Structural and Compositional Changes in Erythrocyte Membrane of Obese Compared to Normal-Weight Adolescents

Unhealthy dietary habits are key determinants of obesity in adolescents. Assuming that dietary fat profile influences membrane lipid composition, the aim of this study was to analyze structural changes in the erythrocyte membrane of obese compared to normal-weight adolescents. The study was conducted in a group of 11 obese and 11 normal-weight adolescent subjects. The lipid profile, lipid peroxidation and acetylcholinesterase enzyme (AChE) activity were analyzed by conventional methods. The structural properties of reconstituted erythrocyte membrane were characterized by X-ray diffraction. Erythrocyte membrane from obese adolescents had a lipid profile characterized by a higher cholesterol/phospholipid ratio, an increase in saturated fatty acid and a decrease in monounsaturated and n-6 polyunsaturated fatty acid concentrations. Differences in lipid content were associated with changes in the structural properties of reconstituted membranes and the oxidative damage of erythrocyte membrane. The lower oxidative level shown in the obese group (0.15 ± 0.04 vs. 0.20 ± 0.06 nmol/mg for conjugated diene concentrations and 2.43 ± 0.25 vs. 2.83 ± 0.31 nmol/mg protein for malondialdehyde levels) was related to a lower unsaturation index. These changes in membrane structural properties were accompanied by a lower AChE activity (1.64 ± 0.13 vs. 1.91 ± 0.24 nmol AChE/[min mg protein]) in the obese group. The consequences of unhealthy dietary habits in adolescents are reflected in the membrane structural properties and may influence membrane-associated protein activities and functions.     

Pollution in mediterranean-climate rivers

This review examines information generated over the past decade on the pollution of rivers in regions with a mediterranean-type climate (med-climate). Pollution has clearly increased in the last 100 years and is correlated with the development of industry, agriculture and human population. Important efforts have been made in some med-climate countries in order to characterise the chemical status of rivers. In addition, the number of chemical substances detectable in mediterranean-climate rivers (med-rivers), as well as the limits of detection, have improved from the development of better analytical methods. New substances detected in rivers are gaining attention. We discuss available knowledge regarding real and potential effects of pollutants on the biota and ecosystems in med-rivers, taking into account natural environmental characteristics of these rivers. The extreme seasonal conditions in med-rivers add to the potential risk because these characteristics can enhance pollutant effects. Efforts and policies to prevent or reduce pollution effects on med-rivers are linked to the knowledge about pollution pressures associated with the degree of economic development. Aquatic communities in med-rivers are more sensitive to pollutants because they are exposed to strong natural and human stressors.     

Science Educational Outreach Programs That Benefit Students and Scientists

Both scientists and the public would benefit from improved communication of basic scientific research and from integrating scientists into education outreach, but opportunities to support these efforts are limited. We have developed two low-cost programs—"Present Your PhD Thesis to a 12-Year-Old" and "Shadow a Scientist”—that combine training in science communication with outreach to area middle schools. We assessed the outcomes of these programs and found a 2-fold benefit: scientists improve their communication skills by explaining basic science research to a general audience, and students'' enthusiasm for science and their scientific knowledge are increased. Here we present details about both programs, along with our assessment of them, and discuss the feasibility of exporting these programs to other universities.     

Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing

Background

Improved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways.

Methods

We sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed repetitive unit typing. Following high-depth paired-end sequencing using the Illumina HiSeq 2000 platform, two independent pipelines were employed for analysis, both employing read mapping onto reference genomes as well as de novo assembly, to control biases in variant detection. In addition to single-nucleotide polymorphisms, the analyses also sought to identify insertions, deletions and structural variants.

Results

Isolates were highly similar, with a distance of 13 variants between the most distant members of the cluster. The most sensitive analysis classified the 22 isolates into 18 groups. Four of the isolates did not appear to share a recent common ancestor with the largest clade; another four isolates had an uncertain ancestral relationship with the largest clade.

Conclusion

Whole genome sequencing, with analysis of single-nucleotide polymorphisms, insertions, deletions, structural variants and subpopulations, enabled the highest possible level of discrimination between cluster members, clarifying likely transmission pathways and exposing the complexity of strain origin. The analysis provides a basis for targeted public health intervention and enhanced classification of future isolates linked to the cluster.     

Pathogen profiling for disease management and surveillance

The usefulness of rapid pathogen genotyping is widely recognized, but its effective interpretation and application requires integration into clinical and public health decision-making. How can pathogen genotyping data best be translated to inform disease management and surveillance? Pathogen profiling integrates microbial genomics data into communicable disease control by consolidating phenotypic identity-based methods with DNA microarrays, proteomics, metabolomics and sequence-based typing. Sharing data on pathogen profiles should facilitate our understanding of transmission patterns and the dynamics of epidemics.     

Immunodominant tuberculosis CD8 antigens preferentially restricted by HLA-B

CD8(+) T cells are essential for host defense to intracellular bacterial pathogens such as Mycobacterium tuberculosis (Mtb), Salmonella species, and Listeria monocytogenes, yet the repertoire and dominance pattern of human CD8 antigens for these pathogens remains poorly characterized. Tuberculosis (TB), the disease caused by Mtb infection, remains one of the leading causes of infectious morbidity and mortality worldwide and is the most frequent opportunistic infection in individuals with HIV/AIDS. Therefore, we undertook this study to define immunodominant CD8 Mtb antigens. First, using IFN-gamma ELISPOT and synthetic peptide arrays as a source of antigen, we measured ex vivo frequencies of CD8(+) T cells recognizing known immunodominant CD4(+) T cell antigens in persons with latent tuberculosis infection. In addition, limiting dilution was used to generate panels of Mtb-specific T cell clones. Using the peptide arrays, we identified the antigenic specificity of the majority of T cell clones, defining several new epitopes. In all cases, peptide representing the minimal epitope bound to the major histocompatibility complex (MHC)-restricting allele with high affinity, and in all but one case the restricting allele was an HLA-B allele. Furthermore, individuals from whom the T cell clone was isolated harbored high ex vivo frequency CD8(+) T cell responses specific for the epitope, and in individuals tested, the epitope represented the single immunodominant response within the CD8 antigen. We conclude that Mtb-specific CD8(+) T cells are found in high frequency in infected individuals and are restricted predominantly by HLA-B alleles, and that synthetic peptide arrays can be used to define epitope specificities without prior bias as to MHC binding affinity. These findings provide an improved understanding of immunodominance in humans and may contribute to a development of an effective TB vaccine and improved immunodiagnostics.     

Prevalence of the C282Y, H63D, and S65C mutations of the HFE gene in 1,146 newborns from a region of Northern Spain

In Spain, 85% of patients with genetic hemochromatosis (GH) are homozygous for the C282Y mutation of the HFE gene. H63D and S65C mutations of HFE may also play some role in the disease. The aim of this study was to establish the prevalence of C282Y, H63D, and S65C mutations of the HFE gene in newborns in Catalonia, Spain. One thousand one hundred forty-six newborn screening cards were selected randomly. DNA from these cards was extracted and HFE mutations were analyzed with the LightCycler equipment (Roche Diagnostics Gmbh, Mannheim, Germany). Sufficient DNA sample was obtained to screen for the three mutations in 1,043 cases (91%). The allelic frequencies of C282Y, H63D, and S65C mutations were 0.03 (IC 95% 0.022-0.037), 0.2 (IC 95% 0.19-0.22), and 0.01 (95% confidence interval [CI] 0.006-0.015), respectively. The frequency of C282Y homozygous newborns was 0.001 (95% CI 0.0005-0.0014). The frequencies of newborns doubly heterozygous for C282Y/H63D and C282Y/S65C were 0.01 (95% CI 0.005-0.02) and 0.002 (95% CI 0.0002-0.01), respectively. The allelic frequency of C282Y mutation is similar to that observed in Southern France, in the Czech Republic and in some areas of Italy. The allelic frequency of H63D mutation in Catalonia is the highest reported to date. Nevertheless, S65C is infrequent. These data should be kept in mind when designing hemochromatosis genotypic screening programs in Catalonia.     

Preparation of a beef-extract as a laboratory reference material for the determination of heterocyclic amines

The present paper describes the preparation of a suitable laboratory reference material (LRM) to validate analytical methods for the determination of heterocyclic amines (HAs) in foods. Three different lots of reference material were prepared using a beef extract which was contaminated with a well-known quantity of amines at different levels ranging from 10 to 75 ng/g. These materials were then lyophilised under determined conditions and, after grinding and sieving, homogenised and, finally, bottled and labelled. Homogeneity and stability studies were performed and no statistical differences were observed in the analysis of variances for within- and between-bottle results, thus demonstrating the homogeneity of the material. Stability at different storage temperatures (-18, +4, +25 and +40 degrees C) and times (1, 2, 3 and 6 months) was also tested. Therefore, the material can be considered homogeneous and stable and can be proposed for use in inter-comparison exercises for the determination of HAs.     

Effects of oleic acid and its congeners,elaidic and stearic acids,on the structural properties of phosphatidylethanolamine membranes

Fatty acid derivatives are abundant in biological membranes, mainly as components of phospholipids and cholesterol esters. Their presence, free or bound to phospholipids, modulates the lipid membrane behavior. The present study shows the differential influence of the C-18 fatty acids (FAs), oleic, elaidic, and stearic acids on the structural properties of phosphatidylethanolamine (PE). X-ray diffraction of PE-FA systems demonstrated that oleic acid (OA) produced important concentration-dependent alterations of the lipid membrane structure: it induced reductions of up to 20-23 degrees C in the lamellar-to-hexagonal transition temperature of 1-palmitoyl-2-oleoyl PE and dielaidoyl PE and regulated the dimensions of the hexagonal lattice. In contrast, elaidic and stearic acids did not markedly alter the phospholipid mesomorphism. The above effects were attributed to the different "molecular shape" of OA (with a kink at the middle of the molecule) with respect to their congeners, elaidic and stearic acids. The effects of free fatty acids (FFAs) on membrane structure are relevant for several reasons: i) some biological membranes contain very high levels of FFAs. ii) Mediterranean diets with high OA intake have been shown to exert protective effects against tumoral and hypertensive pathologies. iii) FFA derivatives have been developed as antitumoral and antihypertensive drugs.     

High glucose augments the angiotensin II-induced activation of JAK2 in vascular smooth muscle cells via the polyol pathway

Angiotensin II (Ang II), protein kinase C (PKC), reactive oxygen species (ROS) generated by NADPH oxidase, the activation of Janus kinase 2 (JAK2), and the polyol pathway play important parts in the hyperproliferation of vascular smooth muscle cells (VSMC), a characteristic feature of diabetic macroangiopathy. The precise mechanism, however, remains unclear. This study investigated the relation between the polyol pathway, PKC-beta, ROS, JAK2, and Ang II in the development of diabetic macroangiopathy. VSMC cultured in high glucose (HG; 25 mm) showed significant increases in the tyrosine phosphorylation of JAK2, production of ROS, and proliferation activities when compared with VSMC cultured in normal glucose (5.5 mm (NG)). Both the aldose reductase specific inhibitor (zopolrestat) or transfection with aldose reductase antisense oligonucleotide blocked the phosphorylation of JAK2, the production of ROS, and proliferation of VSMC induced by HG, but it had no effect on the Ang II-induced activation of these parameters in both NG and HG. However, transfection with PKC-beta antisense oligonucleotide, preincubation with a PKC-beta-specific inhibitor (LY379196) or apocynin (NADPH oxidase-specific inhibitor), or electroporation of NADPH oxidase antibodies blocked the Ang II-induced JAK2 phosphorylation, production of ROS, and proliferation of VSMC in both NG and HG. These observations suggest that the polyol pathway hyperactivity induced by HG contributes to the development of diabetic macroangiopathy through a PKC-beta-ROS activation of JAK2.