New insights into the Enterococcus faecalis CroRS two-component system obtained using a differential-display random arbitrarily primed PCR approach

Using a modified random arbitrarily primed PCR approach, the operon encoding the Enterococcus faecalis JH2-2 CroRS two-component regulatory system was shown to be repressed during stationary phase, and a CroRS-regulated operon (glnQHMP) was identified. Gel retardation assays showed that the CroR regulator binds specifically to the glnQHMP promoter.     

cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites.     

The interaction between RPAP3 and TRBP reveals a possible involvement of the HSP90/R2TP chaperone complex in the regulation of miRNA activity

MicroRNAs silence mRNAs by guiding the RISC complex. RISC assembly occurs following cleavage of pre-miRNAs by Dicer, assisted by TRBP or PACT, and the transfer of miRNAs to AGO proteins. The R2TP complex is an HSP90 co-chaperone involved in the assembly of ribonucleoprotein particles. Here, we show that the R2TP component RPAP3 binds TRBP but not PACT. The RPAP3-TPR1 domain interacts with the TRBP-dsRBD3, and the 1.5 Å resolution crystal structure of this complex identifies key residues involved in the interaction. Remarkably, binding of TRBP to RPAP3 or Dicer is mutually exclusive. Additionally, we found that AGO(1/2), TRBP and Dicer are all sensitive to HSP90 inhibition, and that TRBP sensitivity is increased in the absence of RPAP3. Finally, RPAP3 seems to impede miRNA activity, raising the possibility that the R2TP chaperone might sequester TRBP to regulate the miRNA pathway.     

ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma

Insect stage trypanosomes use an "acetate shuttle" to transfer mitochondrial acetyl-CoA to the cytosol for the essential fatty acid biosynthesis. The mitochondrial acetate sources are acetate:succinate CoA-transferase (ASCT) and an unknown enzymatic activity. We have identified a gene encoding acetyl-CoA thioesterase (ACH) activity, which is shown to be the second acetate source. First, RNAi-mediated repression of ASCT in the ACH null background abolishes acetate production from glucose, as opposed to both single ASCT and ACH mutants. Second, incorporation of radiolabeled glucose into fatty acids is also abolished in this ACH/ASCT double mutant. ASCT is involved in ATP production, whereas ACH is not, because the ASCT null mutant is ～1000 times more sensitive to oligomycin, a specific inhibitor of the mitochondrial F(0)/F(1)-ATP synthase, than wild-type cells or the ACH null mutant. This was confirmed by RNAi repression of the F(0)/F(1)-ATP synthase F(1)β subunit, which is lethal when performed in the ASCT null background but not in the wild-type cells or the ACH null background. We concluded that acetate is produced from both ASCT and ACH; however, only ASCT is responsible, together with the F(0)/F(1)-ATP synthase, for ATP production in the mitochondrion.     

Antibody microarrays for label-free cell-based applications

The recent advances in microtechnologies have shown the interest of developing microarrays dedicated to cell analysis. In this way, miniaturized cell analyzing platforms use several detection techniques requiring specific solid supports for microarray read-out (colorimetric, fluorescent, electrochemical, acoustic, optical…). Real-time and label-free techniques, such as Surface Plasmon Resonance imaging (SPRi), arouse increasing interest for applications in miniaturized formats. Thus, we focused our study on chemical methods for antibody-based microarray fabrication dedicated to the SPRi analysis of cells or cellular activity. Three different approaches were designed and developed for specific applications. In the first case, a polypyrrole-based chemistry was used to array antibody-microarray for specific capture of whole living cells. In the second case, the polypyrrole-based chemistry was complexified in a three molecular level assembly using DNA and antibody conjugates to allow the specific release of cells after their capture. Finally, in the third case, a thiol-based chemistry was developed for long incubation times of biological samples of high complexity. This last approach was focused on the simultaneous study of both cell type characterization and secretory activity (detection of proteins secreted by cells). This paper describes three original methods allowing a rapid and efficient analysis of cellular sample on-chip using immunoaffinity-based assays.     

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).