Trans-Species Polymorphism and Selection in the MHC Class II DRA Genes of Domestic Sheep

Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC) in higher vertebrates. Generally, across vertebrate species the class II MHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries). We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201) differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901), which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T-cells and the induction of protective immunity

Language in international business: a review and agenda for future research

A fast growing number of studies demonstrates that language diversity influences almost all management decisions in modern multinational corporations. Whereas no doubt remains about the practical importance of language, the empirical investigation and theoretical conceptualization of its complex and multifaceted effects still presents a substantial challenge. To summarize and evaluate the current state of the literature in a coherent picture informing future research, we systematically review 264 articles on language in international business. We scrutinize the geographic distributions of data, evaluate the field’s achievements to date in terms of theories and methodologies, and summarize core findings by individual, group, firm, and country levels of analysis. For each of these dimensions, we then put forward a future research agenda. We encourage scholars to transcend disciplinary boundaries and to draw on, integrate, and test a variety of theories from disciplines such as psychology, linguistics, and neuroscience to gain a more profound understanding of language in international business. We advocate more multi-level studies and cross-national research collaborations and suggest greater attention to potential new data sources and means of analysis

General anaesthetic and airway management practice for obstetric surgery in England: a prospective, multi-centre observational study

There are no current descriptions of general anaesthesia characteristics for obstetric surgery, despite recent changes to patient baseline characteristics and airway management guidelines. This analysis of data from the direct reporting of awareness in maternity patients' (DREAMY) study of accidental awareness during obstetric anaesthesia aimed to describe practice for obstetric general anaesthesia in England and compare with earlier surveys and best-practice recommendations. Consenting patients who received general anaesthesia for obstetric surgery in 72 hospitals from May 2017 to August 2018 were included. Baseline characteristics, airway management, anaesthetic techniques and major complications were collected. Descriptive analysis, binary logistic regression modelling and comparisons with earlier data were conducted. Data were collected from 3117 procedures, including 2554 (81.9%) caesarean deliveries. Thiopental was the induction drug in 1649 (52.9%) patients, compared with propofol in 1419 (45.5%). Suxamethonium was the neuromuscular blocking drug for tracheal intubation in 2631 (86.1%), compared with rocuronium in 367 (11.8%). Difficult tracheal intubation was reported in 1 in 19 (95%CI 1 in 16-22) and failed intubation in 1 in 312 (95%CI 1 in 169-667). Obese patients were over-represented compared with national baselines and associated with difficult, but not failed intubation. There was more evidence of change in practice for induction drugs (increased use of propofol) than neuromuscular blocking drugs (suxamethonium remains the most popular). There was evidence of improvement in practice, with increased monitoring and reversal of neuromuscular blockade (although this remains suboptimal). Despite a high risk of difficult intubation in this population, videolaryngoscopy was rarely used (1.9%)

Metrical rules and the notion of ‘maximum’ : a reply to Derek Attridge

Attridge (2003) is in effect an argument against generative metrics, focusing on the notion of ‘stress maximum’. He argues that ‘stress maximum’ is inadequate on its own terms, which I will show is incorrect when we use a fuller definition (based on the original definition from Halle and Keyser, 1966). His solution is to present metre as control directly over the phonetics of the text, but I will show that metre controls metrical elements (maxima) rather than phonetic elements (stressed syllables)

DNA-Dependent Protein Kinase Is a Context Dependent Regulator of Lmx1a and Midbrain Specification

<div><p>The identification of small molecules capable of directing pluripotent cell differentiation towards specific lineages is highly desirable to both reduce cost, and increase efficiency. Within neural progenitors, LIM homeobox transcription factor 1 alpha (Lmx1a) is required for proper development of roof plate and cortical hem structures of the forebrain, as well as the development of floor plate and midbrain dopaminergic neurons. In this study we generated homologous recombinant cell lines expressing either luciferase or β-lactamase under the control of the <i>Lmx1a</i> promoter, and used these cell lines to investigate kinase-mediated regulation of Lmx1a activity during neuronal differentiation. A screen of 143 small molecule tyrosine kinase inhibitors yielded 16 compounds that positively or negatively modulated Lmx1a activity. Inhibition of EGF, VEGF and DNA-dependent protein kinase (DNA-PK) signaling significantly upregulated Lmx1a activity whereas MEK inhibition strongly downregulated its activity. Quantitative FACS analysis revealed that the DNA-PK inhibitor significantly increased the number of Lmx1a+ progenitors while subsequent qPCR showed an upregulation of Notch effectors, the basic helix-loop-helix genes, <i>Hes5</i> and <i>Hey1</i>. FACS further revealed that DNA-PK-mediated regulation of Lmx1a+ cells is dependent on the rapamycin-sensitive complex, mTORC1. Interestingly, this DNA-PK inhibitor effect was preserved in a co-culture differentiation protocol. Terminal differentiation assays showed that DNA-PK inhibition shifted development of neurons from forebrain toward midbrain character as assessed by Pitx3/TH immunolabeling and corresponding upregulation of midbrain (<i>En1</i>), but not forebrain (<i>FoxG1</i>) transcripts. These studies show that Lmx1a signaling in mouse embryonic stem cells contributes to a molecular cascade establishing neuronal specification. The data presented here identifies a novel regulatory pathway where signaling from DNA-PK appears to suppress midbrain-specific Lmx1a expression.</p> </div

Modulation of Lmx1a reporter gene expression by combinations of small molecule kinase inhibitors.

<p>Luciferase activity was assayed on day 8 after exposure of differentiating mESCs to combinations of SMAs from day 4-8. (A) Activity of EGF inhibitor plus other SMAs (B) Activity of VEGF inhibitor plus other SMAs, ***p&lt;0.001 and *p&lt;0.05, one-way ANOVA using Bonferroni’s multiple comparison test. Data expressed as mean ± SEM, n=4.</p

Small molecule inhibitor screens reveal multiple inhibitor classes effect Lmx1a reporter gene expression.

<p>143 kinase inhibitors were screened yielding 16 small molecule agents (SMAs) for further analysis. Panel A shows Luciferase activity on day 8 of differentiation after incubation of <i>Lmx1a</i>-<i>luc</i> mESCs with SMAs from day 4 to day 8. Panel B shows that incubation with inhibitors that promote Lmx1a activity also increases the generation of Lmx1a+ cells. Color coded bars in A represent different SMAs with known similar kinase inhibitory activity, where black arrows indicate SMAs selected for analysis in panel B See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078759#pone.0078759.s001" target="_blank">Figures <b>S1</b></a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078759#pone.0078759.s002" target="_blank"><b>S2</b></a> for compound libraries used. Data expressed as mean ± SEM of at least four independent experiments. **p&lt;0.01 and *p&lt;0.05 one-way ANOVA with Bonferroni’s post-hoc test, n=4. </p

Activation of PI3K and PIKK signaling pathways induces Lmx1a reporter gene expression.

<p>Panel A shows the luciferase activity from <i>Lmx1a</i>-<i>luc</i> mESCs on day 8, after incubation with combinations of SMAs from day 4-8. PD169316 and SB220025 are inhibitors of p38 MAPK pathway whereas U-73122 specifically inhibits PLC-γ signaling. Solid colored columns represent incubations with single SMAs whereas striped columns depict combination incubation of SMAs. Western Blot analysis (B and C) revealed that DNA-PKi induces phosphorylation of mTOR, another member of the PIKK superfamily, whilst Rapamycin inhibited its phosphorylation. Membranes were probed for mTOR specifically phosphorylated at Serine 2448 (Ser2448) and β-actin using primary antibodies. Proteins were visualized using donkey anti-rabbit AlexaFluor 680 and donkey anti-mouse IE-800 for mTOR and β-actin, respectively. Data presented as mean ± SEM, ***p&lt;0.001, **p&lt;0.01 and *p&lt;0.05 for one-way ANOVA compared to vehicle using Bonferroni’s post-hoc test, n=3. Abbreviations: PTENi- Phosphatase tensin homologue inhibitor, JNK- Janus Kinase, MEK- Mitogen-activated protein kinase kinase, PDA- Phorbol di-acetate and mTOR- Mammalian target of rapamycin.</p

Small molecules can promote the specification of specific post-mitotic phenotypes.

<p>Neural progenitors incubated with SMAs gave rise to TH+ (A), Pitx3+ (B) and TH+/Pitx3+ neurons (C) by day 19 of differentiation. Pitx3 expression was determined using from residual GFP expression after fixation of cells produced by differentiating the <i>Pitx3</i>-<i>eGFP</i> mESC line, whereas visualization of TH was achieved using the secondary antibody anti-Rabbit AlexFluor594 binding to a Rabbit anti-TH primary antibody. Column bars show mean ± SEM fraction of Pitx3+ (D) and TH+ (E) cells present in each field of view, expressed as a fraction of the total cell counts (n = 35-36 fields of view from four replicate experiments). ***p&lt;0.001 **p&lt;0.01 and *p&lt;0.05 respectively, using one-way ANOVA compared to vehicle with Dunnett’s multiple comparisons. Scale 100 µm. </p

DNA-PK inhibition promotes regional specification and upregulation of Notch pathway transcripts.

<p>Real-time PCR analyses for transcripts of Notch signaling and regional specification using Lmx1a+ progenitors after isolation by flow cytometry. Cells exposed to DNA-PKi for four days (days 4-8), significantly (p&lt;0.01) upregulated the Notch effector genes <i>Hes5</i> and <i>Hey1</i> as well as the Notch target, and RG marker <i>Pax6</i>, by day 8. Incubation with DNA-PKi during this period also upregulated expression of the midbrain marker Engrailed 1 (En1), however, expression of forebrain marker (FoxG1), also known as brain factor 1 (BF-1), was unchanged in these cultures. DNA-PKi-incubated cells also significantly downregulated the expression of <i>FoxA2</i> and <i>Corin</i>, two markers that demarcate progenitors of the ventral neural tube. Data presented as mean ± SEM of at least four independent experiments ***p&lt;0.001, **p&lt;0.01 and *p&lt;0.05, respectively, using one-way ANOVA with Bonferroni’s post-hoc test, n=3.</p