Mother's smoking habits affects IL10 methylation but not asthma in Ecuadorian children.

There is no evidence evaluating the IL10 epigenetic upregulation among mestizo children in a high-altitude Andean city in Latin America. OBJECTIVE: To identify polymorphisms and methylation profiles in the IL10 gene associated with asthma in children aged 5 to 11. METHODS: A case-control study was conducted with asthmatic and non-asthmatic children aged 5 to 11 years in Cuenca-Ecuador. Data on allergic diseases and risk factors were collected through a questionnaire for parents. Atopy was measured by skin prick test (SPT) to relevant aeroallergens. Three IL10 single nucleotide polymorphisms were evaluated in all participants, and methylation analysis was performed in 54 participants. Association between risk factors, allergic diseases and genetic factors were estimated using multivariate logistic regression. RESULTS: The results of polymorphisms showed no differences between cases and controls when comparing the SNPs rs3024495, rs3024496, rs1800896 allelic and genotypic frequencies. In the methylation analysis, no differences in the IL10 methylation profile were found between cases and controls; however, the multivariate analysis showed an association between the mother's smoking habits and the IL10 methylation profile. CONCLUSION: Smoking habit could be essential as an environmental exposure factor in regulating gene expression in children with asthma.

Regulation of Ebola GP conformation and membrane binding by the chemical environment of the late endosome.

Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH and Ca2+ have been implicated in mediating the GP-membrane interaction. However, the molecular mechanism by which these environmental factors regulate the conformational changes that enable engagement of GP with the target membrane is unknown. Here, we apply fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (smFRET) imaging to elucidate how the acidic pH, Ca2+ and anionic phospholipids in the late endosome promote GP-membrane interaction, thereby facilitating virus entry. We find that bis(monoacylglycero)phosphate (BMP), which is specific to the late endosome, is especially critical in determining the Ca2+-dependence of the GP-membrane interaction. Molecular dynamics (MD) simulations suggested residues in GP that sense pH and induce conformational changes that make the fusion loop available for insertion into the membrane. We similarly confirm residues in the fusion loop that mediate GP's interaction with Ca2+, which likely promotes local conformational changes in the fusion loop and mediates electrostatic interactions with the anionic phospholipids. Collectively, our results provide a mechanistic understanding of how the environment of the late endosome regulates the timing and efficiency of virus entry.

Real-world 6-month outcomes of minimally invasive aortic valve replacement with the EDWARDS INTUITY Elite valve system.

OBJECTIVES: We report on real-world safety and performance outcomes of minimally invasive rapid-deployment aortic valve replacement using the EDWARDS INTUITY Elite aortic valve system. METHODS: The study valve system was used in a European, prospective, multicentre post-market study. Various procedural, haemodynamic and clinical outcomes were evaluated through 6 months of post-implant. RESULTS: A total of 276 patients out of 280 (98.6%) enrolments were successfully implanted with the study valve using a minimally invasive approach between February 2016 and April 2017. Of these 276 patients, 240 (87%) underwent partial sternotomy and 36 (13%) patients underwent right thoracotomy. Mean cross-clamp time was 51.9 [standard deviation (SD): 16.0] min. From baseline to 6 months, the mean effective orifice area increased from 0.8 (SD: 0.3) to 1.8 (SD: 0.6) cm2 and the mean systolic gradient decreased from 46.0 (SD: 14.1) to 8.8 (SD: 3.7) mmHg. After 6 months, 70.7% and 26.4% of patients were in New York Heart Association class I and II, respectively. Freedom from death, major bleeding, major paravalvular leak, reoperation and device explant at 6 months were 96.0%, 98.5%, 98.8%, 99.2% and 99.2%, respectively. CONCLUSIONS: These results demonstrate that the study valve is a safe and effective choice for patients undergoing aortic valve replacement via minimally invasive surgery. NAME AND REGISTRATION OF REGISTRY: MISSION (Assessing clinical outcomes using the EDWARDS INTUITY Elite Valve System in isolated AVR using Minimally InvaSive Surgery In a EurOpean multi-ceNter, active, post-market registry). clinicaltrials.gov ID #NCT02907463.

Relationship of Serum Levels of IL-17, IL-18, TNF-α, and Lung Function Parameters in Patients with COPD, Asthma-COPD Overlap, and Bronchial Asthma.

Determination of markers of systemic inflammation is one of the important directions in the study of pathogenesis and improvement of diagnosis of chronic obstructive pulmonary disease (COPD), asthma-COPD overlap (ACO), and bronchial asthma (BA). The aim of our work was a comparative study of the features of changes in serum levels of IL-17, IL-18, and TNF-α in patients with COPD, ACO, and BA with various severity of the disease, as well as evaluation of the relationship between the level of these cytokines and lung ventilation function. A total of 147 patients with COPD (n = 58), ACO (n = 57), and BA (n = 32) during a stable period have been examined in this study. The control group included 21 healthy nonsmokers with similar sex-age indicators. Serum levels of IL-17, IL-18, and TNF-α were determined by ELISA. The concentrations of these cytokines in the circulation in the studied patients with COPD, ACO, and BA were higher than those in healthy nonsmokers (p ≤ 0.001). IL-17 and IL-18 levels in the blood serum were comparable in all examined patients. The mean TNF-α concentrations in the circulation in COPD and ACO were significantly higher than those in BA (p < 0.001). In patients with COPD, the levels of IL-17 and TNF-α increased progressively against the background of a decrease in numerous spirometric indicators, which allows us to consider these cytokines as systemic biomarkers of disease severity. In BA, the inverse correlations between the level of IL-17 and FEV1/FVC (%) and FEV1 have been found. In patients with ACO, the increase in IL-18 levels was associated with a decrease in FEV1 and TNF-α with FEV1/FVC (%). These findings indicate that IL-17, IL-18, and TNF-α can participate in the mechanisms of systemic inflammation and the genesis of disorders of airway obstruction in COPD, AСO, and BA. An increase in the levels of IL-17 and TNF-α may be associated with impaired bronchial patency in COPD and BA. The established associations of the IL-18 concentration in the blood serum and FEV1 only in patients with ACO allow using the level of IL-18 as a potential marker of the degree of impaired airway obstruction in this disease.

Defining the multiplicity and time of infection for the production of Zaire Ebola virus-like particles in the insect cell-baculovirus expression system.

The Ebola virus disease is a public health challenge. To date, the only available treatments are medical support or the emergency administration of experimental drugs. The absence of licensed vaccines against Ebola virus impedes the prevention of infection. Vaccines based on recombinant virus-like particles (VLP) are a promising alternative. The Zaire Ebola virus serotype (ZEBOV) is the most aggressive with the highest mortality rates. Production of ZEBOV-VLP has been accomplished in mammalian and insect cells by the recombinant coexpression of three structural proteins, the glycoprotein (GP), the matrix structural protein VP40, and the nucleocapsid protein (NP). However, specific conditions to manipulate protein concentrations and improve assembly into VLP have not been determined to date. Here, we used a design of experiments (DoE) approach to determine the best MOI and TOI for three recombinant baculoviruses: bac-GP, bac-VP40 and bac-NP, each coding for one of the main structural proteins of ZEBOV. We identified two conditions where the simultaneous expression of the three recombinant proteins was observed. Interestingly, a temporal and stoichiometric interplay between the three structural proteins was observed. VP40 was required for the correct assembly of ZEBOV-VLP. High NP concentrations reduced the accumulation of GP, which has been reported to be necessary for inducing a protective immune response. Electron microscopy showed that the ZEBOV-VLP produced were morphologically similar to the native virus micrographs previously reported in the literature. A strategy for producing ZEBOV in insect cells, which consists in using a high MOI of bac-VP40 and bac-GP, and reducing expression of NP, either by delaying infection or reducing the MOI of bac-NP, was the most adequate for the production of VLP.

Rotaviruses reach late endosomes and require the cation-dependent mannose-6-phosphate receptor and the activity of cathepsin proteases to enter the cell.

UNLABELLED: Rotaviruses (RVs) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the endosomal sorting complex required for transport (ESCRT), and the formation of endosomal intraluminal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the trans-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UK × RRV reassortant viruses, we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention. IMPORTANCE: Rotavirus is an important etiological agent of severe gastroenteritis in children. In most instances, viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EEs, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EEs to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work, we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated with the virus spike protein VP4. These findings illustrate the elaborate pathway of RV entry that could be used for drug intervention.