Serine 970 of RNA helicase MOV10 is phosphorylated and controls unfolding activity and fate of mRNAs targeted for AGO2-mediated silencing.

MOV10 is an RNA helicase required for organismal development and is highly expressed in postnatal brain. MOV10 is an AGO2-associated protein that is also necessary for AGO2-mediated silencing. AGO2 is the primary effector of the miRNA pathway. MOV10 has been shown to be ubiquitinated, leading to its degradation and release from bound mRNAs, but no other posttranslational modifications with functional implications have been described. Using mass spectrometry, we show that MOV10 is phosphorylated in cells at the C-terminus, specifically at serine 970 (S970). Substitution of S970 to phospho-mimic aspartic acid (S970D) blocked unfolding of an RNA G-quadruplex, similar to when the helicase domain was mutated (K531A). In contrast, the alanine substitution (S970A) of MOV10 unfolded the model RNA G-quadruplex. To examine its role in cells, our RNA-seq analysis showed that the expression of S970D causes decreased expression of MOV10 enhanced Cross-Linking Immunoprecipitation targets compared to WT. Introduction of S970A had an intermediate effect, suggesting that S970 was protective of mRNAs. In whole-cell extracts, MOV10 and its substitutions bound AGO2 comparably; however, knockdown of AGO2 abrogated the S970D-induced mRNA degradation. Thus, MOV10 activity protects mRNA from AGO2; phosphorylation of S970 restricts this activity resulting in AGO2-mediated mRNA degradation. S970 is positioned C-terminal to the defined MOV10-AGO2 interaction site and is proximal to a disordered region that likely modulates AGO2 interaction with target mRNAs upon phosphorylation. In summary, we provide evidence whereby MOV10 phosphorylation facilitates AGO2 association with the 3'UTR of translating mRNAs that leads to their degradation.

Should axis deviation or atrial enlargement be categorised as abnormal in young athletes? The athlete's electrocardiogram: time for re-appraisal of markers of pathology.

AIMS: The 2010 European Society of Cardiology (ESC) guidelines for electrocardiogram (ECG) interpretation in athletes are associated with a relatively high false positive rate and warrant modification to improve the specificity without compromising sensitivity. The aim of this study was to investigate whether non-specific anomalies such as axis deviation and atrial enlargement in isolation require further assessment in highly trained young athletes. METHOD AND RESULTS: Between 2003 and 2011, 2533 athletes aged 14-35 years were investigated with 12-lead ECG and echocardiography. Electrocardiograms were analysed for non-training-related (Group 2) changes according to the 2010 ESC guidelines. Results were compared with 9997 asymptomatic controls. Of the 2533 athletes, 329 (13%) showed Group 2 ECG changes. Isolated axis deviation and isolated atrial enlargement comprised 42.6% of all Group 2 changes. Athletes revealed a slightly higher prevalence of these anomalies compared with controls (5.5 vs. 4.4%; P = 0.023). Echocardiographic evaluation of athletes and controls with isolated axis deviation or atrial enlargement (n = 579) failed to identify any major structural or functional abnormalities. Exclusion of axis deviation or atrial enlargement reduced the false positive rate from 13 to 7.5% and improved specificity from 90 to 94% with a minimal reduction in sensitivity (91-89.5%). CONCLUSION: Isolated axis deviation and atrial enlargement comprise a high burden of Group 2 changes in athletes and do not predict underlying structural cardiac disease. Exclusion of these anomalies from current ESC guidelines would improve specificity and cost-effectiveness of pre-participation screening with ECG.

Increased left ventricular trabeculation in highly trained athletes: do we need more stringent criteria for the diagnosis of left ventricular non-compaction in athletes?

OBJECTIVE: To investigate the prevalence and significance of increased left ventricular (LV) trabeculation in highly trained athletes. DESIGN: Cross sectional echocardiographic study. SETTING: Sports cardiology institutions in the UK and France. SUBJECTS: 1146 athletes aged 14-35 years (63.3% male), participating in 27 sporting disciplines, and 415 healthy controls of similar age. The results of athletes fulfilling conventional criteria for LV non-compaction (LVNC) were compared with 75 patients with LVNC. MAIN OUTCOME MEASURE: Number of athletes with increased LV trabeculation and the number fulfilling criteria for LVNC. RESULTS: Athletes displayed a higher prevalence of increased LV trabeculation compared with controls (18.3% vs 7.0%; p ≤ 0.0001) and 8.1% athletes fulfilled conventional criteria for LVNC. Increased LV trabeculation were more common in athletes of African/Afro-Caribbean origin. A small proportion of athletes (n = 10; 0.9%) revealed reduced systolic function and marked repolarisation changes in association with echocardiographic criteria for LVNC raising the possibility of an underlying cardiomyopathy. Follow-up during the ensuing 48.6 ± 14.6 months did not reveal adverse events. CONCLUSIONS: A high proportion of young athletes exhibit conventional criteria for LVNC highlighting the non-specific nature of current diagnostic criteria if applied to elite athletic populations. Further assessment of such athletes should be confined to the small minority that demonstrate low indices of systolic function and marked repolarisation changes.

Systematic random sampling of the comet assay.

The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory 'tail' DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the 'randomness' of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

Reactive aldehyde modification of thioredoxin-1 activates early steps of inflammation and cell adhesion.

Several lines of evidence suggest that an increase in aldehyde-modified proteins is associated with development of atherosclerosis. Acrolein and 4-hydroxynonenal (HNE) are reactive aldehydes generated during active inflammation as a consequence of lipid peroxidation; both react with protein thiols, including thioredoxin-1 (Trx1), a protein recently found to regulate antioxidant function in endothelial cells. The present study examined whether acrolein or HNE modification of Trx1 could potentiate monocyte adhesion to endothelial cells, an early event of atherosclerosis. We examined the function of acrolein and HNE-modified Trx1 in the regulation of the early events of atherosclerosis using cultured aortic endothelial cells as a vascular model system, for in vitro enzymatic assay, and in mass spectrometry analysis. Our data show that acrolein and HNE at 1:1 ratios with Trx1 modified Cys-73 and inhibited activity. In endothelial cells, adducts were detected at concentrations as low as 1 mumol/L including conditions in which there was no detectable change in glutathione. Acrolein and HNE modification of Trx1 was associated with increased production of reactive oxygen species. Microinjection of acrolein- and HNE-modified Trx1 into endothelial cells stimulated monocyte adhesion. Chemical modification of Trx1 by common environmental and endogenously generated reactive aldehydes can contribute to atherosclerosis development by interfering with antioxidant and redox signaling functions of Trx1.

Phosphoregulation of mixed-lineage kinase 1 activity by multiple phosphorylation in the activation loop.

Mixed-lineage kinase 1 (MLK1) is a mitogen-activated protein kinase kinase kinase capable of activating the c-Jun NH(2)-terminal kinase (JNK) pathway. Full-length MLK1 has 1104 amino acids and a domain structure identical to MLK2 and MLK3. Immunoblot and mass spectrometry show that MLK1 is threonine (and possibly serine) phosphorylated in or near the activation loop. A kinase-dead mutant is not, consistent with autophosphorylation. Mutation to alanine of any of the four serine or threonine residues in the activation loop reduces both the activity of the recombinant kinase domain and JNK pathway activation driven by full-length MLK1 expressed in mammalian cells. Furthermore, the gel mobility of the mutant MLK1s is closer to that of the kinase-dead than wild type, consistent with reduced phosphorylation. Thr312 is the key residue: MLK1[T312A] retains only basal activity (about 1-2% of wild type), and its gel mobility is indistinguishable from kinase-dead. Thr312 does not suffice, however; phosphorylation of multiple sites is necessary for full activation of MLK1. An activation mechanism consistent with these data involves phosphorylation of multiple sites in the activation loop, with phosphorylation of Thr312 required for full phosphorylation. This mechanism is broadly similar to that previously reported for MLK3 [Leung, I. W., and Lassam, N. (2001) J. Biol. Chem. 276, 1961-1967], but the key residue differs.

"La manie d'ecrire": psychology, auto-observation, and case history.

This essay examines the modern psychiatric case study as a scientific method and as a genre of writing about the self. As psychological discourse became a privileged language of selfhood at the fin de siècle, the penultimate signifier of the self came to be found in the realm of sexuality, in the particular contours of an individual's sexual desire. The investigative tool used to uncover these secrets of identity was the case history. This article concerns an influential project of sexual research based on "auto-observations"-autobiographical patient narratives-conducted by Dr. Georges Saint-Paul, who published under the inverted pseudonym "Laupts." The article focuses on the central autobiography of his collection, the "roman d'un inverti," and related sexological literature to suggest how this emphasis on patient stories in psychiatric writing engendered new narrative possibilities for doctors and patients alike. Putting patient stories at the center of psychiatric investigation created a new relationship between patient and doctor, observation and diagnosis, subject and discourse. The tools of psychological observation simultaneously placed a subject's confession more firmly at the center of the investigation and made it more open to interpretation by nonspecialists. Rather than simply confirming the authority of the trained observer, the story of Laupts's enquête suggests that the method actually authorized inquiry by others.

Phosphorylation influences the translation state of FMRP-associated polyribosomes.

Fragile X mental retardation protein, FMRP, is absent in patients with fragile X syndrome, a common form of mental retardation. FMRP is a nucleocytoplasmic RNA binding protein that is primarily associated with polyribosomes. FMRP is believed to be a translational repressor and may regulate the translation of certain mRNAs at the base of dendritic spines in neurons. However, little is known about the regulation of FMRP. Using mass spectrometry and site-directed mutagenesis, we show that FMRP is phosphorylated between residues 483 and 521, N-terminal to the RGG box, both in murine brain and in cultured cells. Primary phosphorylation occurs on the highly conserved serine 499, which triggers hierarchical phosphorylation of nearby serines. FMRP is phosphorylated within 2-4 h of synthesis, however, phosphorylation has no effect on the half-life of the protein. In contrast to the Drosophila ortholog dFxr, the phosphorylation status of mammalian FMRP does not influence its association with specific mRNAs in vivo. However, we find unphosphorylated FMRP associated with actively translating polyribosomes while a fraction of phosphorylated FMRP is associated with apparently stalled polyribosomes. Our data suggest that the phosphorylation may regulate FMRP and that the release of FMRP-induced translational suppression may involve a dephosphorylation signal.