Having Fun in Learning Formal Specifications

There are many benefits in providing formal specifications for our software. However, teaching students to do this is not always easy as courses on formal methods are often experienced as dry by students. This paper presents a game called FormalZ that teachers can use to introduce some variation in their class. Students can have some fun in playing the game and, while doing so, also learn the basics of writing formal specifications in the form of pre- and post-conditions. Unlike existing software engineering themed education games such as Pex and Code Defenders, FormalZ takes the deep gamification approach where playing gets a more central role in order to generate more engagement. This short paper presents our work in progress: the first implementation of FormalZ along with the result of a preliminary users' evaluation. This implementation is functionally complete and tested, but the polishing of its user interface is still future work

A Multi-Scale Test of the Forage Maturation Hypothesis in a Partially Migratory Ungulate Population

The forage maturation hypothesis (FMH) proposes that ungulate migration is driven by selection for high forage quality. Because quality declines with plant maturation, but intake declines at low biomass, ungulates are predicted to select for intermediate forage biomass to maximize energy intake by following phenological gradients during the growing season. We tested the FMH in the Canadian Rocky Mountains by comparing forage availability and selection by both migrant and nonmigratory resident elk (Cervus elaphus) during three growing seasons from 2002-2004. First, we confirmed that the expected trade-off between forage quality and quantity occurred across vegetation communities. Next, we modeled forage biomass and phenology during the growing season by combining ground and remote-sensing approaches. The growing season started 2.2 days earlier every 1 km east of the continental divide, was delayed by 50 days for every 1000-m increase in elevation, and occurred 8 days earlier on south aspects. Migrant and resident selection for forage biomass was then compared across three spatial scales (across the study area, within summer home ranges, and along movement paths) using VHF and GPS telemetry locations from 119 female elk. Migrant home ranges occurred closer to the continental divide in areas of higher topographical diversity, resulting in migrants consistently selecting for intermediate biomass at the two largest scales, but not at the. nest scale along movement paths. In contrast, residents selected maximum forage biomass across all spatial scales. To evaluate the consequences of selection, we compared exposure at telemetry locations of migrant and resident elk to expected forage biomass and digestibility. The expected digestibility for migrant elk in summer was 6.5% higher than for residents, which was corroborated with higher fecal nitrogen levels for migrants. The observed differences in digestibility should increase migrant elk body mass, pregnancy rates, and adult and calf survival rates. Whether bottom-up effects of improved forage quality are realized will ultimately depend on trade-offs between forage and predation. Nevertheless, this study provides comprehensive evidence that montane ungulate migration leads to greater access to higher-quality forage relative to nonmigratory congeners, as predicted by the forage maturation hypothesis, resulting primarily from large-scale selection patterns

Promoting patient utilization of outpatient cardiac rehabilitation: A joint International Council and Canadian Association of Cardiovascular Prevention and Rehabilitation position statement

Background: Cardiac Rehabilitation (CR) is a recommendation in international clinical practice guidelines given its’ benefits, however use is suboptimal. The purpose of this position statement was to translate evidence on interventions that increase CR enrolment and adherence into implementable recommendations. Methods: The writing panel was constituted by representatives of societies internationally concerned with preventive cardiology, and included disciplines that would be implementing the recommendations. Patient partners served, as well as policy-makers. The statement was developed in accordance with AGREE II, among other guideline checklists. Recommendations were based on our update of the Cochrane review on interventions to promote patient utilization of CR. These were circulated to panel members, who were asked to rate each on a 7-point Likert scale in terms of scientific acceptability, actionability, and feasibility of assessment. A web call was convened to achieve consensus and confirm strength of the recommendations (based on GRADE). The draft underwent external review and public comment. Results: The 3 drafted recommendations were that to increase enrolment, healthcare providers, particularly nurses (strong), should promote CR to patients face-to-face (strong), and that to increase adherence part of CR could be delivered remotely (weak). Ratings for the 3 recommendations were 5.95±0.69 (mean ± standard deviation), 5.33±1.12 and 5.64±1.08, respectively. Conclusions: Interventions can significantly increase utilization of CR, and hence should be widely applied. We call upon cardiac care institutions to implement these strategies to augment CR utilization, and to ensure CR programs are adequately resourced to serve enrolling patients and support them to complete programs

The Epstein-Barr Virus Glycoprotein gp150 Forms an Immune-Evasive Glycan Shield at the Surface of Infected Cells

<div><p>Cell-mediated immunity plays a key role in host control of viral infection. This is exemplified by life-threatening reactivations of e.g. herpesviruses in individuals with impaired T-cell and/or iNKT cell responses. To allow lifelong persistence and virus production in the face of primed immunity, herpesviruses exploit immune evasion strategies. These include a reduction in viral antigen expression during latency and a number of escape mechanisms that target antigen presentation pathways. Given the plethora of foreign antigens expressed in virus-producing cells, herpesviruses are conceivably most vulnerable to elimination by cell-mediated immunity during the replicative phase of infection. Here, we show that a prototypic herpesvirus, Epstein-Barr virus (EBV), encodes a novel, broadly acting immunoevasin, gp150, that is expressed during the late phase of viral replication. In particular, EBV gp150 inhibits antigen presentation by HLA class I, HLA class II, and the non-classical, lipid-presenting CD1d molecules. The mechanism of gp150-mediated T-cell escape does not depend on degradation of the antigen-presenting molecules nor does it require gp150’s cytoplasmic tail. Through its abundant glycosylation, gp150 creates a shield that impedes surface presentation of antigen. This is an unprecedented immune evasion mechanism for herpesviruses. In view of its likely broader target range, gp150 could additionally have an impact beyond escape of T cell activation. Importantly, B cells infected with a gp150-null mutant EBV displayed rescued levels of surface antigen presentation by HLA class I, HLA class II, and CD1d, supporting an important role for iNKT cells next to classical T cells in fighting EBV infection. At the same time, our results indicate that EBV gp150 prolongs the timespan for producing viral offspring at the most vulnerable stage of the viral life cycle.</p></div

EBV gp150 is broadly acting, but displays a degree of specificity for antigen-presenting molecules.

<p>Flow cytometric analyses of MJS-CD1d cells (adherent fraction) three days post transduction with gp150-IRES-GFP lentiviruses. A) Total EBV gp150 levels were determined by intracellular staining of permeabilized cells. Surface levels of HLA I, II, CD1d, CD10, and CD54 were determined using Ab staining on non-permeabilized cells. B) Surface levels of CD1d and CD54 are depicted as log MFI values with 95% confidence intervals, for GFP⁺ versus GFP^- MJS-CD1d cells transduced with gp150 or a GFP control. The slopes of the connecting lines reflect the declines in fluorescence (Δlog MFI) and, thus, the downregulation of HLA I, II, CD1d, CD10, and CD54 induced by gp150 (for the GFP control, the Δlog MFI did not significantly differ from 0). C) Cell surface levels of HLA I, II, CD10, CD34, CD44, and CD54 were determined using surface Ab staining. EBV gp150-induced downregulation of these molecules was calculated as in B) and represented as Δlog MFI.</p

Immune evasion by EBV gp150 occurs at the cell surface.

<p>A) The glycosylation status of CD1d and HLA I molecules in lysates of MJS-CD1d-gp150 and control cells was analysed by Western blot. To this end, denatured post-nuclear lysates of different FACS sorted cell populations (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005550#ppat.1005550.s004" target="_blank">S4 Fig</a>) were treated with Endo H or PNGase F to remove N-linked glycans. Endo H digestions served to examine protein transport beyond the cis-Golgi compartment; PNGase F digestions revealed the deglycosylated protein backbone (minus carbohydrates, -CHO). Actin was a loading control. B) Using the same experimental setup as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005550#ppat.1005550.g004" target="_blank">Fig 4C</a>, MJS-A2-GFP cells were transduced with a lentivirus encoding HA-gp150ΔC. Cell surface levels of CD1d, HLA I, and HLA-A2 (BB7.2) and total levels of HLA-A2 (GFP) were compared for (non-permeabilized) gp150^- and gp150⁺ cells. C) Confocal microscopy of non-permeabilized MJS-A2GFP-HA-gp150ΔC cells. Cell surface stains were performed for HLA-A2 (BB7.2) and gp150ΔC (HA). D) Quantification of surface expression of the indicated molecules based on multiple microscopy pictures.</p

gp150 interferes with surface display of Ag-presenting molecules during productive EBV infection.

<p>A) EBV⁺ AKBM-CD1d BL cells were treated for 20 hours with anti-human IgG Ab to induce viral replication. EBV-producing cells were identified by induced expression of the lytic cycle reporter rat CD2-GFP. In the presence of PAA, productive infection is arrested before late protein expression. Surface levels of the EBV late complex gHgL and of the Ag-presenting molecules HLA I, II, and CD1d were determined by flow cytometry. Histograms depict overlays to allow comparison of latently (GFP^-) and lytically (GFP⁺) infected B cells. B) 293T-CD1d cells were transfected with expression vectors encoding late EBV glycoproteins. Glycoproteins known to require a viral interaction partner were transfected together (BMRF2/BDLF2 and gM/gN). EBV protein expression was deduced from coexpression of GFP (BMRF2/BDLF2) or on the basis of a C-terminal tag (gM/gN, gB, gp350, gp150). Cell surface CD1d was stained prior to an intracellular staining for the tagged EBV proteins. Surface levels were compared between non-transfected and transfected cells. C) 293T-CD1d cells were transfected with N-terminally HA-tagged gp150 or an empty IRES-GFP vector control. Total gp150 expression was determined by intracellular staining and was compared to levels in EBV-producing B cells. The other histograms depict surface levels of HA (HA-gp150), HLA I, and CD1d staining for empty vector-transfected and gp150⁺GFP^high 293T-CD1d cells; GFP^high gating as indicated in the dot plots. D) Expression of gp150 was assessed at different time points during productive EBV infection of B cells; the early EBV antigen BGLF5 was taken along as a control (time points 6–10 hours and 12–20 hours are from different experiments).</p

The mechanism of gp150-induced immune evasion relies on sialoglycans shielding surface-exposed Ag-presenting molecules.

<p>A-D) CHO and Lec2 cells expressing human β₂m, HLA I, and CD1d were lentivirally transduced to co-express gp150 or HA-gp150ΔC and GFP (from EF1a and PGK promoters, respectively). A) Lectin (WGA-FITC) binding confirmed the glycosylation defect in Lec2 cells compared to parental CHO cells. B) Migration height of HA-gp150 was visualised by Western blot analysis. C-D) Levels of HLA I and CD1d at the surface of HA-gp150ΔC⁺GFP⁺ (gp150⁺) cells were compared to those on control, non-transduced GFP^- (gp150^-) cells. D) Surface levels of HLA I and CD1d are depicted as log MFI values with 95% confidence intervals, for gp150⁺ versus gp150^- wt CHO cells or sialylation-defective Lec2 cells. The slopes of the connecting lines (Δlog MFI) reflect the downregulation induced by gp150. Statistical analysis was performed using two-way ANOVAs and significance of the interaction term was assessed, as described in the Material and Methods section. One representative experiment of at least six is depicted. * p<0.01. E-L) MJS-CD1d-gp150 cells were generated by transduction with a lentivirus encoding both gp150 and GFP (from a CMV promoter and an IRES sequence, respectively) and were analysed 3 days post-transduction by Western blot and by flow cytometry, as for A-D with the modifications indicated below. E-H) To prevent sialylation, cells were treated with the sialic acid transferase inhibitor (500 μM inhibitor) fluorinated P-3F_ax-Neu5Ac. As a control, cells were treated with the non-fluorinated compound (500 μM, ctrl) for 4 days, starting 1 day prior to transduction. This control treatment was comparable to when cells were left untreated. E) Lectins SNAI, MALII, or PNA were used to detect sialoglycans or desialylated glycans, respectively. G) gp150⁺GFP^high cells were compared to non-transduced cells. H) One representative experiment of two is depicted. * p<0.01. I-L) To remove surface sialylation, intact cells were treated with neuraminidase (1U/μl, 60 min, 37°C) prior to cell lysis or lectin/Ab staining. L) One representative experiment of three is depicted. * p<0.01.</p