TRAP assists membrane protein topogenesis at the mammalian ER membrane

Membrane protein insertion and topogenesis generally occur at the Sec61 translocon in the endoplasmic reticulum membrane. During this process, membrane spanning segments may adopt two distinct orientations with either their N- or C-terminus translocated into the ER lumen. While different topogenic determinants in membrane proteins, such as flanking charges, polypeptide folding, and hydrophobicity, have been identified, it is not well understood how the translocon and/or associated components decode them. Here we present evidence that the translocon-associated protein (TRAP) complex is involved in membrane protein topogenesis in vivo. Small interfering RNA (siRNA)-mediated silencing of the TRAP complex in HeLa cells enhanced the topology effect of mutating the flanking charges of a signal-anchor, but not of increasing signal hydrophobicity. The results suggest a role of the TRAP complex in moderating the ‘positive-inside’ rule.     

Notch-signalling is required for head regeneration and tentacle patterning in Hydra

Local self-activation and long ranging inhibition provide a mechanism for setting up organising regions as signalling centres for the development of structures in the surrounding tissue. The adult hydra hypostome functions as head organiser. After hydra head removal it is newly formed and complete heads can be regenerated. The molecular components of this organising region involve Wnt-signalling and β-catenin. However, it is not known how correct patterning of hypostome and tentacles are achieved in the hydra head and whether other signals in addition to HyWnt3 are needed for re-establishing the new organiser after head removal. Here we show that Notch-signalling is required for re-establishing the organiser during regeneration and that this is due to its role in restricting tentacle activation. Blocking Notch-signalling leads to the formation of irregular head structures characterised by excess tentacle tissue and aberrant expression of genes that mark the tentacle boundaries. This indicates a role for Notch-signalling in defining the tentacle pattern in the hydra head. Moreover, lateral inhibition by HvNotch and its target HyHes are required for head regeneration and without this the formation of the β-catenin/Wnt dependent head organiser is impaired. Work on prebilaterian model organisms has shown that the Wnt-pathway is important for setting up signalling centres for axial patterning in early multicellular animals. Our data suggest that the integration of Wnt-signalling with Notch-Delta activity was also involved in the evolution of defined body plans in animals.     

Genomic Pathology of SLE-Associated Copy-Number Variation at the FCGR2C/FCGR3B/FCGR2B Locus

Reduced FCGR3B copy number is associated with increased risk of systemic lupus erythematosus (SLE). The five FCGR2/FCGR3 genes are arranged across two highly paralogous genomic segments on chromosome 1q23. Previous studies have suggested mechanisms for structural rearrangements at the FCGR2/FCGR3 locus and have proposed mechanisms whereby altered FCGR3B copy number predisposes to autoimmunity, but the high degree of sequence similarity between paralogous segments has prevented precise definition of the molecular events and their functional consequences. To pursue the genomic pathology associated with FCGR3B copy-number variation, we integrated sequencing data from fosmid and bacterial artificial chromosome clones and sequence-captured DNA from FCGR3B-deleted genomes to establish a detailed map of allelic and paralogous sequence variation across the FCGR2/FCGR3 locus. This analysis identified two highly paralogous 24.5 kb blocks within the FCGR2C/FCGR3B/FCGR2B locus that are devoid of nonpolymorphic paralogous sequence variations and that define the limits of the genomic regions in which nonallelic homologous recombination leads to FCGR2C/FCGR3B copy-number variation. Further, the data showed evidence of swapping of haplotype blocks between these highly paralogous blocks that most likely arose from sequential ancestral recombination events across the region. Functionally, we found by flow cytometry, immunoblotting and cDNA sequencing that individuals with FCGR3B-deleted alleles show ectopic presence of FcγRIIb on natural killer (NK) cells. We conclude that FCGR3B deletion juxtaposes the 5′-regulatory sequences of FCGR2C with the coding sequence of FCGR2B, creating a chimeric gene that results in an ectopic accumulation of FcγRIIb on NK cells and provides an explanation for SLE risk associated with reduced FCGR3B gene copy number.     

N-Alkyldeoxynojirimycin derivatives with novel terminal tertiary amide substitution for treatment of bovine viral diarrhea virus (BVDV), Dengue,and Tacaribe virus infections

Novel N-alkyldeoxynojirimycins (NADNJs) with two hydrophobic groups attached to a nitrogen linker on the alkyl chain were designed. A novel NADNJ containing a terminal tertiary carboxamide moiety was discovered that was a potent inhibitor against BVDV. Further optimization resulted in a structurally more stable lead compound 24 with a submicromolar EC₅₀ against BVDV, Dengue, and Tacaribe; and low cytotoxicity.     

Neurocognitive Impairment in Patients Treated with Protease Inhibitor Monotherapy or Triple Drug Antiretroviral Therapy

Background

In patients who remain virologically suppressed in plasma with triple-drug ART a switch to protease inhibitor monotherapy maintains high rates of suppression; however it is unknown if protease inhibitor monotherapy is associated to a higher rate of neurocognitive impairment.

Methods

In this observational, cross-sectional study we included patients with plasma virological suppression (≥1 year) without concomitant major neurocognitive confounders, currently receiving for ≥1 year boosted lopinavir or darunavir as monotherapy or as triple ART. Neurocognitive impairment was defined as per the 2007 consensus of the American Association of Neurology. The association between neurocognitive impairment and protease inhibitor monotherapy, adjusted by significant confounders, was analysed.

Results

Of the 191 included patients - triple therapy: 96, 1–2 years of monotherapy: 40 and >2 years of monotherapy: 55 - proportions (95% CI) with neurocognitive impairment were: overall, 27.2% (20.9–33.6); triple therapy, 31.6% (22.1–41.0); short-term monotherapy, 25.0% (11.3–38.7); long-term monotherapy: 21.4% (10.5–32.3); p = 0.38. In all groups, neurocognitive impairment was mildly symptomatic or asymptomatic by self-report. There were not significant differences in Global Deficit Score by group. In the regression model confounding variables for neurocognitive impairment were years on ART, ethnicity, years of education, transmission category and the HOMA index. Adjusted by these variables the Odds Ratio (95% CI) for neurocognitive impairment of patients receiving short-term monotherapy was 0.85 (0.29–2.50) and for long-term monotherapy 0.40 (0.14–1.15).

Conclusions

Compared to triple drug antiretroviral therapy, monotherapy with lopinavir/ritonavir or darunavir/ritonavir in patients with adequate plasma suppression was not associated with a higher rate of asymptomatic neurocognitive impairment than triple drug ART.     

Increased Frequency of T Follicular Helper Cells and Elevated Interleukin-27 Plasma Levels in Patients with Pemphigus

Pemphigus is an autoimmune disease in which IgG auto-antibodies (auto-ab) against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 cause loss of epidermal keratinocyte adhesion. Aim of this study was to investigate cytokines derived from antigen-presenting cells (APC) and their relation to CD4⁺ T cell subpopulations and to the auto-ab response in pemphigus. In this regard, patients with pemphigus were compared to patients with myasthenia gravis (MG), an unrelated auto-ab–mediated autoimmune disease, and healthy controls. In pemphigus and MG, the plasma concentrations of the APC-derived immunomodulatory cytokine IL-27 were highly increased. Strikingly, IL-27 strongly correlated with Dsg-specific IgG auto-ab titers. T helper (Th) 17 cells were augmented in both pemphigus and MG patients while T follicular helper (Tfh) cells, which are essential in providing B cell help, were increased only in pemphigus along with increasing plasma concentrations of IL-21, a cytokine produced by Th17 and Tfh cells. Moreover, we could detect Dsg3-specific autoreactive T cells producing IL-21 upon ex vivo stimulation with Dsg3. These findings suggest that IL-27 and IL-21-producing T cells, are involved in the pathogenesis of pemphigus. The further characterization of IL-21-producing T cells and of the role of IL-27 will lead to a more defined understanding of the auto-ab response in pemphigus.     

Does AMH Reflect Follicle Number Similarly in Women with and without PCOS?

Context

Increased Anti-Mullerian Hormone in polycystic ovary syndrome, may be due to overactive follicles rather than reflect antral follicle count.

Objective

Does Anti-Mullerian Hormone reflect antral follicle count similarly in women with or without polycystic ovary syndrome or polycystic ovarian morphology?

Design

Cross-sectional, case-control.

Setting

Women who delivered preterm in 1999–2006. For each index woman, a woman with a term delivery was identified.

Patients

Participation rate was 69%. Between 2006–2008, 262 women were included, and diagnosed to have polycystic ovary syndrome, polycystic ovarian morphology or to be normal controls.

Intervention(s)

Blood tests, a clinical examination and vaginal ultrasound.

Main Outcome Measure(s)

Anti-Mullerian Hormone / antral follicle count -ratio, SHBG, androstenedione and insulin, to test potential influence on the Anti-Mullerian Hormone / antral follicle count -ratio.

Results

Mean Anti-Mullerian Hormone / antral follicle count ratio in women with polycystic ovary syndrome or polycystic ovarian morphology was similar to that of the controls (polycystic ovary syndrome: 1,2 p = 0,10 polycystic ovarian morphology: 1,2, p = 0,27 Controls 1,3). Anti-Mullerian Hormone showed a positive linear correlation to antral follicle count in all groups. Multivariate analysis did not change the results.

Conclusions

We confirmed the positive correlation between AMH and follicle count. Anti-Mullerian Hormone seems to be a reliable predictor of antral follicle count, independent of polycystic ovary syndrome diagnosis or ovarian morphology.