High-performance liquid chromatographic analysis of ubiquinones from new Legionella species

Abstract The ubiquinone composition of Legionella feeleii, L. oakridgensis, L. rubrilucens, L. sainthelensi, L. wadsworthii and some environmental legionella isolates was investigated by high performance liquid chromatography. The results of the present study indicate ubiquinones are valuable criteria for the rapid identification of Legionella species and the recognition of new taxa.     

Effect of acetabular labral tears,repair and resection on hip cartilage strain: A 7 T MR study

BackgroundTears of the acetabular labrum are frequently present in patients with groin pain. While it is clear that the labrum contributes to the surface area articulating with the femoral head, it is not clear whether labral repair yields different load distribution in the hip compared to labral resection.PurposeDetermine whether labral repair reduces cartilage strain more effectively than labral resection.MethodsSix human cadaveric hips (mean age 37 years) were loaded in a simulated single-leg stance within the bore of a 7 T MR scanner. After cartilage had reached a steady-state thickness distribution, a scan of the cartilage was acquired with a voxel size of 0.1×0.1×0.3 mm. This method was repeated for each of six specimens when the labrum was intact, after a surgically simulated labral tear, after an arthroscopic labral repair and after labral resection. Cartilage thickness and strain in an anterosuperior region of interest were measured from the MR scans. A paired t-test was used to compare mean and maximum cartilage strain when the labrum was intact vs. torn, torn vs. repaired and repaired vs. resected. Three-dimensional patterns of cartilage strain distribution were qualitatively compared for the different labral conditions.ResultsFor the number of specimens tested we found no change in mean and maximum cartilage strain, and little obvious change in the pattern of cartilage strain distribution after a simulated labral tear. Labral repair caused a 2% decrease in mean cartilage strain compared to a torn labrum (p=0.014). Labral resection caused a 4% and 6% increase in mean and maximum cartilage strain, respectively, compared to labral repair (p=0.02), and the cartilage strain distribution was elevated throughout the region of interest.ConclusionBased on our ex vivo findings of increased cartilage strain after labral resection when compared to labral repair, we have demonstrated the associated consequences to the mechanical environment of the cartilage following surgical treatment of the labrum.     

Persistent Difficulties in Switching to Second-Line ART in Sub-Saharan Africa — A Systematic Review and Meta-Analysis

Objectives

Switching to second-line antiretroviral therapy (ART) largely depends on careful clinical assessment and access to biological measurements. We performed a systematic review and meta-analysis to estimate the incidence of switching to second-line ART in sub-Saharan Africa and its main programmatic determinants.

Methods

We searched 2 databases for studies reporting the incidence rate of switching to second-line ART in adults living in sub-Saharan Africa. Data on the incidence rate of switching were pooled, and random-effect models were used to evaluate the effect of factors measured at the programme level on this incidence rate.

Results

Nine studies (157,340 patients) in 21 countries were included in the meta-analysis. All studies considered patients under first-line ART and conditions to initiate ART were similar across studies. Overall, 3,736 (2.4%) patients switched to second-line ART. Incidence rate of switch was in mean 2.65 per 100 person-years (PY) (95% confidence interval: 2.01–3.30); it ranged from 0.42 to 4.88 per 100 PY and from 0 to 4.80 per 100 PY in programmes with and without viral load monitoring, respectively. No factors measured at the programme level were associated with the incidence rate of switching to second-line ART.

Conclusion

The low incidence rate of switching to second-line ART suggests that the monitoring of patients under ART is challenging and that access to second-line ART is ineffective; efforts should be made to increase access to second-line ART to those in need by providing monitoring tools, education and training, as well as a more convenient regimen.     

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding.Immunoglobulins are key players of the human immune system. Immunoglobulin G (IgG)¹ is the most abundant representative of this group, with serum concentrations of ∼10 mg/ml (1). It consists of two heavy chains (γ-chains) made up of three constant regions (C_H1, C_H2, and C_H3) and one variable region (V_H). Attached to each heavy chain is a light chain (λ or κ). Based on chemical and biological properties, different regions can be distinguished in the IgG molecule: two antigen binding fragments (obtained as F(ab′)₂ by IdeS treatment; herein referred to as Fab) and a crystallizable fragment (Fc). The structure of IgG is schematically presented in Fig. 1.Open in a separate windowFig. 1.Schematic representation of IgG with the heavy γ chains (dark blue), light chains (lighter blue), and N-glycans. In the top right-hand corner of the Fc and Fab areas, the percentages of galactosylation, sialylation, bisection, and fucosylation are depicted. The inset represents the stable heptasaccharide core with possible extensions.IgGs are glycoproteins, and N-glycans are present at Asn297 of the C_H2 domain. These glycans consist of a constant heptasaccharide core that is often modified by a core fucose and is in part decorated with bisecting N-acetylglucosamine (GlcNAc), galactose(s), and sialic acid(s) (Fig. 1) (1). The Fc glycans have been extensively studied, and glycosylation changes have been found to be associated with disease (e.g. rheumatoid arthritis) (2, 3) and aging (4–6). Several immune regulatory properties have been demonstrated for IgG Fc glycans (7–13). For example, Fc-linked glycans influence the IgG effector function by altering the three-dimensional structure of the protein, and thereby the binding to Fcγ-receptors (12, 13). Additionally, glycan–glycan interactions occur between IgG and Fcγ-receptor-IIIa (8), with the presence of a core fucose decreasing this affinity by ∼2 orders of magnitude (7).The Fab portion consists of the heavy chain C_H1 and V_H regions combined with a light chain and exhibits the antigen binding sites formed by the variable and hypervariable regions of those two chains. N-glycans are known to occur on 15% to 25% of the IgG Fab portions (1, 14, 15). The Fab N-glycans can be involved in immunomodulation, because they influence the affinity and avidity of antibodies for antigens (16–19), as well as antibody half-life (17, 20). The glycans of the Fab have been described as biantennary complex-type structures that are, in contrast to Fc glycans, highly sialylated (21–23). Additionally, high-mannose-type structures have been said to be located on the Fab portion (23).Pregnancy is known to be associated with overall changes in IgG glycosylation. Indeed, a marked increase of galactosylation and sialylation has been observed in IgG Fc glycosylation during pregnancy (3, 24, 25). In addition, lectin binding studies suggest changes in Fab glycosylation of IgG during pregnancy (26), which may be caused by increased levels of progesterone (27). Changes in glycosylation during pregnancy could be one of the mechanisms that contribute to acceptance of the fetal allograft by the maternal immune system (26).Our knowledge on the Fab glycosylation of IgGs from peripheral blood is scarce, which is in part due to difficulty detecting the glycans in a Fab-region-specific manner. Because of the polyclonal nature of serum IgG, one may expect Fab glycans to be attached to a large variety of sequence motifs arising from somatic rearrangements and mutations (28), making the analysis of Fab glycopeptides from polyclonal serum IgG very demanding, if feasible at all. Therefore, study of the Fab glycosylation of polyclonal serum IgG has mainly been pursued at the level of released glycans (14, 23). Difficulties lie in the purification of IgG and the separation of Fc and Fab glycosylation, which is essential for the assignment of the glycans to either part of the IgG molecule.Here we present a high-throughput method for studying Fab glycosylation at the level of released glycans obtained from serum-derived polyclonal IgG. Using state-of-the-art affinity capturing beads and enzymes, we were able to obtain Fab and Fc separately, which, after glycan release, resulted in Fc- and Fab-specific glycan pools. The released glycans were subjected to a novel derivatization protocol resulting in linkage-specific modification of sialic acids, followed by HILIC sample purification and MALDI-TOF-MS. Finally, because marked changes in glycosylation during pregnancy have been described, the technique was applied to consecutive serum samples from a cohort of pregnant women. This approach was chosen to determine the usefulness of this technique in a clinical setting. The method proved to be able to demonstrate pregnancy-related changes in glycosylation of the Fab portion, in addition to the already known changes in Fc glycosylation (3, 24, 25).     

Innate Immune Responses and Rapid Control of Inflammation in African Green Monkeys Treated or Not with Interferon-Alpha during Primary SIVagm Infection

Chronic immune activation (IA) is considered as the driving force of CD4⁺ T cell depletion and AIDS. Fundamental clues in the mechanisms that regulate IA could lie in natural hosts of SIV, such as African green monkeys (AGMs). Here we investigated the role of innate immune cells and IFN-α in the control of IA in AGMs. AGMs displayed significant NK cell activation upon SIVagm infection, which was correlated with the levels of IFN-α. Moreover, we detected cytotoxic NK cells in lymph nodes during the early acute phase of SIVagm infection. Both plasmacytoid and myeloid dendritic cell (pDC and mDC) homing receptors were increased, but the maturation of mDCs, in particular of CD16⁺ mDCs, was more important than that of pDCs. Monitoring of 15 cytokines showed that those, which are known to be increased early in HIV-1/SIVmac pathogenic infections, such as IL-15, IFN-α, MCP-1 and CXCL10/IP-10, were significantly increased in AGMs as well. In contrast, cytokines generally induced in the later stage of acute pathogenic infection, such as IL-6, IL-18 and TNF-α, were less or not increased, suggesting an early control of IA. We then treated AGMs daily with high doses of IFN-α from day 9 to 24 post-infection. No impact was observed on the activation or maturation profiles of mDCs, pDCs and NK cells. There was also no major difference in T cell activation or interferon-stimulated gene (ISG) expression profiles and no sign of disease progression. Thus, even after administration of high levels of IFN-α during acute infection, AGMs were still able to control IA, showing that IA control is independent of IFN-α levels. This suggests that the sustained ISG expression and IA in HIV/SIVmac infections involves non-IFN-α products.     

“NiCo Buster”: engineering <Emphasis Type="Italic">E. coli</Emphasis> for fast and efficient capture of cobalt and nickel

Background

Metal contamination is widespread and results from natural geogenic and constantly increasing anthropogenic sources (mainly mining and extraction activities, electroplating, battery and steel manufacturing or metal finishing). Consequently, there is a growing need for methods to detoxify polluted ecosystems. Industrial wastewater, surface water and ground water need to be decontaminated to alleviate the contamination of soils and sediments and, ultimately, the human food chain. In nuclear power plants, radioactive metals are produced; these metals need to be removed from effluents before they are released into the environment, not only for pollution prevention but also for waste minimization. Many physicochemical methods have been developed for metal removal from aqueous solutions, including chemical coagulation, adsorption, extraction, ion exchange and membrane separation; however, these methods are generally not metal selective. Bacteria, because they contain metal transporters, provide a potentially competitive alternative to the current use of expensive and high-volume ion-exchange resins.

Results

The feasibility of using bacterial biofilters as efficient tools for nickel and cobalt ions specific remediation was investigated. Among the factors susceptible to genetic modification in Escherichia coli, specific efflux and sequestration systems were engineered to improve its metal sequestration abilities. Genomic suppression of the RcnA nickel (Ni) and cobalt (Co) efflux system was combined with the plasmid-controlled expression of a genetically improved version of a specific metallic transporter, NiCoT, which originates from Novosphingobium aromaticivorans. The resulting strain exhibited enhanced nickel (II) and cobalt (II) uptake, with a maximum metal ion accumulation of 6 mg/g bacterial dry weight during 10 min of treatment. A synthetic adherence operon was successfully introduced into the plasmid carrying the improved NiCoT transporter, conferring the ability to form thick biofilm structures, especially when exposed to nickel and cobalt metallic compounds.

Conclusions

This study demonstrates the efficient use of genetic engineering to increase metal sequestration and biofilm formation by E. coli. This method allows Co and Ni contaminants to be sequestered while spatially confining the bacteria to an abiotic support. Biofiltration of nickel (II) and cobalt (II) by immobilized cells is therefore a promising option for treating these contaminants at an industrial scale.

     

Last Generation Triazoles for Imported Eumycetoma in Eleven Consecutive Adults

Background

Optimal management of eumycetoma, a severely debilitating chronic progressive fungal infection of skin, disseminating to bone and viscera, remains challenging. Especially, optimal antifungal treatment and duration are ill defined.

Methodology/Principal Findings

We conducted a monocentric retrospective study of 11 imported cases of eumycetoma treated by voriconazole or posaconazole for at least 6 months. Response to treatment was assessed through evolution of clinical and magnetic resonance imaging (MRI). (1→3) ß-D-glucan (BG) and positron emission tomography using [18F] fluorodeoxyglucose (PET/CT) results were also assessed. Identified species were Fusarium solani complex (n = 3); Madurella mycetomatis, (n = 3), and Exophiala jeanselmei, (n = 1). Moreover, two coelomycetes and one phaeohyphomycetes strains without species identification were retrieved. Serum BG and PET/CT were abnormal in 7/8 and 6/6 patients tested, respectively. Patients received last generation azoles for a mean duration of 25.9±18 months. Complete response (major clinical and MRI improvement) was observed in 5/11 patients, partial response (minor MRI improvement or stable MRI findings) in 5 and failure (MRI evidence of disease progression) in one, with a 73±39 [6–132] months mean follow-up. Relapse occurred in 2 patients after treatment discontinuation. Optimal outcome was associated with fungal species, initiation of last generation triazole therapy (<65 months since first symptoms), negative serum BG and PET/CT normalization.

Conclusions/Significance

MRI, PET/CT and serum BG appear as promising tools to assess optimal time of antifungal treatment for eumycetoma.