Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation

Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 A resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close to the C terminus, and each epitope comprises residues from both Cgamma3 domains. A central role in the unusually hydrophilic epitope is played by the side-chain of Arg355, accounting for the subclass specificity of RF61, which recognizes IgG1,-2, and -3 in preference to IgG4, in which the corresponding residue is Gln355. Compared with a previously determined complex of a lower affinity RF (RF-AN) bound to IgG4 Fc, in which only residues at the very edge of the antibody combining site were involved in binding, the epitope bound by RF61 is centered in classic fashion on the axis of the V(H):V(L) beta-barrel. The complementarity determining region-H3 loop plays a key role, forming a pocket in which Arg355 is bound by two salt-bridges. The antibody contacts also involve two somatically mutated V(H) residues, reinforcing the suggestion of a process of antigen-driven maturation and selection for IgG Fc during the generation of this RF autoantibody.     

Intestinal helminthiases and schistosomiasis among school children in an urban center and some rural communities in southwest Nigeria

Intestinal helminths and schistosomiasis among school children were investigated in an urban and some rural communities of Ogun State, southwest Nigeria. Fecal samples of 1,059 subjects (524 males, 535 females) aged 3-18 years were examined using direct smear and brine concentration methods between June 2005 and November 2006. The pooled prevalence of infection was 66.2%. Ascaris lumbricoides showed the highest prevalence (53.4%) (P < 0.001) followed by hookworms (17.8%), Trichuris trichiura (10.4%), Taenia sp. (9.6%), Schistosoma mansoni (2.3%), Strongyloides stercoralis (0.7%), Schistosoma haematobium (0.6%), and Enterobius vermicularis (0.3%). The prevalences of A. lumbricoides, hookworms, Taenia sp., S. mansoni, and S. stercoralis in the urban centre were similar (P > 0.05) to those in the rural communities. The fertile and infertile egg ratios of A. lumbricoides in the urban centre and the rural communities were 13: 1 and 3.7: 1, respectively. Each helminth had similar prevalences among both genders (P > 0.05). The prevalence of A. lumbricoides increased significantly with age (P < 0.001). The commonest double infections were Ascaris and hookworms, while the commonest triple infections were Ascaris, hookworms, and Trichuris. The study demonstrates the need for urgent intervention programmes against intestinal helminthiases and schistosomiasis in the study area.     

Separation and quantification of two diastereomers of a Drug Candidate in rat plasma by ultra-high pressure liquid chromatography/mass spectrometry

Ultra-high pressure liquid chromatography (UHPLC) is a relatively new technology which utilizes chromatographic media with a 1.7 microm particle size. This technology has the potential to offer significant advantages in resolution, speed, and sensitivity for analytical determinations, particularly when coupled with mass spectrometric detection. Drug Candidate A, under development at Merck Research Laboratories, contains two chiral centers which have the absolute configuration R, S. Under in vivo and ex vivo conditions, one of the chiral centers readily epimerizes to produce the R, R diastereomer. Initially, a traditional high performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method was developed to separate and quantify these two diastereomers in rat plasma. The lower limit of quantification (LOQ) of the two analytes was 2 ng/mL, and a chromatographic run time of approximately 11 min was needed to separate R, S-(A) and R, R-(A). In this study, we explored a simple and robust UHPLC-MS/MS method in order to increase sample throughput and productivity. We were able to achieve a two-fold reduction in the lower limit of quantification and a three-fold reduction in retention time utilizing the UHPLC method, while keeping the same sample extraction procedure and similar MS/MS methodology. The new method exhibited good intra- and inter-day accuracy and precision, and was linear over a dynamic range of 1-500 ng/mL for each diastereomer. The method was successfully applied for the determination of R, S-(A) and R, R-(A) concentrations for in vitro and in vivo studies of epimerization of A in Sprague-Dawley rats.     

Alamethicin interaction with lipid membranes: a spectroscopic study on synthetic analogues

Alamethicin (Alm) is one of the most extensively studied membrane-active antibiotic peptides, but several aspects of its mechanism of action are still under debate. In this study, synthetic analogues of natural Alm F50/5 (Alm-N), labeled with a 9H-fluoren-9-yl group at the N- (F-Alm) or C-terminus (Alm-F), were employed to investigate the position and orientation of this peptide in the membrane environment. Depth-dependent fluorescence quenching and polarized ATR-FT-IR experiments demonstrated that, in the absence of a transmembrane potential, Alm inserts its N-terminus into the membrane, while the C-terminus is exposed to the outer aqueous phase. We also found that the peptaibol populates different orientations with respect to the membrane normal. Furthermore, fluorescence resonance-energy transfer (FRET) indicated that no peptide translocation to the inner leaflet of lipid bilayers occurs. The mechanism of action of Alm is discussed on the basis of these findings. Two other Alm analogues, Alm-P and Alm-S, were exploited to investigate the role of specific Alm residues in terms of membrane-perturbing activity. Substitution of two or three Gln (E) residues (the only polar amino acids in the alamethicin sequence) by gamma-methyl glutamate (Glu(OMe)) residues induced marked variations in the aggregation and partition behaviors of the peptaibols, which, in turn, modulate their membrane activity. In particular, substitution of Gln(18) and Gln(19) caused a six-fold increase in membrane-perturbing activity, thus demonstrating that these residues are not essential for the stabilization of Alm pores.     

Long‐term plant community trajectories suggest divergent responses of native and non‐native perennials and annuals to vegetation removal and seeding treatments

Land managers frequently apply vegetation removal and seeding treatments to restore ecosystem function following woody plant encroachment, invasive species spread, and wildfire. However, the long‐term outcome of these treatments is unclear due to a lack of widespread monitoring. We quantified how vegetation removal (via wildfire or management) with or without seeding and environmental conditions related to plant community composition change over time in 491 sites across the intermountain western United States. Most community metrics took over 10 years to reach baseline conditions posttreatment, with the slowest recovery observed for native perennial cover. Total cover was initially higher in sites with seeding after vegetation removal than sites with vegetation removal alone, but increased faster in sites with vegetation removal only. Seeding after vegetation removal was associated with rapidly increasing non‐native perennial cover and decreasing non‐native annual cover. Native perennial cover increased in vegetation removal sites irrespective of seeding and was suppressed by increasing non‐native perennial cover. Seeding was associated with higher non‐native richness across the monitoring period as well as initially higher, then declining, total and native species richness. Several cover and richness recovery metrics were positively associated with mean annual precipitation and negatively associated with mean annual temperature, whereas relationships with weather extremes depended on the lag time and season. Our results suggest that key plant groups, such as native perennials and non‐native annuals, respond to restoration treatments at divergent timescales and with different sensitivities to climate and weather variation.     

Obesity-Induced Cellular Senescence Drives Anxiety and Impairs Neurogenesis

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Relative extended haplotype homozygosity signals across breeds reveal dairy and beef specific signatures of selection

Background

A number of methods are available to scan a genome for selection signatures by evaluating patterns of diversity within and between breeds. Among these, “extended haplotype homozygosity” (EHH) is a reliable approach to detect genome regions under recent selective pressure. The objective of this study was to use this approach to identify regions that are under recent positive selection and shared by the most representative Italian dairy and beef cattle breeds.

Results

A total of 3220 animals from Italian Holstein (2179), Italian Brown (775), Simmental (493), Marchigiana (485) and Piedmontese (379) breeds were genotyped with the Illumina BovineSNP50 BeadChip v.1. After standard quality control procedures, genotypes were phased and core haplotypes were identified. The decay of linkage disequilibrium (LD) for each core haplotype was assessed by measuring the EHH. Since accurate estimates of local recombination rates were not available, relative EHH (rEHH) was calculated for each core haplotype. Genomic regions that carry frequent core haplotypes and with significant rEHH values were considered as candidates for recent positive selection. Candidate regions were aligned across to identify signals shared by dairy or beef cattle breeds. Overall, 82 and 87 common regions were detected among dairy and beef cattle breeds, respectively. Bioinformatic analysis identified 244 and 232 genes in these common genomic regions. Gene annotation and pathway analysis showed that these genes are involved in molecular functions that are biologically related to milk or meat production.

Conclusions

Our results suggest that a multi-breed approach can lead to the identification of genomic signatures in breeds of cattle that are selected for the same production goal and thus to the localisation of genomic regions of interest in dairy and beef production.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0113-9) contains supplementary material, which is available to authorized users.