Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients

Fragile X‐associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55–200 CGG repeats in the 5′‐UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA‐binding proteins by the expanded CGG repeats. Sam68 is an RNA‐binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA‐binding proteins sequentially, first Sam68, then hnRNP‐G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing‐regulatory function. Consequently, Sam68‐responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain‐of‐function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.     

The Extracellular Domains of IgG1 and T Cell-Derived IL-4/IL-13 Are Critical for the Polyclonal Memory IgE Response In Vivo

IgE-mediated activation of mast cells and basophils contributes to protective immunity against helminths but also causes allergic responses. The development and persistence of IgE responses are poorly understood, which is in part due to the low number of IgE-producing cells. Here, we used next generation sequencing to uncover a striking overlap between the IgE and IgG1 repertoires in helminth-infected or OVA/alum-immunized wild-type BALB/c mice. The memory IgE response after secondary infection induced a strong increase of IgE⁺ plasma cells in spleen and lymph nodes. In contrast, germinal center B cells did not increase during secondary infection. Unexpectedly, the memory IgE response was lost in mice where the extracellular part of IgG1 had been replaced with IgE sequences. Adoptive transfer studies revealed that IgG1⁺ B cells were required and sufficient to constitute the memory IgE response in recipient mice. T cell-derived IL-4/IL-13 was required for the memory IgE response but not for expansion of B cells from memory mice. Together, our results reveal a close relationship between the IgE and IgG1 repertoires in vivo and demonstrate that the memory IgE response is mainly conserved at the level of memory IgG1⁺ B cells. Therefore, targeting the generation and survival of allergen-specific IgG1⁺ B cells could lead to development of new therapeutic strategies to treat chronic allergic disorders.     

Maternal Filaggrin Mutations Increase the Risk of Atopic Dermatitis in Children: An Effect Independent of Mutation Inheritance

Epidemiological studies suggest that allergy risk is preferentially transmitted through mothers. This can be due to genomic imprinting, where the phenotype effect of an allele depends on its parental origin, or due to maternal effects reflecting the maternal genome''s influence on the child during prenatal development. Loss-of-function mutations in the filaggrin gene (FLG) cause skin barrier deficiency and strongly predispose to atopic dermatitis (AD). We investigated the 4 most prevalent European FLG mutations (c.2282del4, p.R501X, p.R2447X, and p.S3247X) in two samples including 759 and 450 AD families. We used the multinomial and maximum-likelihood approach implemented in the PREMIM/EMIM tool to model parent-of-origin effects. Beyond the known role of FLG inheritance in AD (R1_{meta-analysis} = 2.4, P = 1.0 x 10⁻³⁶), we observed a strong maternal FLG genotype effect that was consistent in both independent family sets and for all 4 mutations analysed. Overall, children of FLG-carrier mothers had a 1.5-fold increased AD risk (S1 = 1.50, P_{meta-analysis} = 8.4 x 10⁻⁸). Our data point to two independent and additive effects of FLG mutations: i) carrying a mutation and ii) having a mutation carrier mother. The maternal genotype effect was independent of mutation inheritance and can be seen as a non-genetic transmission of a genetic effect. The FLG maternal effect was observed only when mothers had allergic sensitization (elevated allergen-specific IgE antibody plasma levels), suggesting that FLG mutation-induced systemic immune responses in the mother may influence AD risk in the child. Notably, the maternal effect reported here was stronger than most common genetic risk factors for AD recently identified through genome-wide association studies (GWAS). Our study highlights the power of family-based studies in the identification of new etiological mechanisms and reveals, for the first time, a direct influence of the maternal genotype on the offspring’s susceptibility to a common human disease.     

miRNA-197 and miRNA-223 Predict Cardiovascular Death in a Cohort of Patients with Symptomatic Coronary Artery Disease

Background

Circulating microRNAs (miRNAs) have been described as potential diagnostic biomarkers in cardiovascular disease and in particular, coronary artery disease (CAD). Few studies were undertaken to perform analyses with regard to risk stratification of future cardiovascular events. miR-126, miR-197 and miR-223 are involved in endovascular inflammation and platelet activation and have been described as biomarkers in the diagnosis of CAD. They were identified in a prospective study in relation to future myocardial infarction.

Objectives

The aim of our study was to further evaluate the prognostic value of these miRNAs in a large prospective cohort of patients with documented CAD.

Methods

Levels of miR-126, miR-197 and miR-223 were evaluated in serum samples of 873 CAD patients with respect to the endpoint cardiovascular death. miRNA quantification was performed using real time polymerase chain reaction (RT-qPCR).

Results

The median follow-up period was 4 years (IQR 2.78–5.04). The median age of all patients was 64 years (IQR 57–69) with 80.2% males. 38.9% of the patients presented with acute coronary syndrome (ACS), 61.1% were diagnosed with stable angina pectoris (SAP). Elevated levels of miRNA-197 and miRNA-223 reliably predicted future cardiovascular death in the overall group (miRNA-197: hazard ratio (HR) 1.77 per one standard deviation (SD) increase (95% confidence interval (CI) 1.20; 2.60), p = 0.004, C-index 0.78; miRNA-223: HR 2.23 per one SD increase (1.20; 4.14), p = 0.011, C-index 0.80). In ACS patients the prognostic power of both miRNAs was even higher (miRNA-197: HR 2.24 per one SD increase (1.25; 4.01), p = 0.006, C-index 0.89); miRA-223: HR 4.94 per one SD increase (1.42; 17.20), p = 0.012, C-index 0.89).

Conclusion

Serum-derived circulating miRNA-197 and miRNA-223 were identified as predictors for cardiovascular death in a large patient cohort with CAD. These results reinforce the assumption that circulating miRNAs are promising biomarkers with prognostic value with respect to future cardiovascular events.     

Biophysical and enzymatic properties of aminoglycoside adenylyltransferase AadA6 from Pseudomonas aeruginosa

The gene coding for the aminoglycoside adenylyltransferase (aadA6) from a clinical isolate of Pseudomonas aeruginosa was cloned and expressed in Escherichia coli strain BL21(DE3)pLysS. The overexpressed enzyme (AadA6, 281 amino-acid residues) and a carboxy-terminal truncated variant molecule ([1-264]AadA6) were purified to near homogeneity and characterized. Light scattering experiments conducted under low ionic strength supported equilibrium between monomeric and homodimeric arrangements of the enzyme subunits. Circular Dichroism spectropolarimetry indicated a close structural relation to adenylate kinases. Both forms modified covalently the aminoglycosides streptomycin and spectinomycin. The enzyme required at least 5 mM MgCl₂ for normal Michaelis–Menten kinetics. Streptomycin exhibited a strong substrate inhibition effect at 1 mM MgCl₂. The truncated 17 residues at the C-terminus have little influence on protein folding, whereas they have a positive effect on the enzymic activity and stabilize dimers at high protein concentrations (>100 μM). Homology modelling and docking based on known crystal structures yielded models of the central ternary complex of monomeric AadA6 with ATP and streptomycin or spectinomycin.     

Unikaryon phyllotretae sp. n. (Protista,Microspora), a new microsporidian pathogen of Phyllotreta undulata (Coleoptera; Chrysomelidae)

The microsporidium Unikaryon phyllotretae sp. n., a new pathogen of Phyllotreta undulata, is described based on light microscopic and ultrastructural characteristics. Microscopic examination of parasitized individuals revealed two types of spores. The majority of the spores were of the first type, which are oval and measured 2.74±0.17×1.93±0.17 μm when fresh. Fresh spores of the second type (very rare) are elongated and measured 4.39±0.18×1.61±0.20 μm. All life stages have single nuclei. Sporogony ends with uninucleate single sporoblasts and spores. The spores were only observed in Malpighian tubules. The isofilar polar filament of the parasite has six to eight coils, and a well-developed polaroplast was of the lamellated type, with closely packed anterior lamellae and loosely packed posterior lamellae.