Parental Self-Efficacy to Support Teens During a Suicidal Crisis and Future Adolescent Emergency Department Visits and Suicide Attempts

This study of adolescents seeking emergency department (ED) services and their parents examined parents’ self-efficacy beliefs to engage in suicide prevention activities, whether these beliefs varied based on teens’ characteristics, and the extent to which they were associated with adolescents’ suicide-related outcomes. Participants included 162 adolescents (57% female, 81.5% Caucasian), ages 13–17, and their parents. At index visit, parents rated their self-efficacy to engage in suicide prevention activities and their expectations regarding their teen’s future suicide risk. Adolescents’ ED visits for suicide-related concerns and suicide attempts were assessed 4 months later. Parents endorsed high self-efficacy to engage in most suicide prevention activities. At the same time, they endorsed considerable doubt in being able to keep their child safe if the teen has thoughts of suicide and in their child not attempting suicide in the future. Parents whose teens experienced follow-up suicide-related outcomes endorsed, at clinically meaningful effect sizes, lower self-efficacy for recognizing suicide warning signs, for obtaining the teen’s commitment to refrain from suicide, and for encouraging their teen to cope, as well as lower confidence that their teen will not attempt suicide; self-efficacy to recognize warning signs was at trend level. Despite endorsing high self-efficacy for the majority of suicide prevention activities, parents of high-risk teens expressed less confidence in their capacity to influence their teen’s suicidal behavior, which could undermine parents’ effort to implement these strategies. The relationship between parental self-efficacy and youth suicide-related outcomes points to its potential value in guiding clinical decision making and interventions.     

A novel ECEL1 mutation expands the phenotype of distal arthrogryposis multiplex congenita type 5D to include pretibial vertical skin creases

Arthrogryposis multiplex congenita (AMC) is a heterogeneous disorder characterized by multiple joint contractures often in association with other congenital abnormalities. Pretibial linear vertical creases are a rare finding associated with arthrogryposis, and the etiology of the specific condition is unknown. We aimed to genetically and clinically characterize a boy from a consanguineous family, presenting with AMC and pretibial vertical linear creases on the shins. Whole exome sequencing and variant analysis revealed homozygous novel missense variants of ECEL1 (c.1163T > C, p.Leu388Pro, NM_004826) and MUSK (c.2572C > T, p.Arg858Cys, NM_005592). Both variants are predicted to have deleterious effects on the protein function, with amino acid positions highly conserved among species. The variants segregated in the family, with healthy mother, father, and sister being heterozygous carriers and the index patient being homozygous for both mutations. We report on a unique patient with a novel ECEL1 homozygous mutation, expanding the phenotypic spectrum of Distal AMC Type 5D to include vertical linear skin creases. The homozygous mutation in MUSK is of unknown clinical significance. MUSK mutations have previously shown to cause congenital myasthenic syndrome, a neuromuscular disorder with defects in the neuromuscular junction.     

C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

The variant rs26232, in the first intron of the chromosome 5 open reading frame 30 (C5orf30) locus, has recently been associated with both risk of developing rheumatoid arthritis (RA) and severity of tissue damage. The biological activities of human C5orf30 are unknown, and neither the gene nor protein show significant homology to any other characterized human sequences. The C5orf30 gene is present only in vertebrate genomes with a high degree of conservation, implying a central function in these organisms. Here, we report that C5orf30 is highly expressed in the synovium of RA patients compared with control synovial tissue, and that it is predominately expressed by synovial fibroblast (RASF) and macrophages in the lining and sublining layer of the tissue. These cells play a central role in the initiation and perpetuation of RA and are implicated in cartilage destruction. RASFs lacking C5orf30 exhibit increased cell migration and invasion in vitro, and gene profiling following C5orf30 inhibition confirmed up-regulation of genes involved in cell migration, adhesion, angiogenesis, and immune and inflammatory pathways. Importantly, loss of C5orf30 contributes to the pathology of inflammatory arthritis in vivo, because inhibition of C5orf30 in the collagen-induced arthritis model markedly accentuated joint inflammation and tissue damage. Our study reveal C5orf30 to be a previously unidentified negative regulator of tissue damage in RA, and this protein may act by modulating the autoaggressive phenotype that is characteristic of RASFs.Rheumatoid arthritis is a chronic systemic autoimmune disease characterized by a symmetrical, inflammatory arthropathy that frequently results in damage to synovial-lined joints with consequent pain, stiffness, and reduced functional capacity. The prevalence of RA is 0.8–1% in Western Europe and North America, and it is believed to arise from an interplay between genetics and the environment. Smoking is known to be a major risk factor particularly for anticitrullinated protein antibody-positive RA (1), whereas consumption of alcohol reduces both the risk and the severity of RA (2). The severity of RA varies from a mild condition with little joint damage to an unremitting condition that leads to extensive bone and cartilage damage. The radiological severity of damage to the hands and feet is widely used to measure outcome of RA and has been shown to have a significant genetic component (3, 4). Loci genetically associated with radiological damage include DRB1 (5), CD40 (6) and TRAF1/C5 (7), IL-4 (8), and IL-15 (9).A genome-wide association study involving 12,277 RA cases and 28,975 controls, all of European descent, reported association of rs26232 in the first intron of chromosome 5 open reading frame 30 (C5orf30) with risk of RA (10). Importantly linkage disequilibrium did not extend to genes in the flanking regions, indicating that the association was arising from C5orf30. This association was subsequently replicated in a British study of 6,108 RA cases and 13,009 controls (11). In a study of three large European RA populations (n = 1,884), we reported an allele dose association of rs26232 with radiological damage (12).The biological activities of human C5orf30 are unknown, and the precise roles it plays in RA have not yet been reported. There is indirect evidence linking human C5orf30 with immune function via its association with intracellular UNC119 (13); the latter increasing both T-cell activation by up-regulating Lck/Fyn activity and Src kinases regulating macrophages activation (14, 15). There are, however, no studies of the biological functions of human C5orf30 and, in view of the genetic association with RA susceptibility and severity, we have undertaken in silico analysis and both in vitro and in vivo experiments to determine its functional activities in RA. Here, we report C5orf30 to be a yet unidentified negative regulator of tissue damage in RA, acting by modulating the autoaggressive phenotype that is characteristic of RA synovial fibroblasts (RASF). It is highly expressed in the synovium of RA patients compared with healthy and osteoarthritis (OA) predominately by RASF in the lining and sublining layer. These cells play an important role in the initiation and perpetuation of RA and are implicated in cartilage destruction (16). Targeting C5orf30 expression by using siRNA technology resulted in increased invasiveness, proliferation and migration of RASFs in vitro, and modulated expression of genes in RA-relevant pathways including migration and adhesion. Importantly, loss of C5orf30 contributes to the pathology of inflammatory arthritis in vivo, because inhibition of C5orf30 in the collagen-induced arthritis (CIA) model mice markedly accentuated joint inflammation and cartilage destruction. These data confirm C5orf30 as a previously unidentified regulator of tissue destruction in RA.     

β-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR

Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly β-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified β-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of β-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a β-helical architecture, in which 18 β-strand segments are arranged in six consecutive windings of a β-helix.Similar to eukaryotes, bacteria use a number of different cytoskeletal elements to ensure the proper temporal and spatial organization of their cellular machinery. Various studies proved the existence of bacterial homologs of eukaryotic cytoskeleton proteins including tubulin homologs such as FtsZ (1), actin homologs such as MreB (2), and intermediate filament (IF)-like proteins (3), which together have important roles in cell division, morphogenesis, polarity determination, and DNA segregation (4–7). In addition, several groups of polymer-forming proteins that are limited to the bacterial domain have been described (6).A recent addition to these bacteria-specific cytoskeletal proteins are the so-called bactofilins (8), a class of proteins that is widespread among most bacterial lineages and involved in a variety of different cellular processes. In the prosthecate α-proteobacterium Caulobacter crescentus, for instance, the two bactofilin paralogues BacA and BacB (Fig. 1A) assemble into membrane-associated polymeric sheets that are specifically localized to the cell pole carrying the stalk (8), a thin protrusion of the cell body involved in cell attachment and nutrient acquisition (9). These assemblies serve as spatial landmarks mediating the polar localization of a cell wall biosynthetic enzyme, PbpC, involved in stalk biogenesis (8) and organization (10). The δ-proteobacterial species Myxococcus xanthus, by contrast, possesses four bactofilin paralogues (BacMNOP) with, at least partly, distinct functions. BacM was shown to form cable- or rod-like structures that are critical for proper cell shape (8, 11). BacP, on the other hand, assembles into short filamentous structures that emanate from the cell poles, recruiting and thus controlling a small GTPase involved in type IV pili-dependent motility (12). As another well-characterized example, the ε-proteobacterium Helicobacter pylori, a human pathogen notorious for causing peptic ulcers, was shown to depend on a bactofilin homolog (CcmA) for maintaining its characteristic helical cell shape, a feature required for cells to efficiently colonize the gastric mucus (13). Moreover, in the γ-proteobacterium Proteus mirabilis, a homolog of the bactofilin CcmA has been implicated in cell shape and swarming motility (14).Open in a separate windowFig. 1.High-resolution ssNMR spectra of BacA filaments. (A) Selected bacterial bactofilins. The total number of amino acids is indicated in parentheses. (B) Amino acid sequence of C. crescentus BacA, together with a synthetic linker peptide (of 17 residues, in red) and a His₆-tag (in blue) attached at the C terminus. The DUF583 domain is highlighted in magenta, and prolines are shown in green. (C) Carbon–carbon 2D correlation spectrum of uniformly [¹³C, ¹⁵N]-labeled BacA. The carbon–carbon magnetization transfer is achieved by PDSD. A short PDSD mixing time of 20 ms was applied, optimal for intraresidue transfer. In the spectrum, a trace through Ile60 is shown to illustrate sensitivity and line width.Bactofilins are usually small proteins (∼20 kDa) that are composed of a central conserved domain of unknown function (DUF583) and flanking N- and C-terminal regions of variable length and sequence. A characteristic of bactofilins is their ability to polymerize spontaneously in the absence of nucleotides or other cofactors (8). Native BacM protofilaments have been isolated from M. xanthus whole-cell lysates by sucrose density centrifugation (11). Moreover, polymers of C. crescentus BacA and BacB were obtained after heterologous expression in Escherichia coli, a species that lacks chromosomally encoded bactofilin homologs (8). Similarly, polymerization was observed for heterologously produced M. xanthus BacN, BacO, and BacP (8, 12). In all cases, the filamentous structures formed were biochemically inert and resistant to nonphysiological salt concentrations and pH values. This behavior is reminiscent of IFs, although there is no evolutionary relationship between these two groups of cytoskeletal elements (6). In particular, bactofilins lack predicted coiled-coil regions, which are a key feature of IF proteins.Up to now, the molecular structure of bactofilins and the mechanism(s) underlying their assembly have remained unknown. This is in large part due to the spontaneous formation, inertness, and insolubility of bactofilin polymers, which makes them difficult substrates for crystallography studies as well as conventional liquid-state NMR methods. We therefore resorted to the use of solid-state NMR (ssNMR) spectroscopy, a technique that has recently been adapted to obtain high-resolution structural information on insoluble and noncrystalline protein assemblies, including functional oligomeric assemblies (15–17), disease-related amyloid fibrils (18–21), and membrane proteins in a lipid bilayer environment (22–26). In this study, we have applied state-of-the-art magic-angle spinning ssNMR spectroscopy in combination with a range of other biophysical methods to filaments of the C. crescentus bactofilin BacA (161 residues). We show that the DUF583 domain serves as a polymerization module that forms the rigid core of BacA filaments, whereas the terminal regions of the protein remain flexible. The core domain folds exclusively into β-sheets, but with an arrangement different from that typically found in amyloids. On the basis of the diffraction pattern of 2D crystalline assemblies observed by transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM) measurements that yield a value for the mass per length (MPL), and the β-strand segments identified by ssNMR, we propose a β-helical arrangement of the BacA subunit. Finally, we performed a mutational analysis of conserved residues in the β-strand segments, which suggests that the folding and/or polymerization of bactofilins are mediated by an extensive and redundant network of hydrophobic interactions. These findings provide for the first time, to our knowledge, insight into the atomic structure of a bacteria-specific cytoskeletal filament and highlight ssNMR as a powerful technique for the analysis of cytoskeletal elements that are unamenable to standard structural biological approaches.     

From the Cover: A simple nutrient-dependence mechanism for predicting the stoichiometry of marine ecosystems

It is widely recognized that the stoichiometry of nutrient elements in phytoplankton varies within the ocean. However, there are many conflicting mechanistic explanations for this variability, and it is often ignored in global biogeochemical models and carbon cycle simulations. Here we show that globally distributed particulate P:C varies as a linear function of ambient phosphate concentrations, whereas the N:C varies with ambient nitrate concentrations, but only when nitrate is most scarce. This observation is consistent with the adjustment of the phytoplankton community to local nutrient availability, with greater flexibility of phytoplankton P:C because P is a less abundant cellular component than N. This simple relationship is shown to predict the large-scale, long-term average composition of surface particles throughout large parts of the ocean remarkably well. The relationship implies that most of the observed variation in N:P actually arises from a greater plasticity in the cellular P:C content, relative to N:C, such that as overall macronutrient concentrations decrease, N:P rises. Although other mechanisms are certainly also relevant, this simple relationship can be applied as a first-order basis for predicting organic matter stoichiometry in large-scale biogeochemical models, as illustrated using a simple box model. The results show that including variable P:C makes atmospheric CO₂ more sensitive to changes in low latitude export and ocean circulation than a fixed-stoichiometry model. In addition, variable P:C weakens the relationship between preformed phosphate and atmospheric CO₂ while implying a more important role for the nitrogen cycle.Nutrient elements are used by phytoplankton to synthesize molecules, in order to accomplish biochemical functions. Some of these molecules are absolutely necessary, and the nutrient elements have no substitutes. Examples are P in nucleic acids, N in amino acids, and Fe in the photosynthetic apparatus (1). However, there is a degree of plasticity in the molecular assemblage required per phytoplankton cell, which varies between species and between clades (2, 3). Furthermore, there is a capacity for plasticity in molecular composition of even a given species, as shown in culture experiments (4, 5). Such plasticity leads to variability in the elemental ratios of nutrients in marine phytoplankton, widely documented in laboratory and field measurements (2, 6, 7). Recent analyses of global nutrient and particulate observations have shown that N:P, the most commonly discussed ratio, varies regionally, including low N:P in the high-latitude Southern Ocean and high N:P in the oligotrophic regions (7–9). Explanations of high N:P in oligotrophic waters have often invoked an enhanced reliance on N-rich proteins for gathering scarce resources (1, 10), whereas low N:P in the Southern Ocean has been variously attributed to the abundance of P-rich molecules in cold, fast-growing plankton (11), or to the availability of Si, which supports P-rich diatom communities (8, 12).Despite an abundant literature on stoichiometric variability and its potential causes, no simple predictive relationship has been widely adopted in global biogeochemical models. Instead, the vast majority of global biogeochemical models assumes fixed C:N:P in organic matter, including most participants in the recent Coupled Model Intercomparison Project, CMIP5 (13). Thus, the potential impact of changes in organic matter stoichiometry on ocean carbon storage and oxygen consumption remain largely unexplored. The neglect of stoichiometric variability is due, at least in part, to the lack of a clear predictive framework.Here, it is argued that the concentration of a nutrient element in seawater can provide a suitable predictive framework, because it is a critical determinant of the rate at which that element will tend to be taken up by the organisms in the local community. This hypothesis builds on classic resource competition theory (14), which argues that if the concentration of an element is low, such that uptake is difficult, the community will be dominated by organisms that are well adapted to a low cellular quota of that nutrient (10). If, on the other hand, the concentration is high, facilitating high uptake rates, the community will be dominated by organisms that are capable of taking advantage of that nutrient to grow faster. This suggestion leads to clear predictions with significant biogeochemical consequences, as outlined below.     

From the Cover: PNAS Plus: The essential gene set of a photosynthetic organism

Synechococcus elongatus PCC 7942 is a model organism used for studying photosynthesis and the circadian clock, and it is being developed for the production of fuel, industrial chemicals, and pharmaceuticals. To identify a comprehensive set of genes and intergenic regions that impacts fitness in S. elongatus, we created a pooled library of ∼250,000 transposon mutants and used sequencing to identify the insertion locations. By analyzing the distribution and survival of these mutants, we identified 718 of the organism’s 2,723 genes as essential for survival under laboratory conditions. The validity of the essential gene set is supported by its tight overlap with well-conserved genes and its enrichment for core biological processes. The differences noted between our dataset and these predictors of essentiality, however, have led to surprising biological insights. One such finding is that genes in a large portion of the TCA cycle are dispensable, suggesting that S. elongatus does not require a cyclic TCA process. Furthermore, the density of the transposon mutant library enabled individual and global statements about the essentiality of noncoding RNAs, regulatory elements, and other intergenic regions. In this way, a group I intron located in tRNA^Leu, which has been used extensively for phylogenetic studies, was shown here to be essential for the survival of S. elongatus. Our survey of essentiality for every locus in the S. elongatus genome serves as a powerful resource for understanding the organism’s physiology and defines the essential gene set required for the growth of a photosynthetic organism.Determining the sets of genes necessary for survival of diverse organisms has helped to identify the fundamental processes that sustain life across an array of environments (1). This research has also served as the starting point for efforts by synthetic biologists to design organisms from scratch (2, 3). Despite the importance of essential gene sets, they have traditionally been challenging to gather because of the difficulty of observing mutations that result in lethal phenotypes. More recently, the pairing of transposon mutagenesis with next generation sequencing, referred to collectively as transposon sequencing (Tn-seq), has resulted in a dramatic advance in the identification of essential gene sets (4–7). The key characteristic of Tn-seq is the use of high-throughput sequencing to screen for the fitness of every transposon mutant in a pooled population to measure each mutation’s impact on survival. These data can be used to quantitatively ascertain the effect of loss-of-function mutations at any given locus, intragenic or intergenic, in the conditions under which the library is grown (8). Essential gene sets for 42 diverse organisms distributed across all three domains have now been defined, largely through the use of Tn-seq (9). A recently developed variation on Tn-seq, random barcode transposon site sequencing (RB-TnSeq) (10), further minimizes the library preparation and sequencing costs of whole-genome mutant screens.Despite the proliferation of genome-wide essentiality screens, a complete essential gene set has yet to be defined for a photosynthetic organism. A collection of phenotyped Arabidopsis thaliana mutants has been created but extends to only one-tenth of Arabidopsis genes (11). In algae, efforts are underway to produce a Tn-seq–like system in Chlamydomonas reinhardtii; however, the mutant library currently lacks sufficient saturation to determine gene essentiality (12). To date, the essential genes for photoautotrophs have only been estimated by indirect means, such as by comparative genomics (13). The absence of experimentally determined essential gene sets in photosynthetic organisms, despite their importance to the environment and industrial production, is largely because of the difficulty and time required for genetic modification of these organisms.Cyanobacteria comprise an extensively studied and ecologically important photosynthetic phylum. They are responsible for a large portion of marine primary production and have played a foundational role in research to decipher the molecular components of photosynthesis (14, 15). Synechococcus elongatus PCC 7942 is a particularly well-studied member of this phylum because of its genetic tractability and streamlined genome (16). As a result, it has been developed as a model photosynthetic organism and a production platform for a number of fuel products and high-value chemicals (17). Despite the importance of S. elongatus for understanding photosynthesis and industrial production, 40% of its genes have no functional annotation, and only a small portion of those that do have been studied experimentally.Here, we use RB-TnSeq, a method that pairs high-density transposon mutagenesis and pooled mutant screens, to probe the S. elongatus genome for essential genes and noncoding regions. We categorized 96% of 2,723 genes in S. elongatus as either essential (lethal when mutated), beneficial (growth defect when mutated), or nonessential (no phenotype when mutated) under standard laboratory conditions. Furthermore, we determined the genome-wide essentiality of noncoding RNAs (ncRNAs), regulatory regions, and intergenic regions. Our investigation has produced an extensive analysis of the loci essential for the growth of a photosynthetic organism and developed a powerful genomic tool that can be used for additional screens under a wide array of ecologically and industrially relevant growth conditions.     

Renal Arcuate Vein Microthrombi-Associated AKI

Backgrounds and objectives

This report describes six patients with AKI stages 2–3 (median admission creatinine level, 2.75 mg/dl [range, 1.58–5.44 mg/dl]), hematuria (five with hemoproteinuria), and unremarkable imaging with an unusual and unexplained histologic diagnosis on renal biopsy.

Design, setting, participants, & measurements

The patients were young adults who presented to two neighboring United Kingdom nephrology centers over a 40-month period (between July 2010 and November 2013). Four were male, and the median age was 22.5 years (range, 18–27 years). Their principal symptoms were flank pain or lower back pain. All had consumed alcohol in the days leading up to admission.

Results

Renal biopsy demonstrated microthrombi in the renal arcuate veins with a corresponding stereotypical, localized inflammatory infiltrate at the corticomedullary junction. All patients recovered to baseline renal function with supportive care (median, 17 days; range, 6–60 days), and none required RRT. To date, additional investigations have not revealed an underlying cause for these histopathologic changes. Investigations have included screening for thrombophilic tendencies, renal vein Doppler ultrasonographic studies, and testing for recreational drugs and alcohol (including liquid chromatography–mass spectrometry of urine) to look for so-called designer drugs. Inquiries to the United Kingdom National Poisons Information Centre have identified no other cases with similar presentation or histologic findings.

Conclusions

Increased awareness and additional study of future cases may lead to a greater understanding of the underlying pathophysiologic mechanisms that caused AKI in these patients.