The impact of CFNS-causing EFNB1 mutations on ephrin-B1 function

BACKGROUND: Mutations of EFNB1 cause the X-linked malformation syndrome craniofrontonasal syndrome (CFNS). CFNS is characterized by an unusual phenotypic pattern of inheritance, because it affects heterozygous females more severely than hemizygous males. This sex-dependent inheritance has been explained by random X-inactivation in heterozygous females and the consequences of cellular interference of wild type and mutant EFNB1-expressing cell populations. EFNB1 encodes the transmembrane protein ephrin-B1, that forms bi-directional signalling complexes with Eph receptor tyrosine kinases expressed on complementary cells. Here, we studied the effects of patient-derived EFNB1 mutations predicted to give rise to truncated ephrin-B1 protein or to disturb Eph/ephrin-B1 reverse ephrin-B1 signalling. Five mutations are investigated in this work: nonsense mutation c.196C > T/p.R66X, frameshift mutation c.614_615delCT, splice-site mutation c.406 + 2T > C and two missense mutations p.P54L and p.T111I. Both missense mutations are located in the extracellular ephrin domain involved in Eph-ephrin-B1 recognition and higher order complex formation. METHODS: Nonsense mutation c.196C > T/p.R66X, frameshift mutation c.614_615delCT and splice-site mutation c.406+2T > C were detected in the primary patient fibroblasts by direct sequencing of the DNA and were further analysed by RT-PCR and Western blot analyses.The impact of missense mutations p.P54L and p.T111I on cell behaviour and reverse ephrin-B1 cell signalling was analysed in a cell culture model using NIH 3T3 fibroblasts. These cells were transfected with the constructs generated by in vitro site-directed mutagenesis. Investigation of missense mutations was performed using the Western blot analysis and time-lapse microscopy. RESULTS AND DISCUSSION: Nonsense mutation c.196C > T/p.R66X and frameshift mutation c.614_615delCT escape nonsense-mediated RNA decay (NMD), splice-site mutation c.406+2T > C results in either retention of intron 2 or activation of a cryptic splice site in exon 2. However, c.614_615delCT and c.406+2T > C mutations were found to be not compatible with production of a soluble ephrin-B1 protein. Protein expression of the p.R66X mutation was predicted unlikely but has not been investigated.Ectopic expression of p.P54L ephrin-B1 resists Eph-receptor mediated cell cluster formation in tissue culture and intracellular ephrin-B1 Tyr324 and Tyr329 phosphorylation. Cells expressing p.T111I protein show similar responses as wild type expressing cells, however, phosphorylation of Tyr324 and Tyr329 is reduced. CONCLUSIONS: Pathogenic mechanisms in CFNS manifestation include impaired ephrin-B1 signalling combined with cellular interference

An e-learning reproductive health module to support improved student learning and interaction: a prospective interventional study at a medical school in Egypt

<p>Abstract</p> <p>Background</p> <p>The Public Health (PH) course at the medical college of Cairo University is based on traditional lectures. Large enrollment limits students' discussions and interactions with instructors.</p> <p>Aim</p> <p>Evaluate students' learning outcomes as measured by improved knowledge acquisition and opinions of redesigning the Reproductive Health (RH) section of the PH course into e-learning and assessing e-course utilization.</p> <p>Methods</p> <p>This prospective interventional study started with development of an e-learning course covering the RH section, with visual and interactive emphasis, to satisfy students' diverse learning styles. Two student groups participated in this study. The first group received traditional lecturing, while the second volunteered to enroll in the e-learning course, taking online course quizzes. Both groups answered knowledge and course evaluation questionnaires and were invited to group discussions. Additionally, the first group answered another questionnaire about reasons for non-participation.</p> <p>Results</p> <p>Students participating in the e-learning course showed significantly better results, than those receiving traditional tutoring. Students who originally shunned the e-course expressed eagerness to access the course before the end of the academic year. Overall, students using the redesigned e-course reported better learning experiences.</p> <p>Conclusions</p> <p>An online course with interactivities and interaction, can overcome many educational drawbacks of large enrolment classes, enhance student's learning and complement pit-falls of large enrollment traditional tutoring.</p

Evidence for Limited Genetic Compartmentalization of HIV-1 between Lung and Blood

BACKGROUND:HIV-1 is frequently detected in the lungs of infected individuals and is likely important in the development of pulmonary opportunistic infections. The unique environment of the lung, rich in alveolar macrophages and with specialized local immune responses, may contribute to differential evolution or selection of HIV-1. METHODOLOGY AND FINDINGS:We characterized HIV-1 in the lung in relation to contemporaneous viral populations in the blood. The C2-V5 region of HIV-1 env was sequenced from paired lung (induced sputum or bronchoalveolar lavage) and blood (plasma RNA and proviral DNA from sorted or unsorted PBMC) from 18 subjects. Compartmentalization between tissue pairs was assessed using 5 established tree or distance-based methods, including permutation tests to determine statistical significance. We found statistical evidence of compartmentalization between lung and blood in 10/18 subjects, although lung and blood sequences were intermingled on phylogenetic trees in all subjects. The subject showing the greatest compartmentalization contained many nearly identical sequences in BAL sample, suggesting clonal expansion may contribute to reduced viral diversity in the lung in some cases. However, HIV-1 sequences in lung were not more homogeneous overall, nor were we able to find a lung-specific genotype associated with macrophage tropism in V3. In all four subjects in whom predicted X4 genotypes were found in blood, predicted X4 genotypes were also found in lung. CONCLUSIONS:Our results support a picture of continuous migration of HIV-1 between circulating blood and lung tissue, with perhaps a very limited degree of localized evolution or clonal replication

A genome-wide association scan implicates <i>DCHS2, RUNX2, GLI3, PAX1</i> and <i>EDAR</i> in human facial variation

We report a genome-wide association scan for facial features in ∼6,000 Latin Americans. We evaluated 14 traits on an ordinal scale and found significant association (P values−8) at single-nucleotide polymorphisms (SNPs) in four genomic regions for three nose-related traits: columella inclination (4q31), nose bridge breadth (6p21) and nose wing breadth (7p13 and 20p11). In a subsample of ∼3,000 individuals we obtained quantitative traits related to 9 of the ordinal phenotypes and, also, a measure of nasion position. Quantitative analyses confirmed the ordinal-based associations, identified SNPs in 2q12 associated to chin protrusion, and replicated the reported association of nasion position with SNPs in PAX3. Strongest association in 2q12, 4q31, 6p21 and 7p13 was observed for SNPs in the EDAR, DCHS2, RUNX2 and GLI3 genes, respectively. Associated SNPs in 20p11 extend to PAX1. Consistent with the effect of EDAR on chin protrusion, we documented alterations of mandible length in mice with modified Edar function

Influences motivating smokers in a radon-affected area to quit smoking

Domestic radon gas concentrations in parts of the UK are sufficiently high to increase lung cancer risk among residents, and recent studies have confirmed that the risk of smokers developing lung cancer is significantly enhanced by the presence of radon. Despite campaigns encouraging residents of radon-affected areas (RAEs) to test and remediate their homes, public response to the risks posed by radon remains relatively modest, particularly among smokers and young families, limiting the health benefits and cost-effectiveness achievable by remediation. The observation that smokers, who are most at risk from radon, are not explicitly targeted by current radon remediation campaigns prompted an assessment of the value of smoking-cessation initiatives in reducing radon-induced lung cancers by reaching at-risk subgroups of the population hitherto uninfluenced by radon-awareness programmes. This study addresses the motivation of current quitters in a designated RAE using a postal questionnaire administered around one year after the cessation attempt. Residents of the Northamptonshire RAE who had joined the smoking-cessation programme between July and September 2006 and who remained verifiably tobacco free at four weeks, were subsequently invited to participate in a questionnaire-based investigation into factors affecting their decision to cease smoking. From an initial population of 445 eligible individuals, 205 of those contacted by telephone after 12 months agreed to complete postal questionnaires, and unsolicited questionnaires were sent to a further 112 participants for whom telephone contact had proved impossible. One hundred and three completed questionnaires were returned and analysed, the principal tools being c2, Mann-Whitney and Kruskal-Wallis tests. Individuals decide to quit smoking from self-interest, principally on health grounds, and regard the effects of their smoke on others, particularly children and unborn babies, as less significant. The risk of developing respiratory, coronary/cardiac or cancerous conditions provides the greatest motivation to the decision to quit, with knowledge of radon among the lowest-ranked influences. This study confirms that quitters place risks to their personal health as the highest factors influencing their decision to quit, and health professionals should be aware of this when designing smoking-cessation initiatives. As radon risk is ranked very low by quitters, there would appear to be the potential to raise radon awareness through smoking-cessation programmes, with the objective of increasing the uptake and success rate of such programmes and encouraging participation in radon-remediation programmes

Synthesis of carboxylated derivatives of poly(isobutylene-co-isoprene) by azide–alkyne “click” chemistry

The final publication is available at Springer via https://dx.doi.org/10.1038/s41428-018-0130-yThe synthesis of carboxylated derivatives of poly(isobutylene-co-isoprene) (isobutylene–isoprene rubber, IIR) with substitution levels ranging from 1 to 4 mol% and different spacer lengths was accomplished through azide–alkyne Huisgen cycloaddition. Azido-functionalized IIR was first prepared by reacting brominated IIR with sodium azide to full conversion in a 90:10 tetrahydrofuran/N,N-dimethylacetamide mixture. The click reaction of azido-functionalized IIR with acetylenic acids, which was carried out using the copper(I) bromide/N,N,N′,N″,N″-pentamethyldiethylenetriamine catalyst system in tetrahydrofuran, yielded carboxylated IIRs. The products were characterized by 1H NMR and FT-IR spectroscopy, and their molecular weight was determined by size exclusion chromatography analysis. The conversion to carboxylated groups reached up to 100% as determined by NMR spectroscopy but was highly dependent on the type of solvent and the amounts of catalysts and reactants used in the procedures.ARLANXEO Canada Inc.Natural Sciences and Engineering Research Council (NSERC) of Canad

Adenosine Deaminase Acting on RNA-1 (ADAR1) Inhibits HIV-1 Replication in Human Alveolar Macrophages

While exploring the effects of aerosol IFN-γ treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-γ-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-γ induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro . Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-γ suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages