When is an obscurin variant pathogenic? The impact of Arg4344Gln and Arg4444Trp variants on protein-protein interactions and protein stability.

Obscurin is a giant muscle protein that connects the sarcomere with the sarcoplasmic reticulum, and has poorly understood structural and signalling functions. Increasingly, obscurin variants are implicated in the pathophysiology of cardiovascular diseases. The Arg4344Gln variant (R4344Q) in obscurin domain Ig58, initially discovered in a patient with hypertrophic cardiomyopathy, has been reported to reduce binding to titin domains Z8-Z9, impairing obscurin's Z-disc localization. An R4344Q knock-in mouse developed a cardiomyopathy-like phenotype with abnormal Ca2+-handling and arrhythmias, which were attributed to an enhanced affinity of a putative interaction between obscurin Ig58 and phospholamban (PLN) due to the R4344Q variant. However, the R4344Q variant is found in 15% of African Americans, arguing against its pathogenicity. To resolve this apparent paradox, we quantified the influence of the R4344Q variant (alongside another potentially pathogenic variant: Arg4444Trp (R4444W)) on binding to titin Z8-Z9, novex-3 and PLN using pull-down assays and microscale thermophoresis and characterized the influence on domain stability using differential scanning fluorimetry. We found no changes in titin binding and thermostability for both variants and modestly increased affinities of PLN for R4344Q and R4444W. While we could not confirm the novex-3/obscurin interaction, the PLN/obscurin interaction relies on the transmembrane region of PLN and is not reproducible in mammalian cells, suggesting it is an in vitro artefact. Without clear clinical evidence for disease involvement, we advise against classifying these obscurin variants as pathogenic.

Ion-Pairing Chromatography and Amine Derivatization Provide Complementary Approaches for the Targeted LC-MS Analysis of the Polar Metabolome.

Liquid chromatography coupled to mass spectrometry is a key metabolomics/metabonomics technology. Reversed-phase liquid chromatography (RPLC) is very widely used as a separation step, but typically has poor retention of highly polar metabolites. Here, we evaluated the combination of two alternative methods for improving retention of polar metabolites based on 6-aminoquinoloyl-N-hydroxysuccinidimyl carbamate derivatization for amine groups, and ion-pairing chromatography (IPC) using tributylamine as an ion-pairing agent to retain acids. We compared both of these methods to RPLC and also to each other, for targeted analysis using a triple-quadrupole mass spectrometer, applied to a library of ca. 500 polar metabolites. IPC and derivatization were complementary in terms of their coverage: combined, they improved the proportion of metabolites with good retention to 91%, compared to just 39% for RPLC alone. The combined method was assessed by analyzing a set of liver extracts from aged male and female mice that had been treated with the polyphenol compound ampelopsin. Not only were a number of significantly changed metabolites detected, but also it could be shown that there was a clear interaction between ampelopsin treatment and sex, in that the direction of metabolite change was opposite for males and females.

Making sense of missense variants in TTN-related congenital myopathies.

Mutations in the sarcomeric protein titin, encoded by TTN, are emerging as a common cause of myopathies. The diagnosis of a TTN-related myopathy is, however, often not straightforward due to clinico-pathological overlap with other myopathies and the prevalence of TTN variants in control populations. Here, we present a combined clinico-pathological, genetic and biophysical approach to the diagnosis of TTN-related myopathies and the pathogenicity ascertainment of TTN missense variants. We identified 30 patients with a primary TTN-related congenital myopathy (CM) and two truncating variants, or one truncating and one missense TTN variant, or homozygous for one TTN missense variant. We found that TTN-related myopathies show considerable overlap with other myopathies but are strongly suggested by a combination of certain clinico-pathological features. Presentation was typically at birth with the clinical course characterized by variable progression of weakness, contractures, scoliosis and respiratory symptoms but sparing of extraocular muscles. Cardiac involvement depended on the variant position. Our biophysical analyses demonstrated that missense mutations associated with CMs are strongly destabilizing and exert their effect when expressed on a truncating background or in homozygosity. We hypothesise that destabilizing TTN missense mutations phenocopy truncating variants and are a key pathogenic feature of recessive titinopathies that might be amenable to therapeutic intervention.

A large-scale genetic screen identifies genes essential for motility in Agrobacterium fabrum.

The genetic and molecular basis of flagellar motility has been investigated for several decades, with innovative research strategies propelling advances at a steady pace. Furthermore, as the phenomenon is examined in diverse bacteria, new taxon-specific regulatory and structural features are being elucidated. Motility is also a straightforward bacterial phenotype that can allow undergraduate researchers to explore the palette of molecular genetic tools available to microbiologists. This study, driven primarily by undergraduate researchers, evaluated hundreds of flagellar motility mutants in the Gram-negative plant-associated bacterium Agrobacterium fabrum. The nearly saturating screen implicates a total of 37 genes in flagellar biosynthesis, including genes of previously unknown function.

The effects of gender composition on women's experience in math work groups.

The present studies tested a model outlining the effects of group gender composition on self- and others' perceptions of women's math ability in a truly interactive setting with groups composed entirely of naïve participants (N = 158 4-person groups across 3 studies). One woman in each group was designated to be the "expert" by having her complete a tutorial that gave her task-relevant knowledge for a subsequent group task. Group gender composition was hypothesized to influence perceptions of women's math ability through intrapersonal processes (stereotype threat effects on performance) and interpersonal processes (social cohesion between the expert and other group members). Group composition affected the experts' performance in the group math task, but importantly, it also affected their social cohesion with group members. Moreover, both of these effects-lowered performance and poorer social cohesion in male-dominated groups-made independent contributions in accounting for group gender composition effects on perceptions of women's math ability (Studies 1 and 2). Boundary conditions were examined in a 3rd study. Women who had a history of excelling in math and had chosen a math-intensive STEM major were selected to be the designated experts. We predicted and found this would be sufficient to eliminate the effect of group gender composition on interpersonal processes, and correspondingly the effect on women's perceived math ability. Interestingly (and consistent with past work on stereotype threat effects among highly domain-identified individuals), there were continued performance differences indicative of effects on intrapersonal processes. (PsycINFO Database Record

Caregivers' distress: youth with attention-deficit/hyperactivity disorder and comorbid disorders assessed via telemental health.

OBJECTIVE: This article evaluates the additive effects of children's comorbid conditions with attention-deficit/hyperactivity disorder (ADHD) in relation to caregivers' distress, in a clinical trial conducted through telemental health (TMH). METHODS: The Children's ADHD Telemental Health Treatment Study (CATTS) is examining the effectiveness of treatment delivered via TMH for children with ADHD who are living in underserved communities. The CATTS trial recruited 223 children (µ=9.53±2.06 years) and their caregivers. Diagnoses of ADHD and comorbid oppositional defiant disorder (ODD) and anxiety disorders (ADs) were established with the Child Behavior Checklist and the Computerized Diagnostic Interview Schedule for Children. We took advantage of rich baseline data from the CATTS trial to investigate associations between caregivers' distress and children's comorbid mental health conditions. Caregivers' distress was assessed with the Patient Health Questionnaire-9, Parenting Stress Index, and Caregiver Strain Questionnaire. ANOVAs were used to compare children with ADHD alone with children having one comorbid condition (ODD or ADs) and children having two comorbid conditions (ODD and ADs). RESULTS: Three quarters (75.3%) of participants met criteria for ODD and/or AD comorbid with ADHD: 24.7% had neither comorbidity; 47.5% had ODD or AD; and 27.8% had both ODD and AD comorbidities. The parents of children with multiple comorbid conditions experienced the highest levels of depression, stress, and burden of care. CONCLUSIONS: The CATTS sample that was recruited from underserved communities provided evidence of additive effects of child psychiatric comorbidities with caregivers' distress, echoing earlier findings from the Multi-modal Treatment of ADHD (MTA) study that was conducted with a metropolitan sample of youth. Results indicate that caregivers' distress should be addressed in developing treatment models for children with ADHD. CLINICAL TRIALS REGISTRY: http://clinicaltrials.gov/show/NCT00830700 .