Early life stress enhances the vulnerability to chronic psychosocial stress and experimental colitis in adult mice

Early life stress enhances the vulnerability to both mood and chronic inflammatory disorders, suggesting a link between these stress-related disorders. To study this, we exposed male C57BL/6 mice to early life stress [maternal separation (MS), 3 h/d, d 1-14] and to adult chronic psychosocial stress [chronic subordinate colony housing (CSC)] and measured changes in neuroendocrine parameters and in the severity of a chemically induced colitis. In both unseparated and MS mice, 19 d of CSC exposure resulted in a transient decrease in body weight gain, increased anxiety-related behavior, and decreased vasopressin mRNA expression in the hypothalamic paraventricular nucleus compared with respective nonstressed mice. However, only CSC-stressed MS mice showed elevated CRH mRNA expression in the paraventricular nucleus and reduced plasma corticosterone. Subsequent treatment with dextran sulfate sodium (1%, 7 d) resulted in a more severe colonic inflammation in MS compared with unseparated mice. This was indicated by an increased histological damage score and increased TNF secretion (nonstressed MS mice), more severe body weight loss and inflammatory reduction in colon length (CSC-stressed MS mice), and increased interferon-gamma secretion (nonstressed and CSC-stressed MS mice). In conclusion, early life stress and subsequent exposure to chronic psychosocial stress in adulthood induced neuroendocrine abnormalities, which likely contributed to enhanced vulnerability to chemically induced colitis. The combined use of MS and CSC represents a potential animal model providing novel (patho)physiological insights into the complex interactions between neuroendocrine and inflammatory actions upon chronic stress exposure. These findings may further help to reveal mechanisms of hypocortisolemic disorders.     

Deaths following methotrexate overdoses by medical staff

Methotrexate (MTX) is an effective disease modifying antirheumatic drug (DMARD) with a relatively safe profile, and it is widely used to treat neoplastic diseases and dermatologic and rheumatologic disorders. As indications for use of MTX increase, more accidental overdoses are noted to occur. Typical problems include deficiencies in labeling, instructions, or packaging, as well as erroneous use. We describe 5 fatal cases of repeated oral overdose of MTX prescribed by physicians in the treatment of rheumatoid arthritis to focus attention on the design of the underlying system and the organizational practices as sources of problems.     

Expression of Phosphofructokinase in Skeletal Muscle Is Influenced by Genetic Variation and Associated With Insulin Sensitivity

Using an integrative approach in which genetic variation, gene expression, and clinical phenotypes are assessed in relevant tissues may help functionally characterize the contribution of genetics to disease susceptibility. We sought to identify genetic variation influencing skeletal muscle gene expression (expression quantitative trait loci [eQTLs]) as well as expression associated with measures of insulin sensitivity. We investigated associations of 3,799,401 genetic variants in expression of >7,000 genes from three cohorts (n = 104). We identified 287 genes with cis-acting eQTLs (false discovery rate [FDR] <5%; P < 1.96 × 10⁻⁵) and 49 expression–insulin sensitivity phenotype associations (i.e., fasting insulin, homeostasis model assessment–insulin resistance, and BMI) (FDR <5%; P = 1.34 × 10⁻⁴). One of these associations, fasting insulin/phosphofructokinase (PFKM), overlaps with an eQTL. Furthermore, the expression of PFKM, a rate-limiting enzyme in glycolysis, was nominally associated with glucose uptake in skeletal muscle (P = 0.026; n = 42) and overexpressed (Bonferroni-corrected P = 0.03) in skeletal muscle of patients with T2D (n = 102) compared with normoglycemic controls (n = 87). The PFKM eQTL (rs4547172; P = 7.69 × 10⁻⁶) was nominally associated with glucose uptake, glucose oxidation rate, intramuscular triglyceride content, and metabolic flexibility (P = 0.016–0.048; n = 178). We explored eQTL results using published data from genome-wide association studies (DIAGRAM and MAGIC), and a proxy for the PFKM eQTL (rs11168327; r² = 0.75) was nominally associated with T2D (DIAGRAM P = 2.7 × 10⁻³). Taken together, our analysis highlights PFKM as a potential regulator of skeletal muscle insulin sensitivity.     

Head impact exposure in youth football—Are current interventions hitting the target?

Restrictions on heading in youth football have been implemented in some countries to limit head impact exposure. However, current interventions remain poorly guided by evidence. Our objective was to quantify heading exposure in youth football, assessing the effects of sex and age. Football matches played during an international youth football tournament with no heading restrictions were directly observed, including players from both sexes (11-19 years). The elite senior level was included for comparison, using video analysis. All heading events were registered, classified, and assigned to individual players. Heading rates were calculated for each sex and age group. We observed a total of 267 matches, corresponding to 4011 player hours (1927 player hours for females, 2083 player hours for males). Males headed more frequently than females (2.7 vs 1.8 headers/player hour; P < .001). Heading rates increased with age (ANOVA, P < .001), approaching the elite senior level for players 16 years and older. There was substantial variation within teams for all age and sex groups, with the widest range (1-18 headers) observed for girls aged 19. Girls younger than 12 years had the lowest exposure, with an average of <2 players per team heading the ball, each with 1-2 headers. In conclusion, age and sex influence head impact exposure in youth football, and warrants careful consideration when introducing injury prevention measures. Males are more frequently exposed than females, heading rates increase with age, and there is substantial variation between players. Heading is a rare event in the youngest age groups, especially among females.     

DEAD-box protein CYT-19 is activated by exposed helices in a group I intron RNA

DEAD-box proteins are nonprocessive RNA helicases and can function as RNA chaperones, but the mechanisms of their chaperone activity remain incompletely understood. The Neurospora crassa DEAD-box protein CYT-19 is a mitochondrial RNA chaperone that promotes group I intron splicing and has been shown to resolve misfolded group I intron structures, allowing them to refold. Building on previous results, here we use a series of tertiary contact mutants of the Tetrahymena group I intron ribozyme to demonstrate that the efficiency of CYT-19–mediated unfolding of the ribozyme is tightly linked to global RNA tertiary stability. Efficient unfolding of destabilized ribozyme variants is accompanied by increased ATPase activity of CYT-19, suggesting that destabilized ribozymes provide more productive interaction opportunities. The strongest ATPase stimulation occurs with a ribozyme that lacks all five tertiary contacts and does not form a compact structure, and small-angle X-ray scattering indicates that ATPase activity tracks with ribozyme compactness. Further, deletion of three helices that are prominently exposed in the folded structure decreases the ATPase stimulation by the folded ribozyme. Together, these results lead to a model in which CYT-19, and likely related DEAD-box proteins, rearranges complex RNA structures by preferentially interacting with and unwinding exposed RNA secondary structure. Importantly, this mechanism could bias DEAD-box proteins to act on misfolded RNAs and ribonucleoproteins, which are likely to be less compact and more dynamic than their native counterparts.DEAD-box proteins constitute the largest family of RNA helicases and function in all stages of RNA metabolism (1, 2). In vivo, many DEAD-box proteins have been implicated in assembly and conformational rearrangements of large structured RNAs and ribonucleoproteins (RNPs), including the ribosome, spliceosome, and self-splicing introns (3). Thus, it is important to establish how these proteins use their basic mechanisms of RNA binding and helix unwinding to interact with and remodel higher-order RNA structures.Structural and mechanistic studies have elucidated the basic steps of the ATPase cycle of DEAD-box proteins and have provided an understanding of the coupling between ATPase and duplex unwinding activities (4–11). The conserved helicase core consists of two flexibly linked RecA-like domains that contain at least 12 conserved motifs, including the D-E-A-D sequence in the ATP-binding motif II (3, 12). Binding of ATP and double-stranded RNA to domains 1 and 2, respectively, induces domain closure, which completes the formation of an ATPase active site at the domain interface and introduces steric clashes in the RNA binding site, leading to the displacement of one of the RNA strands (6, 7). ATP hydrolysis and inorganic phosphate release are then thought to regenerate the open enzyme conformation (4, 8, 13). Unlike conventional helicases, DEAD-box proteins have not been found to translocate, limiting the unwinding activity to short helices that can be disrupted in a single cycle of ATP binding and hydrolysis (4, 8, 9, 14–16). This mechanism is compatible with the physiological roles of DEAD-box proteins, because cellular RNAs rarely contain continuous base-paired regions that are longer than one or two helical turns.The interactions of DEAD-box proteins with structured RNAs have been extensively studied using two homologous proteins that function as general RNA chaperones: CYT-19 from Neurospora crassa and Mss116 from Saccharomyces cerevisiae. In vivo, CYT-19 is required for efficient splicing of several mitochondrial group I introns and can promote splicing of group I and group II introns in yeast mutants that lack functional Mss116 (17, 18). Both proteins have been shown to act as general RNA chaperones during group I and group II intron folding in vitro and are thought to act primarily by reversing misfolding of the intron RNAs, although additional mechanisms may be used for some substrates (17–23). Importantly, the chaperone activities of these and other DEAD-box proteins correlate with their ATP-dependent helix unwinding activities, suggesting that DEAD-box proteins function by lowering the energy barriers for transitions between alternative structures that involve disruption of base pairs (24, 25).In vitro studies using the group I intron ribozyme from Tetrahymena thermophila have been instrumental in probing the chaperone mechanism of CYT-19 (17, 26–28). This ∼400-nt RNA folds into a compact, globular structure composed of a conserved core and a series of peripheral elements that encircle the core by forming long-range tertiary contacts (Fig. 1) (29–31). Upon addition of Mg²⁺ ions, the majority of the ribozyme population becomes trapped in a long-lived misfolded conformation, which then slowly refolds to the native state (32). The misfolded intermediate is remarkably similar to the native ribozyme, forming a complete native network of secondary and tertiary interactions and a globally compact fold (33, 34). Despite these similarities, refolding to the native state requires extensive unfolding, including disruption of all five peripheral tertiary contacts and the core helix P3 (33, 35). To explain these results, a topological error has been proposed, wherein two single-stranded joining elements are crossed incorrectly in the core of the misfolded ribozyme, and transient disruption of the surrounding native structure is required for refolding (33, 35).Open in a separate windowFig. 1.The Tetrahymena group I intron ribozyme. (A) Secondary structure and mutations. Peripheral elements are colored and thick arrows mark the long-range peripheral tertiary contacts. Paired regions (P) and loops that were mutated in this study (L) are labeled based on group I intron nomenclature in ref. 31. The mutated regions are enclosed in dashed boxes and labeled in bold, with sequence substitutions indicated nearby. Sequences that were deleted to construct the helix truncation mutants (Fig. 6) are enclosed in gray dashed boxes and the replacement nucleotides are shown in gray italic font. (B) Tertiary structure model of the ribozyme (31). Peripheral elements (colored surface) and the locations of the long-range peripheral tertiary contacts (circles) are highlighted using the same color scheme as in A. The ribozyme core is shown in silver. The block arrows indicate the approximate positions of tertiary contacts not visible in each respective view of the ribozyme. The figures were prepared using PyMOL.Given the structural similarity between the native and misfolded ribozyme, it is interesting that CYT-19 can accelerate refolding of the misfolded intermediate by at least an order of magnitude without detectably unfolding the native ribozyme (26). Insights into this apparent preference for the misfolded ribozyme came from studies of two ribozyme mutants in which the tertiary structure was destabilized, making the stability of the native ribozyme comparable to that of the misfolded intermediate (28). CYT-19 unfolded the native and misfolded conformers of these mutants with comparable efficiencies, suggesting that the efficiency of chaperone-mediated unfolding depended on the stability of ribozyme tertiary structure. However, the mutations studied were concentrated in one region of the ribozyme, leaving open the possibility that CYT-19 recognizes local disruptions rather than global stability.Here we investigate the roles of RNA stability in CYT-19-mediated unfolding of the Tetrahymena ribozyme by using a series of ribozyme mutants with disruptions of each of the five peripheral tertiary contacts. We observe a strong correlation between CYT-19 activity and global stability of ribozyme tertiary structure. Further, we find that the RNA-dependent ATPase activity of CYT-19 depends on the accessibility of secondary structure in the ribozyme. Our results lead to a general model for recognition and remodeling of unstable or incorrectly folded RNAs by a DEAD-box protein.     

Dissatisfied patients after total knee arthroplasty: A registry study involving 114 patients with 8–13 years of follow-up

Background and purpose

In 2003, an enquiry by the Swedish Knee Arthroplasty Register (SKAR) 2–7 years after total knee arthroplasty (TKA) revealed patients who were dissatisfied with the outcome of their surgery but who had not been revised. 6 years later, we examined the dissatisfied patients in one Swedish county and a matched group of very satisfied patients.

Patients and methods

118 TKAs in 114 patients, all of whom had had their surgery between 1996 and 2001, were examined in 2009–2010. 55 patients (with 58 TKAs) had stated in 2003 that they were dissatisfied with their knees and 59 (with 60 TKAs) had stated that they were very satisfied with their knees. The patients were examined clinically and radiographically, and performed functional tests consisting of the 6-minute walk and chair-stand test. All the patients filled out a visual analog scale (VAS, 0–100 mm) regarding knee pain and also the Hospital and Anxiety and Depression scale (HAD).

Results

Mean VAS score for knee pain differed by 30 mm in favor of the very satisfied group (p < 0.001). 23 of the 55 patients in the dissatisfied group and 6 of 59 patients in the very satisfied group suffered from anxiety and/or depression (p = 0.001). Mean range of motion was 11 degrees better in the very satisfied group (p < 0.001). The groups were similar with regard to clinical examination, physical performance testing, and radiography.

Interpretation

The patients who reported poor response after TKA continued to be unhappy after 8–13 years, as demonstrated by VAS pain and HAD, despite the absence of a discernible objective reason for revision.The results of TKA are regarded as being favorable (Robertsson et al. 2000, Kane et al. 2005, Nilsdotter et al. 2009, Carr et al. 2012) with few surgical complications and a revision rate of less than 5% after 10 years (Vessely et al. 2006, Robertsson et al. 2010). Poor outcome after primary TKA, apart from the revision, is between 6% and 14% (Anderson et al. 1996, Hawker et al. 1998, Heck et al. 1998, Robertsson et al. 2000, Robertsson and Dunbar 2001, Brander et al. 2003, Noble et al. 2006, Fisher et al. 2007, Wylde et al. 2008, Kim et al. 2009, Bourne et al. 2010, Scott et al. 2010). The reason for poor outcome after TKA may be related to problems with the knee surgery itself, although it has been suggested that extra-articular causes such as hip disease, spine disorder, vascular disease, or reflex sympathetic dystrophy may contribute. Some studies have suggested that factors not primarily related to structural tissue changes, but of psychological nature instead, may be involved (Wylde et al. 2007, Rolfson et al. 2009).The Swedish Knee Arthroplasty Register (SKAR) registers primary arthroplasties performed in Sweden as well as revisions, and has been estimated to capture 97% of the surgeries performed (SKAR 2012). The SKAR sends questionnaires regarding satisfaction to patients who were operated on during certain time periods (Robertsson et al. 2000, and Dunbar 2001). We used the SKAR to identify patients who had not undergone revision surgery and who were dissatisfied with their outcome 2–7 years after TKA surgery. As a reference we chose an age-, sex-, date-of-surgery-, and hospital-matched control group of highly satisfied patients who were operated during the same period. Our aim was to assess the differences between these 2 patient groups.     

Fetal alcohol spectrum disorders

Prenatal alcohol exposure (PAE) is one of the most prevalent and modifiable risk factors for somatic, behavioral, and neurological abnormalities. Affected individuals exhibit a wide range of such features referred to as fetal alcohol spectrum disorders (FASD). These are characterized by a more or less specific pattern of minor facial dysmorphic features, growth deficiency and central nervous system symptoms. Nevertheless, whereas the diagnosis of the full-blown fetal alcohol syndrome does not pose a major challenge, only a tentative diagnosis of FASD can be reached if only mild features are present and/or maternal alcohol consumption during pregnancy cannot be verified. The respective disorders have lifelong implications. The teratogenic mechanisms induced by PAE can lead to various additional somatic findings and structural abnormalities of cerebrum and cerebellum. At the functional level, cognition, motor coordination, attention, language development, executive functions, memory, social perception and emotion processing are impaired to a variable extent. The long-term development is characterized by disruption and failure in many domains; an age-adequate independency is frequently not achieved. In addition to primary prevention, individual therapeutic interventions and tertiary prevention are warranted; provision of extensive education to affected subjects and their caregivers is crucial. Protective environments are often required to prevent negative consequences such as delinquency, indebtedness or experience of physical/sexual abuse.     

How ‘love’ and ‘hate’ differ from ‘sleep’: Using combined electro/magnetoencephalographic data to reveal the sources of early cortical responses to emotional words

Emotional words—as symbols for biologically relevant concepts—are preferentially processed in brain regions including the visual cortex, frontal and parietal regions, and a corticolimbic circuit including the amygdala. Some of the brain structures found in functional magnetic resonance imaging are not readily apparent in electro‐ and magnetoencephalographic (EEG; MEG) measures. By means of a combined EEG/MEG source localization procedure to fully exploit the available information, we sought to reduce these discrepancies and gain a better understanding of spatiotemporal brain dynamics underlying emotional‐word processing. Eighteen participants read high‐arousing positive and negative, and low‐arousing neutral nouns, while EEG and MEG were recorded simultaneously. Combined current‐density reconstructions (L2‐minimum norm least squares) for two early emotion‐sensitive time intervals, the P1 (80–120 ms) and the early posterior negativity (EPN, 200–300 ms), were computed using realistic individual head models with a cortical constraint. The P1 time window uncovered an emotion effect peaking in the left middle temporal gyrus. In the EPN time window, processing of emotional words was associated with enhanced activity encompassing parietal and occipital areas, and posterior limbic structures. We suggest that lexical access, being underway within 100 ms, is speeded and/or favored for emotional words, possibly on the basis of an “emotional tagging” of the word form during acquisition. This gives rise to their differential processing in the EPN time window. The EPN, as an index of natural selective attention, appears to reflect an elaborate interplay of distributed structures, related to cognitive functions, such as memory, attention, and evaluation of emotional stimuli. Hum Brain Mapp 35:875–888, 2014. © 2012 Wiley Periodicals, Inc.     

Beta‐band activity in auditory pathways reflects speech localization and recognition in bilateral cochlear implant users

In normal‐hearing listeners, localization of auditory speech involves stimulus processing in the postero‐dorsal pathway of the auditory system. In quiet environments, bilateral cochlear implant (CI) users show high speech recognition performance, but localization of auditory speech is poor, especially when discriminating stimuli from the same hemifield. Whether this difficulty relates to the inability of the auditory system to translate binaural electrical cues into neural signals, or to a functional reorganization of auditory cortical pathways following long periods of binaural deprivation is unknown. In this electroencephalography study, we examined the processing of auditory syllables in postlingually deaf adults with bilateral CIs and in normal‐hearing adults. Participants were instructed to either recognize (“recognition” task) or localize (“localization” task) the syllables. The analysis focused on event‐related potentials and oscillatory brain responses. N1 amplitudes in CI users were larger in the localization compared with recognition task, suggesting an enhanced stimulus processing effort in the localization task. Linear beamforming of oscillatory activity in CI users revealed stronger suppression of beta‐band activity after 200 ms in the postero‐dorsal auditory pathway for the localization compared with the recognition task. In normal‐hearing adults, effects for longer latency event‐related potentials were found, but no effects were observed for N1 amplitudes or beta‐band responses. Our study suggests that difficulties in speech localization in bilateral CI users are not reflected in a functional reorganization of cortical auditory pathways. New signal processing strategies of cochlear devices preserving unambiguous binaural cues may improve auditory localization performance in bilateral CI users. Hum Brain Mapp 35:3107–3121, 2014. © 2013 Wiley Periodicals, Inc .