more' program on efficiency-related outcomes of office employees.

BACKGROUND: Few studies have examined the impact of 'sit less, move more' interventions on workplace performance. This study assessed the short and mid-term impacts of and patterns of change within, a 19-week workplace web-based intervention (Walk@WorkSpain; W@WS; 2010-11) on employees´ presenteeism, mental well-being and lost work performance. METHODS: A site randomised control trial recruited employees at six Spanish university campuses (n = 264; 42 ± 10 years; 171 female), assigned by worksite and campus to an Intervention (IG; used W@WS; n = 129; 87 female) or an active Comparison group (A-CG; pedometer, paper diary and self-reported sitting time; n = 135; 84 female). A linear mixed model assessed changes between the baseline, ramping (8 weeks), maintenance (11 weeks) and follow-up (two months) phases for the IG versus A-CG on (i) % of lost work productivity (Work Limitations Questionnaire; WLQ); (ii) three scales for presenteeism (WLQ) assessing difficulty meeting scheduling demands (Time), performing cognitive and inter-personal tasks (Mental-Interpersonal) and decrements in meeting the quantity, quality and timeliness of completed work (Output); and (iii) mental well-being (Warwick-Edinburgh Mental Well-being Scale). T-tests assessed differences between groups for changes on the main outcomes. In the IG, a multivariate logistic regression model identified patterns of response according to baseline socio-demographic variables, physical activity and sitting time. RESULTS: There was a significant 2 (group) × 2 (program time points) interaction for the Time (F [3]=8.69, p = 0.005), Mental-Interpersonal (F [3]=10.01, p = 0.0185), Output scales for presenteeism (F [3]=8.56, p = 0.0357), and for % of lost work performance (F [3]=10.31, p = 0.0161). Presenteeism and lost performance rose significantly in both groups across all study time points; after baseline performance was consistently better in the IG than in the A-CG. Better performance was linked to employees being more active (Time, p = 0.041) and younger (Mental-interpersonal, p = 0.057; Output, p = 0.017). Higher total sitting time during nonworking days (Mental-interpersonal, p = 0.019) and lower sitting time during workdays (WLQ Index, p = 0.013) also improved performance. CONCLUSION: Versus an active comparison condition, a 'sit less, move more` workplace intervention effectively reduced an array of markers of lost workday productivity. TRIAL REGISTRATION: NCT02960750 ; Date of registration: 07/11/2016

The nature and fate of natural resins in the geosphere XIII: a probable pinaceous resin from the early Cretaceous (Barremian), Isle of Wight

Terpenoid resin is produced by all families and most genera of the order Coniferales (the conifers), and the distribution of terpenes present in most conifer resins is characteristic of the originating family. Analyses of early Cretaceous (Barremian) amber (fossil resin) from the English Wealden, Isle of Wight, southern England, by pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS), indicate a terpene distribution dominated by abietane- and labdane-type terpenes. Similar distributions are observed in some species of the extant family Pinaceae. The Pinaceae are well represented within the Wealden deposits of southern England, by only one (known) species, Pityites solmsii (Seward) Seward, whereas the macro-fossil record of these deposits is dominated by the extinct conifer family Cheirolepidiaceae, for which no resin chemistry has been reported. By analogy with modern materials, it is probable that the ambers found in these deposits are derived from an extinct member of the Pinaceae, but given the absence of evidence concerning the chemotaxonomy of the Cheirolepidiaceae, this family cannot be excluded a priori as a possible paleobotanical source. These ambers may therefore be assigned to either the Pinaceae or to the Cheirolepidiaceae. These samples are the oldest ambers to date to yield useful chemotaxonomic data