Gene transfer of programmed death ligand-1.Ig prolongs cardiac allograft survival.

BACKGROUND: The CD28 homologue programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2 (which are homologous to B7), constitute an inhibitory pathway of T cell costimulation. The PD-1 pathway is of interest for immune-mediated diseases given that PD-1-deficient mice develop autoimmune diseases. We have evaluated the effect of local overexpression of a PD-L1.Ig fusion protein on cardiac allograft survival. METHODS: Adenovirus-mediated PD-L1.Ig gene transfer was performed in F344 rat donor hearts placed in the abdominal position in Lewis recipients. Inflammatory cell infiltrates in the grafts were assessed by immunohistochemistry. RESULTS: Allografts transduced with the PD-L1.Ig gene survived for longer periods of time compared with those receiving noncoding adenovirus or virus dilution buffer alone: median survival time (MST), 17 (range: 16-20) days vs. 11 (8-14) and 9 (8-13) days, respectively (P < 0.001). PD-L1.Ig gene transfer combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone: MST, 25 (15-42) vs. 15 (13-19) days (P < 0.05). PD-L1.Ig gene transfer was associated with decreased numbers of CD4 cells and monocytes/macrophages infiltrating the graft (P < 0.05). CONCLUSIONS: Localized PD-L1.Ig expression in donor hearts attenuates acute allograft rejection in a rat model. The effect is additive to that of a subtherapeutic regimen of CsA. These results suggest that targeting of PD-1 by gene therapy may inhibit acute cardiac allograft rejection in vivo.

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors.

Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancer-blocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.

Music Performance Anxiety (MPA) : cardiorespiratory activity in high- and low-anxious professional music students before a performance situation

Descriptors: music performance anxiety, respiration, hyperventilation Surveys indicate that high-anxious musicians may suffer from hyperventilation (HV) before or during performance. Reported symptoms include shortness of breath, fast/ deep breathing and thumping heart. However, no study has yet tested if these selfreported symptoms reflect actual cardiorespiratory activity. Themain goal of this study was to determine if MPA is manifested physiologically in specific correlates of cardiorespiratory activity associated with HV.We studied 74 professional music students from Swiss Music Academies. In this study, we compared the most anxious students (highanxious; n 5 20) with the least anxious students (low-anxious; n 5 23) based on their self-reported performance anxiety. We measured cardiorespiratory patterns with the Lifeshirt system, end-tidal CO2 with a capnograph (EtCO2, a good non-invasive estimator of HV), self-perceived physiological activation and affective experience in three situations on different days: baseline, performance without audience, and performance with audience. Comparing measures for the private vs. the public concert, high- compared to low-anxious students showed a significant drop in EtCO2 before the public concert and reported larger increases in anxiety, tension, palpitations and breathing difficulties. In contrast, heart rate, respiratory rate and volume did not differ significantly between groups. The results of this study support the hypothesis thatMPA may be associated with a tendency to hyperventilate and, thus, point to a potential hyperventilation problem in high-anxious music students.

Laser scanning evaluation of atrophy after autologous free muscle transfer.

Denervated muscle tissue undergoes morphologic changes that result in atrophy. The amount of muscle atrophy after denervation following free muscle transfer has not been measured so far. Therefore, the amount of muscle atrophy in human free muscle transfer for lower extremity reconstruction was measured in a series of 10 patients. Three-dimensional laser surface scanning was used to measure flap volume changes 2 weeks as well as 6 and 12 months after the operation. None of the muscles transferred was re-innervated.All muscles healed uneventfully without signs of compromised perfusion resulting in partial flap loss. The muscle volume decreased to 30 ± 4% and 19 ± 4% 6 and 12 months, respectively, after the operation, ie, the volume decreased by approximately 80% within a 12-month period.Denervated free muscle flap tissue undergoes massive atrophy of approximately 80%, mostly within the first 6 months.

Speaking in the CLIL classroom: to what extent do teachers and students use english in the classroom?

L'objectiu principal d'aquesta investigació va ser determinar els efectes del CLIL en els professors i estudiants en les seves produccions orals. El punt de partida de la meva investigació era de la teoria de Dalton-Puffer (2007), que estableix que la funció de parlar en les classes CLIL és mínima, la meva recerca s'ha basat en la recopilació recollir informació des de diferents fonts, amb la finalitat de veure si aquest fet també passa a les nostres escoles. Eventualment, he planejat algunes estratègies i activitats per incrementar la quantitat d’oportunitats per parlar en llengua anglesa els alumnes.

Teaching Motivational Interviewing to Medical Students to Improve Behavior Change Counseling Skills - Results of a Pilot Test

BACKGROUND: Patient behavior accounts for half or more of the variance in health, disease, mortality and treatment outcome and costs. Counseling using motivational interviewing (MI) effectively improves the substance use and medical compliance behavior of patients. Medical training should include substantial focus on this key issue of health promotion. The objective of the study is to test the efficacy of teaching MI to medical students. METHODS: Thirteen fourth-year medical students volunteered to participate. Seven days before and after an 8-hour interactive MI training workshop, each student performed a video-recorded interview with two standardized patients: a 60 year-old alcohol dependent female consulting a primary care physician for the first time about fatigue and depression symptoms; and a 50 year-old male cigarette smoker hospitalized for myocardial infarction. All 52 videos (13 students×2 interviews before and after training) were independently coded by two blinded clinicians using the Motivational Interviewing Training Integrity (MITI, 3.0). MITI scores consist of global spirit (Evocation, Collaboration, Autonomy/Support), global Empathy and Direction, and behavior count summary scores (% Open questions, Reflection to question ratio, % Complex reflections, % MI-adherent behaviors). A "beginning proficiency" threshold (BPT) is defined for each of these 9 scores. The proportion of students reaching BPT before and after training was compared using McNemar exact tests. Inter-rater reliability was evaluated by comparing double coding, and test-retest analyses were conducted on a sub-sample of 10 consecutive interviews by each coder. Weighted Kappas were used for global rating scales and intra-class correlations (ICC) were computed for behavior count summary scores. RESULTS: The percent of counselors reaching BPT before and after MI training increased significantly for Evocation (15% to 65%, p<.001), Collaboration (27% to 77%, p=.001), Autonomy/Support (15% to 54%, p=.006), and % Open questions (4% to 38%, p=.004). Proportions increased, but were not statistically significant for Empathy (38% to 58%, p=.18), Reflection to question ratio (0% to 15%, p=.12), % Complex reflection (35% to 54%, p=.23), and % MI-adherent behaviors (8% to 15%, p=.69). There was virtually no change for the Direction scale (92% to 88%, p=1.00). The reliability analyses produced mixed results. Weighted kappas for inter-rater reliability ranged from .14 for Direction to .51 for Collaboration, and from .27 for Direction to .80 for Empathy for test-retest. ICCs ranged from .20 for Complex reflections to .89 for Open questions (inter-rater), and from .67 for Complex reflections to .99 for Reflection to question ratio (test-retest). CONCLUSION: This pilot study indicates that a single 8-hour training in motivational interviewing for voluntary fourth-year medical students results in significant improvement of some MI skills. A larger sample of randomly selected medical students observed over longer periods should be studied to test if MI training generalizes to medical students. Inter-rater reliability and test-retest findings indicate a need for caution when interpreting the present results, as well as for more intensive training to help appropriately capture more dimensions of the process in future studies.

Needs, expectations, and concerns of medical students regarding end-of-life issues before the introduction of a mandatory undergraduate palliative care curriculum.

BACKGROUND: In the past, implementation of effective palliative care curricula has emerged as a priority in medical education. In order to gain insight into medical students' needs and expectations, we conducted a survey before mandatory palliative care education was introduced in our faculty. METHODS: Seven hundred nine students answered a questionnaire mainly consisting of numeric rating scales (0-10). RESULTS: Participants attributed a high importance to palliative care for their future professional life (mean, 7.51 ± 2.2). For most students, symptom control was crucial (7.72 ± 2.2). However, even higher importance was assigned to ethical and legal issues (8.16 ± 1.9). "Self-reflection regarding their own role as a physician caring for the terminally ill along with psychological support" was also regarded as highly important (7.25 ± 2.4). Most students were moderately concerned at the prospect of being confronted with suffering and death (5.13 ± 2.4). This emotional distress was rated significantly higher by female students (5.4 ± 2.4 versus 4.6 ± 2.4; p < 0.001). Seventeen percent of all students rated their distress as being 7 of 10 or higher, which indicates a considerable psychological strain in terms of dealing with end-of-life issues in the future. Professional or personal experience with terminally ill persons lowered these anxieties significantly (4.99 ± 2.34 versus 5.47 ± 2.5, p < 0.05). CONCLUSIONS: Medical students stated a remarkably high interest in learning palliative care competencies. Responding to their specific concerns and needs-especially with regard to the acquisition of emotional coping skills-may be key for the development of successful palliative care curricula.

Use of phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow cytometry: advantages and limitations.

BACKGROUND: This study validates the use of phycoerythrin (PE) and allophycocyanin (APC) for fluorescence energy transfer (FRET) analyzed by flow cytometry. METHODS: FRET was detected when a pair of antibody conjugates directed against two noncompetitive epitopes on the same CD8alpha chain was used. FRET was also detected between antibody conjugate pairs specific for the two chains of the heterodimeric alpha (4)beta(1) integrin. Similarly, the association of T-cell receptor (TCR) with a soluble antigen ligand was detected by FRET when anti-TCR antibody and MHC class I/peptide complexes (&lt;&lt;tetramers&gt;&gt;) were used. RESULTS: FRET efficiency was always less than 10%, probably because of steric effects associated with the size and structure of PE and APC. Some suggestions are given to take into account this and other effects (e.g., donor and acceptor concentrations) for a better interpretation of FRET results obtained with this pair of fluorochromes. CONCLUSIONS: We conclude that FRET assays can be carried out easily with commercially available antibodies and flow cytometers to study arrays of multimolecular complexes.

A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system.

BACKGROUND: The genome of Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs). RESULTS: On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit. CONCLUSIONS: A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for Chlamydiales.

Establishing Load Transfer in Existing Jointed Concrete Pavements, HR-1041, 1985

In this paper are described the results of a research project that had the objective of developing construction procedures for restoring load transfer in existing jointed concrete pavements and of evaluating the effectiveness of the restoration methods. A total of 28 test sections with various load transfer devices were placed. The devices include split pipe, figure eight, vee, double vee, and dowel bars. Patching materials used on the project included three types of fast-setting grouts, three brands of polymer concrete, and plain portland cement concrete. The number and spacing of the devices and dowel bars were also variables in the project. Dowel bars and double vee devices were used on the major portion of the project. Performance evaluations were based on deflection tests conducted with a 20,000-lb axle load. Horizontal joint movement measurements and visual observations were also made. The short-term performance data indicate good results with the dowel bar installations regardless of patching materials. The sections with split pipe, figure eight, and vee devices failed in bond during the first winter cycle. The results with the double vee sections indicate the importance of the patching material to the success or failure of the load transfer system: some sections are performing well and other sections are performing poorly with double vee devices. Horizontal joint movement measurements indicate that neither the dowel bars nor the double vee devices are restricting joint movement.

Efficient lentiviral gene transfer into corneal stroma cells using a femtosecond laser.

We investigated a new procedure for gene transfer into the stroma of pig cornea for the delivery of therapeutic factors. A delimited space was created at 110 mum depth with a LDV femtosecond laser in pig corneas, and a HIV1-derived lentiviral vector expressing green fluorescent protein (GFP) (LV-CMV-GFP) was injected into the pocket. Corneas were subsequently dissected and kept in culture as explants. After 5 days, histological analysis of the explants revealed that the corneal pockets had closed and that the gene transfer procedure was efficient over the whole pocket area. Almost all the keratocytes were transduced in this area. Vector diffusion at right angles to the pocket's plane encompasses four (endothelium side) to 10 (epithelium side) layers of keratocytes. After 21 days, the level of transduction was similar to the results obtained after 5 days. The femtosecond laser technique allows a reliable injection and diffusion of lentiviral vectors to efficiently transduce stromal cells in a delimited area. Showing the efficacy of this procedure in vivo could represent an important step toward treatment or prevention of recurrent angiogenesis of the corneal stroma.

Fate of the herbicide glyphosate and its metabolite AMPA in soils and their transfer to surface waters: A multi-scale approach in the Lavaux vineyards, western Switzerland

The use of herbicides in agriculture may lead to environmental problems, such as surface water pollution, with a potential risk for aquatic organisms. The herbicide glyphosate is the most used active ingredient in the world and in Switzerland. In the Lavaux vineyards it is nearly the only molecule applied. This work aimed at studying its fate in soils and its transfer to surface waters, using a multi-scale approach: from molecular (10-9 m) and microscopic scales (10-6 m), to macroscopic (m) and landscape ones (103 m). First of all, an analytical method was developed for the trace level quantification of this widely used herbicide and its main by-product, aminomethylphosphonic acid (AMPA). Due to their polar nature, their derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was done prior to their concentration and purification by solid phase extraction. They were then analyzed by ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The method was tested in different aqueous matrices with spiking tests and validated for the matrix effect correction in relevant environmental samples. Calibration curves established between 10 and 1000ng/l showed r2 values above 0.989, mean recoveries varied between 86 and 133% and limits of detection and quantification of the method were as low as 5 and 10ng/l respectively. At the parcel scale, two parcels of the Lavaux vineyard area, located near the Lutrive River at 6km to the east of Lausanne, were monitored to assess to which extent glyphosate and AMPA were retained in the soil or exported to surface waters. They were equipped at their bottom with porous ceramic cups and runoff collectors, which allowed retrieving water samples for the growing seasons 2010 and 2011. Results revealed that the mobility of glyphosate and AMPA in the unsaturated zone was likely driven by the precipitation regime and the soil characteristics, such as slope, porosity structure and layer permeability discrepancy. Elevated glyphosate and AMPA concentrations were measured at 60 and 80 cm depth at parcel bottoms, suggesting their infiltration in the upper parts of the parcels and the presence of preferential flow in the studied parcels. Indeed, the succession of rainy days induced the gradual saturation of the soil porosity, leading to rapid infiltration through macropores, as well as surface runoff formation. Furthermore, the presence of more impervious weathered marls at 100 cm depth induced throughflows, the importance of which for the lateral transport of the herbicide molecules was determined by the slope steepness. Important rainfall events (>10 mm/day) were clearly exporting molecules from the soil top layer, as indicated by important concentrations in runoff samples. A mass balance showed that total loss (10-20%) mainly occurred through surface runoff (96%) and, to a minor extent, by throughflows in soils (4%), with subsequent exfiltration to surface waters. Observations made in the Lutrive River revealed interesting details of glyphosate and AMPA dynamics in urbanized landscapes, such as the Lavaux vineyards. Indeed, besides their physical and chemical properties, herbicide dynamics at the catchment level strongly depend on application rates, precipitation regime, land use and also on the presence of drains or constructed channels. Elevated concentrations, up to 4970 ng/l, observed just after the application, confirmed the diffuse export of these compounds from the vineyard area by surface runoff during main rain events. From April to September 2011, a total load of 7.1 kg was calculated, with 85% coming from vineyards and minor urban sources and 15% from arable crops. Small vineyard surfaces could generate high concentrations of herbicides and contribute considerably to the total load calculated at the outlet, due to their steep slopes (~10%). The extrapolated total amount transferred yearly from the Lavaux vineyards to the Lake of Geneva was of 190kg. At the molecular scale, the possible involvement of dissolved organic matter (DOM) in glyphosate and copper transport was studied using UV/Vis fluorescence spectroscopy. Combined with parallel factor (PARAFAC) analysis, this technique allowed characterizing DOM of soil and surface water samples from the studied vineyard area. Glyphosate concentrations were linked to the fulvic-like spectroscopic signature of DOM in soil water samples, as well as to copper, suggesting the formation of ternary complexes. In surface water samples, its concentrations were also correlated to copper ones, but not in a significant way to the fulvic-like signature. Quenching experiments with standards confirmed field tendencies in the laboratory, with a stronger decrease in fluorescence intensity for fulvic-like fluorophore than for more aromatic ones. Lastly, based on maximum concentrations measured in the river, an environmental risk for these compounds was assessed, using laboratory tests and ecotoxicity data from the literature. In our case and with the methodology applied, the risk towards aquatic species was found negligible (RF<1).