Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination

Some human cancers maintain their telomeres using the alternative lengthening of telomeres (ALT) mechanism; a process thought to involve recombination. Different types of recombinational telomere elongation pathways have been identified in yeasts. In senescing yeast telomerase deletion (ter1-Δ) mutants with very short telomeres, it has been hypothesized that copying a tiny telomeric circle (t-circle) by a rolling circle mechanism is the key event in telomere elongation. In other cases more closely resembling ALT cells, such as the stn1-M1 mutant of Kluyveromyces lactis, the telomeres appear to be continuously unstable and routinely reach very large sizes. By employing two-dimensional gel electrophoresis and electron microscopy, we show that stn1-M1 cells contain abundant double stranded t-circles ranging from ∼100 to 30 000 bp in size. We also observed small single-stranded t-circles, specifically composed of the G-rich telomeric strand and tailed circles resembling rolling circle replication intermediates. The t-circles most likely arose from recombination events that also resulted in telomere truncations. The findings strengthen the possibility that t-circles contribute to telomere maintenance in stn1-M1 and ALT cells.     

Superoxidedismutase-mimetic copper(II) complexes containing saccharinate and 4-aminopyridine/4-cyanopyridine

Two copper(II) complexes, [Cu(sac)₂(4-cypy)₂(H₂O)], 1 and [Cu(sac)₂(4-Ampy)₂(H₂O)], 2 (4-cypy: 4-cyanopyridine; 4-Ampy: 4-aminopyridine) were prepared. Physicochemical properties of the complexes were studied by spectroscopic (solution UV–vis, diffuse reflectance and IR) techniques. Structural X-ray diffraction data could be obtained only for [Cu(sac)₂(4-cypy)₂(H₂O)] that it crystallized in the tetragonal space group P4cc with a=b=15.313(1), c=13.240(1) Å, and Z=4 molecules per unit cell. The complex was cited on a crystallographic C₂-axis with the Cu(II) ion in a square–pyramidal environment, coordinated at the pyramid basis to the nitrogen atom of two saccharine anions [d(Cu–N)=2.011(3) Å] and the pyridine N-atom of two 4-cyanopyridine ligands [d(Cu–N)=2.038(4) Å]. The coordination was completed by a water molecule at the pyramid apex [d(Cu–Ow)=2.189(5) Å]. Elemental and spectroscopic analyses revealed an O-saccharinate coordination mode for complex 2 and a square–pyramidal structure. Only complex 2 retained its structure in methanolic solution. However, both complexes were able to catalyze the dismutation of superoxide anion (O₂^?) (pH 7.5) at micromolar concentrations. Therefore, these complexes behaved as useful SOD-mimetic compounds.     

High Antibody Responses against Plasmodium falciparum in Immigrants after Extended Periods of Interrupted Exposure to Malaria

Background

Malaria immunity is commonly believed to wane in the absence of Plasmodium falciparum exposure, based on limited epidemiological data and short-lived antibody responses in some longitudinal studies in endemic areas.

Methods

A cross-sectional study was conducted among sub-Saharan African adults residing in Spain for 1 up to 38 years (immigrants) with clinical malaria (n=55) or without malaria (n=37), naïve adults (travelers) with a first clinical malaria episode (n=20) and life-long malaria exposed adults from Mozambique (semi-immune adults) without malaria (n=27) or with clinical malaria (n=50). Blood samples were collected and IgG levels against the erythrocytic antigens AMA-1 and MSP-1₄₂ (3D7 and FVO strains), EBA-175 and DBL-α were determined by Luminex. IgG levels against antigens on the surface of infected erythrocytes (IEs) were measured by flow cytometry.

Results

Immigrants without malaria had lower IgG levels than healthy semi-immune adults regardless of the antigen tested (P≤0.026), but no correlation was found between IgG levels and time since migration. Upon reinfection, immigrants with malaria had higher levels of IgG against all antigens than immigrants without malaria. However, the magnitude of the response compared to semi-immune adults with malaria depended on the antigen tested. Thus, immigrants had higher IgG levels against AMA-1 and MSP-1₄₂ (P≤0.015), similar levels against EBA-175 and DBL-α, and lower levels against IEs (P≤0.016). Immigrants had higher IgG levels against all antigens tested compared to travelers (P≤0.001), both with malaria.

Conclusions

Upon cessation of malaria exposure, IgG responses to malaria-specific antigens were maintained to a large extent, although the conservation and the magnitude of the recall response depended on the nature of the antigen. Studies on immigrant populations can shed light on the factors that determine the duration of malaria specific antibody responses and its effect on protection, with important implications for future vaccine design and public health control measures.     

Agreement among Healthcare Professionals in Ten European Countries in Diagnosing Case-Vignettes of Surgical-Site Infections

Objective

Although surgical-site infection (SSI) rates are advocated as a major evaluation criterion, the reproducibility of SSI diagnosis is unknown. We assessed agreement in diagnosing SSI among specialists involved in SSI surveillance in Europe.

Methods

Twelve case-vignettes based on suspected SSI were submitted to 100 infection-control physicians (ICPs) and 86 surgeons in 10 European countries. Each participant scored eight randomly-assigned case-vignettes on a secure online relational database. The intra-class correlation coefficient (ICC) was used to assess agreement for SSI diagnosis on a 7-point Likert scale and the kappa coefficient to assess agreement for SSI depth on a three-point scale.

Results

Intra-specialty agreement for SSI diagnosis ranged across countries and specialties from 0.00 (95%CI, 0.00–0.35) to 0.65 (0.45–0.82). Inter-specialty agreement varied from 0.04 (0.00–0.62) in to 0.55 (0.37–0.74) in Germany. For all countries pooled, intra-specialty agreement was poor for surgeons (0.24, 0.14–0.42) and good for ICPs (0.41, 0.28–0.61). Reading SSI definitions improved agreement among ICPs (0.57) but not surgeons (0.09). Intra-specialty agreement for SSI depth ranged across countries and specialties from 0.05 (0.00–0.10) to 0.50 (0.45–0.55) and was not improved by reading SSI definition.

Conclusion

Among ICPs and surgeons evaluating case-vignettes of suspected SSI, considerable disagreement occurred regarding the diagnosis, with variations across specialties and countries.     

Construction of a fully active Cys-less elongation factor Tu: functional role of conserved cysteine 81

In order to study the structural and functional requirements of the essential translational GTPase elongation factor (EF) Tu for efficient and accurate ribosome-dependent protein synthesis, construction of a cysteine-free (Cys-less) mutant variant allowing for the site-directed introduction of fluorescent and non-fluorescent labels is of great importance. However, previous reports suggest that a cysteine residue in position 81 of EF-Tu from Escherichia coli is essential for its function. To study the functional role of cysteine 81 and to construct a fully active Cys-less EF-Tu, we have analyzed 125 bacterial sequences with respect to sequence variations in this position revealing that in a small number of sequences alanine and methionine can be found. Here we report the detailed comparative biochemical analysis of three Cys-less variants of EF-Tu containing these substitutions as well as the isosteric amino acid serine. By characterizing nucleotide binding, EF-Ts interaction, aminoacyl-tRNA binding, and delivery to the ribosome, we demonstrate that only alanine (or cysteine) can be tolerated in this position and that the serine and methionine substitutions significantly impair aminoacyl-tRNA, but not nucleotide binding. Our findings suggest a critical functional role of the amino acid residue in position 81 of EF-Tu with respect to aminoacyl-tRNA binding. Based on structural considerations, we suggest that position 81 indirectly contributes to aminoacyl-tRNA binding through the accurate positioning of helix B.     

Structural and functional peculiarities of the lipopolysaccharide of Azospirillum brasilense SR55, isolated from the roots of Triticum durum

Azospirillum brasilense SR55, isolated from the rhizosphere of Triticum durum, was classified as serogroup II on the basis of serological tests. Such serogroup affiliation is uncharacteristic of wheat-associated Azospirillum species. The lipid A of A. brasilense SR55 lipopolysaccharide contained 3-hydroxytetradecanoic, 3-hydroxyhexadecanoic, hexadecanoic and octadecenoic fatty acids. The structure of the lipopolysaccharide's O polysaccharide was established, with the branched octasaccharide repeating unit being represented by l-rhamnose, l-3-O-Me-rhamnose, d-galactose and d-glucuronic acid. The SR55 lipopolysaccharide induced deformations of wheat root hairs. The lipopolysaccharide was not involved in bacterial cell aggregation, but its use to pretreat wheat roots was conducive to cell adsorption. This study shows that Azospirillum bacteria can utilise their own lipopolysaccharide as a carbon source, which may give them an advantage in competitive natural environments.