Search

The aim of the present study was to investigate the effects of fatty fish consumption on cognitive functioning in a group of inpatients characterized by antisocial behavior. Eighty-three male forensic inpatients participated in this study. Participants were randomly assigned into a Fish or a Control group (e.g., meat, chicken, pork). One decision-making task, the Iowa Gambling Task (IGT), and one planning task, the Tower of Hanoi (ToH), were administered before (pre-test) and at the end of the intervention period (post-test). For the IGT the Fish group showed improved performance from pre- to post- test. Moreover, the Fish group showed significantly better performance than the Control group on the IGT at post-test. The Fish group also demonstrated improved performance from pre- to post-test on the ToH; however, this was limited to participants with a history of substance abuse. Further, the improvement was only significant for tasks with high working memory load (5-7 move problems), and not for tasks with low working memory load (1-4 move problems). The Control group showed no improvement on any of the tasks regardless of alcohol or drug abuse history. The present study suggests that regular fatty fish consumption may improve executive functions in forensic inpatients with antisocial traits and a history of substance abuse. Thus, the current results may have important implications with regard to health care interventions.

Introduction We created a standardized workshop to engage residents in quality improvement (QI) using the root cause analysis model. The workshop allows for a robust learning experience while providing solutions derived from clinicians to address important local problems. No prerequisite knowledge or experience is required. Methods The workshop is facilitated by one or more moderators, ideally with experience in QI. An interdisciplinary group of residents, medical students, nurses, and other attendees comprise an audience which actively engages in workshop activities. Facilitators follow a scripted model to teach important patient safety concepts with frequent break-outs for hands-on application of QI tools. During the workshop, participants create a process map and fishbone diagram, as well as develop and critically evaluate novel interventions. Results Over the course of one academic year, the workshop has been implemented 17 times with roughly 25 internal medicine residents in attendance at each workshop. In addition, the workshop was run online for 126 participants with varied exposure to QI techniques. Forty percent of these participants completed a survey indicating that over 89% learned something new, 87% felt they could apply the material to their work, and 95% would recommend the workshop to a colleague. Discussion This 60-minute workshop can provide hands-on QI experience in a standardized format to achieve the dual objectives of teaching QI to clinicians and allowing them to generate innovations. The module can be used for internal case development and trainee participation, but prepared cases are provided for facilitators without the resources for local case development.

Trichuris trichiura (whipworm) is one of the four major soil-transmitted helminth infections of man, affecting an estimated 465 million people worldwide. An effective vaccine that induces long-lasting protective immunity against T. trichiura would alleviate the morbidity associated with this intestinal-dwelling parasite, however the lack of known host protective antigens has hindered vaccine development. Here, we show that vaccination with ES products stimulates long-lasting protection against chronic infection in male C57BL/6 mice. We also provide a framework for the identification of immunogenic proteins within T. muris ES, and identify eleven candidates with direct homologues in T. trichiura that warrant further study. Given the extensive homology between T. muris and T. trichiura at both the genomic and transcriptomic levels, this work has the potential to advance vaccine design for T. trichiura.

Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1–D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.

To understand the mucociliary clearance system, mucins were visualized by light, confocal and electron microscopy, and mucus was stained by Alcian blue and tracked by video microscopy on tracheal explants of newborn piglets. We observed long linear mucus bundles that appeared at the submucosal gland openings and were transported cephalically. The mucus bundles were shown by mass spectrometry and immunostaining to have a core made of MUC5B mucin and were coated with MUC5AC mucin produced by surface goblet cells. The transport speed of the bundles was slower than the airway surface liquid flow. We suggest that the goblet cell MUC5AC mucin anchors the mucus bundles and thus controls their transport. Normal clearance of the respiratory tree of pigs and humans, both rich in submucosal glands, is performed by thick and long mucus bundles., Highlights • Submucosal glands in the piglet trachea form bundles of MUC5B mucin. • The mucus bundles are coated with MUC5AC mucin produced by surface goblet cells. • The mucus bundles are transported 10-times slower than the airway surface liquid. • The surface goblet cells are suggested to control the mucus bundle movement.

This study investigated the effects of fatty fish on sleep, daily functioning and biomarkers such as heart rate variability (HRV), vitamin D status (serum 25-hydroxyvitamin D (25OHD), and eicosapentaenoic acid (EPA, 20:5n-3) + docosahexaenoic acid (DHA, 22:6n-3) in red blood cells. Moreover the relationship among sleep, daily functioning, HRV, vitamin D status, and levels of EPA+DHA was investigated.Ninety-five male forensic patients from a secure forensic inpatient facility in the USA were randomly assigned into a Fish or a Control group. The Fish group received Atlantic salmon three times per week from September to February, and the Control group was provided an alternative meal (e.g., chicken, pork, beef), but with the same nutritional value as their habitual diet, three times per week during the same period. Sleep (sleep latency, sleep efficiency, actual sleep time, and actual wake time), self-perceived sleep quality and daily functioning, as well as vitamin D status, EPA+DHA, and HRV, were assessed pre- and post-intervention period.There was a significant increase in sleep latency from pre- to post-test in the Control group. The Fish group reported better daily functioning than the Control group during post-test. Fish consumption throughout the wintertime had also an effect on resting HRV and EPA+DHA, but not on vitamin D status. However, at post-test, the vitamin D status in the Fish group was still closer to the level regarded as optimal compared to the Control group. Vitamin D status correlated negatively with actual wake time and positively with sleep efficiency during pre-test, as well as positively with daily functioning and sleep quality during post-test. Finally, HRV correlated negatively with sleep latency and positively with daily functioning.Fish consumption seemed to have a positive impact on sleep in general and also on daily functioning, which may be related to vitamin D status and HRV.

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

We have used millisecond pulsars (MSPs) from the southern High Time Resolution Universe (HTRU) intermediate latitude survey area to simulate the distribution and total population of MSPs in the Galaxy. Our model makes use of the scale factor method, which estimates the ratio of the total number of MSPs in the Galaxy to the known sample. Using our best fit value for the z-height, z=500 pc, we find an underlying population of MSPs of 8.3(\pm 4.2)*10^4 sources down to a limiting luminosity of L_min=0.1 mJy kpc^2 and a luminosity distribution with a steep slope of d\log N/d\log L = -1.45(\pm 0.14). However, at the low end of the luminosity distribution, the uncertainties introduced by small number statistics are large. By omitting very low luminosity pulsars, we find a Galactic population above L_min=0.2 mJy kpc^2 of only 3.0(\pm 0.7)*10^4 MSPs. We have also simulated pulsars with periods shorter than any known MSP, and estimate the maximum number of sub-MSPs in the Galaxy to be 7.8(\pm 5.0)*10^4 pulsars at L=0.1 mJy kpc^2. In addition, we estimate that the high and low latitude parts of the southern HTRU survey will detect 68 and 42 MSPs respectively, including 78 new discoveries. Pulsar luminosity, and hence flux density, is an important input parameter in the model. Some of the published flux densities for the pulsars in our sample do not agree with the observed flux densities from our data set, and we have instead calculated average luminosities from archival data from the Parkes Telescope. We found many luminosities to be very different than their catalogue values, leading to very different population estimates. Large variations in flux density highlight the importance of including scintillation effects in MSP population studies., Comment: 13 pages, 5 figures, 5 tables. Accepted for publication in MNRAS

The body's mucosal surfaces are protected from pathogens and physical and chemical attack by the gel-like extracellular matrix, mucus. The framework of this barrier is provided by polymeric, gel-forming mucins. These enormous O-linked glycoproteins are synthesised, stored and secreted by goblet cells that are also the source of other protective factors. Immune regulation of goblet cells during the course of infection impacts on mucin production and properties and ultimately upon barrier function. The barrier function of mucins in protection of the host is well accepted as an important aspect of innate defence. However, it is becoming increasingly clear that mucins have a much more direct role in combating pathogens and parasites and are an important part of the coordinated immune response to infection. Of particular relevance to this review is the finding that mucins are essential anti-parasitic effector molecules. The current understanding of the roles of these multifunctional glycoproteins, and other goblet cell products, in mucosal defence against intestinal dwelling nematodes is discussed.

BACKGROUND AND OBJECTIVES: Empiric antibiotic therapy for presumed urinary tract infection (UTI) leads to unnecessary antibiotic exposure in many children whose urine culture results fail to confirm the diagnosis. The objective of this quality improvement study was to improve follow-up management of negative urine culture results in the off-campus urgent care network of Nationwide Children’s Hospital to reduce inappropriate antibiotic exposure in children. METHODS: A multidisciplinary task force developed and implemented a protocol for routine nurse and clinician follow-up of urine culture results, discontinuation of unnecessary antibiotics, and documentation in the electronic medical record. Monthly antibiotic discontinuation rates were tracked in empirically treated patients with negative urine culture results from July 2013 through December 2015. Statistical process control methods were used to track improvement over time. Fourteen-day return visits for UTIs were monitored as a balancing measure. RESULTS: During the study period, 910 patients received empiric antibiotic therapy for UTIs but had a negative urine culture result. The antibiotic discontinuation rate increased from 4% to 84%, avoiding 3429 (40%) of 8648 antibiotic days prescribed. Among patients with discontinued antibiotics, none was diagnosed with a UTI within 14 days of the initial urgent care encounter. CONCLUSIONS: Implementation of a standard protocol for urine culture follow-up and discontinuation of unnecessary antibiotics was both effective and safe in a high-volume pediatric urgent care network. Urine culture follow-up management is an essential opportunity for improved antimicrobial stewardship in the outpatient setting that will affect many patients by avoiding a substantial number of antibiotic days.

BACKGROUND AND OBJECTIVE: Immunoglobullin E (IgE)-mediated food allergies affect 5% to 8% of children. Serum IgE levels assist in diagnosing food allergies but have low positive predictive value. This can lead to misinterpretation, overdiagnosis, and unnecessary dietary elimination. Use of IgE food allergen panels has been associated with increased cost and burden. The scale of use of these panels has not been reported in the medical literature. METHODS: We conducted a retrospective review of a commercial laboratory database associated with a tertiary care pediatric academic medical center for food IgE tests ordered by all provider types during 2013. RESULTS: A total of 10 794 single-food IgE tests and 3065 allergen panels were ordered. Allergists ordered the majority of single-food IgE tests (58.2%) whereas 78.8% of food allergen panels were ordered by primary care providers (PCPs) (P < .001). Of all IgE tests ordered by PCPs, 45.1% were panels compared with 1.2% of orders placed by allergists (P < .001). PCPs in practice for >15 years ordered a higher number of food allergen panels (P < .05) compared with PCPs with less experience. Compared with allergists, PCPs ordered more tests for unlikely causes of food allergies (P < .001). Total cost of IgE testing and cost per patient were higher for PCPs compared with allergists. CONCLUSIONS: Review of food allergen IgE testing through a high volume outpatient laboratory revealed PCPs order significantly more food allergen panels, tests for uncommon causes of food allergy, and generate higher cost per patient compared with allergists. These results suggest a need for increased education of PCPs regarding proper use of food IgE tests.

Stuttering is linked to sensorimotor deficits related to internal modeling mechanisms. This study compared spectral power and oscillatory activity of EEG mu (μ) rhythms between persons who stutter (PWS) and controls in listening and auditory discrimination tasks. EEG data were analyzed from passive listening in noise and accurate (same/different) discrimination of tones or syllables in quiet and noisy backgrounds. Independent component analysis identified left and/or right μ rhythms with characteristic alpha (α) and beta (β) peaks localized to premotor/motor regions in 23 of 27 people who stutter (PWS) and 24 of 27 controls. PWS produced μ spectra with reduced β amplitudes across conditions, suggesting reduced forward modeling capacity. Group time-frequency differences were associated with noisy conditions only. PWS showed increased μ-β desynchronization when listening to noise and early in discrimination events, suggesting evidence of heightened motor activity that might be related to forward modeling deficits. PWS also showed reduced μ-α synchronization in discrimination conditions, indicating reduced sensory gating. Together these findings indicate spectral and oscillatory analyses of μ rhythms are sensitive to stuttering. More specifically, these analyses can reveal stuttering-related sensorimotor processing differences in passive listening and auditory discrimination tasks which may be influenced by basal ganglia deficits.

We report a case of a child with a negative qualitative opioid result on a urine drug screen using our routine cutoff but a positive confirmation for heroin. A 3-month-old male presented to the emergency department after an unexplained episode of hypoxia. The patient was formula-fed with a history of multidrug neonatal abstinence syndrome (NAS)3 after fetal exposure to opioids and no medical care following discharge from the hospital after his birth. The patient was revived at home and transported to the emergency department by ambulance and was stable upon admission to the hospital. All results were within normal limits except alanine aminotransferase, which was 74 U/L (reference

Better understanding of the role of sensorimotor processing in speech and non-speech segmentation can be achieved with more temporally precise measures. Twenty adults made same/different discriminations of speech and non-speech stimuli pairs, with and without segmentation demands. Independent component analysis of 64-channel EEG data revealed clear sensorimotor mu components, with characteristic alpha and beta peaks, localized to premotor regions in 70% of participants. Time-frequency analyses of mu components from accurate trials showed that (1) segmentation tasks elicited greater event-related synchronization immediately following offset of the first stimulus, suggestive of inhibitory activity; (2) strong late event-related desynchronization in all conditions, suggesting that working memory/covert replay contributed substantially to sensorimotor activity in all conditions; (3) stronger beta desynchronization in speech versus non-speech stimuli during stimulus presentation, suggesting stronger auditory-motor transforms for speech versus non-speech stimuli. Findings support the continued use of oscillatory approaches for helping understand segmentation and other cognitive tasks.

Summary The prevalence of aspergillosis in CF patients has until recently been underestimated, but increasing evidence suggests that it may play an important role in the progression of CF lung disease. In healthy airways, Aspergillus fumigatus can be efficiently removed from the lung by mechanisms such as mucociliary clearance and cough. However, these mechanisms are defective in CF, allowing pathogens such as A. fumigatus to germinate and establish chronic infections within the airways. The precise means by which A. fumigatus contributes to CF lung disease remain largely unclear. As the first point of contact within the lung, and an important component of the innate immune system, it is likely that the mucus barrier plays an important role in this process. Study of the functional interplay between this vital protective barrier, and in particular its principal structural components, the polymeric gel‐forming mucins, and CF pathogens such as A. fumigatus, is at an early stage. A. fumigatus protease activity has been shown to upregulate mucus production by inducing mucin mRNA and protein expression, and A. fumigatus proteases and glycosidases are able to degrade mucins. This may allow A. fumigatus to alter mucus barrier properties to promote fungal colonization of the airways and/or utilize mucins as a nutrient source. Moreover, conidial surface lectin binding to mucin glycans is a key aspect of clearance of Aspergillus from the lung in health but may be an important aspect of colonization, where mucociliary clearance is compromised, as in the CF lung. Here we discuss the nature of the mucus barrier and its mucin components in CF, and how they may be implicated in A. fumigatus infection. Pediatr Pulmonol 2017;52:548–555. © 2016 The Authors. Pediatric Pulmonology. Published by Wiley Periodicals, Inc.

The detection of five new fast radio bursts (FRBs) found in the High Time Resolution Universe high latitude survey is presented. The rate implied is 6$^{+4}_{-3}\times~10^3$ (95%) FRBs sky$^{-1}$ day$^{-1}$ above a fluence of between 0.13 and 5.9 Jy ms for FRBs between 0.128 and 262 ms in duration. One of these FRBs has a clear two-component profile, each component is similar to the known population of single component FRBs and are separated by 2.4(4) ms. All the FRB components appear to be unresolved following deconvolution with a scattering tail and accounting for intra-channel smearing. The two-component FRB also has the highest dispersion measure (1629 pc cm$^{-3}$) of any FRB to-date. Many of the proposed models to explain FRBs use a single high energy event involving compact objects (such as neutron star mergers) and therefore cannot easily explain a two-component FRB. Models that are based on extreme versions of flaring, pulsing or orbital events however could produce multiple component profiles. The compatibility of these models and the FRB rate implied by these detections is discussed., Comment: 5 pages, 1 figure, 1 table, submitted to MNRAS

Introduction: Mucus hypersecretion is a major contributor to asthma pathology and occurs as part of a spectrum of structural changes termed airway wall remodeling. Transforming growth factor (TGF)-β is proposed to play a key role in regulating airway matrix remodeling although less is known about the specific action of TGF-β isoforms in regulating mucus production. Methods: Primary human bronchial epithelial (HBE) cells cultured at air–liquid interface were treated with exogenous TGF-β1, TGF-β2, and/or a Th2 cytokine, interleukin (IL)-13. Expression and production of respiratory mucins, MUC5AC and MUC5B, were analyzed by real-time PCR, agarose gel electrophoresis, and western blotting. A murine-transformed Clara cell line (mtCC1-2) transfected with a luciferase reporter driven by the Muc5ac promoter containing Smad4 site-mutated cis sequences was used to determine whether exogenous TGF-β2 affects Muc5ac promoter function. Results: Surprisingly, TGF-β1 showed no measurable effect on MUC5AC or MUC5B...

Pseudomonas aeruginosa is a common cause of chronic respiratory infection in cystic fibrosis (CF) patients. Infection is established within the lung epithelial mucus layer through adhesion to mucins. Terminal residues on mucin oligosaccharide chains are highly sulfated and sialylated, which increases their resistance to degradation by bacterial enzymes. However, a number of microbes, including P. aeruginosa, display mucin sulfatase activity. Using ion chromatography, the levels of sulfation on different respiratory mucins and the availability of inorganic sulfate to pathogens in sputum from CF patients were quantified. The ability of clinical isolates of P. aeruginosa to desulfate mucin was tested by providing mucin as a sole sulfur source for growth. All tested P. aeruginosa strains isolated from the lungs of CF patients were able to use human respiratory mucin as a source of sulfur for growth, whereas other non-clinical species of the genus Pseudomonas were not. However, measured levels of inorganic sulfate in sputum from CF patients suggested that bacteria resident in the lung have sufficient inorganic sulfate for growth and are unlikely to require access to mucin sulfur as a sulfur source during chronic infection. This was confirmed when expression of sulfate-repressed P. aeruginosa genes atsK and msuE was found to be repressed in the sputum of CF patients, which was detected by using quantitative RT-PCR. These results indicate that sulfate starvation is unlikely to occur in pathogens residing in the sputum of CF patients and, therefore, mucin desulfation may have an alternative purpose in the association between P. aeruginosa and the airways of CF patients.

The ubiquitous filamentous fungus Aspergillus fumigatus secretes a number of allergens with protease activity and has been linked to a variety of allergic conditions such as Severe Asthma with Fungal Sensitization (SAFS) and Allergic Bronchopulmonary Aspergillosis (ABPA). However, it is unclear which allergen proteases are being secreted during fungal invasion and whether the local biological environment regulates their expression. Understanding the dynamic expression of allergen proteases during growth of A. fumigatus may lead to further characterisation of the pathogenesis of these disorders as well as improved standardisation in the commercial production of these allergens. Secretion of proteases during germination and early growth of A. fumigatus was investigated in response to various complex protein sources (pig lung homogenate, mucin or casein). Protease inhibitor studies demonstrated that A. fumigatus (AF293 strain) secretes predominately serine proteases during growth in pig lung based medium and mainly metalloproteases during growth in casein based medium but suppressed protease secretion in unmodified Vogel's minimal medium and secreted both types in mucin based medium. Analysis of gene transcription and protein identification by mass spectrometry showed that the matrix metalloprotease, Mep/Asp f 5 and the serine protease, Alp1/Asp f 13, were upregulated and secreted during growth in pig lung medium, whereas Alp1 was predominately expressed and secreted in mucin based medium. In casein medium, the matrix metalloprotease, Lap1, was also upregulated and secreted in addition to Mep and Alp1. These findings suggest that A. fumigatus is able to detect different complex proteins available as substrates in its environment and regulate protease secretion accordingly. There is a requirement for the standardisation of A. fumigatus allergen extracts used both in clinical diagnosis of A. fumigatus allergy and in research studies., Highlights ► Aspergillus fumigatus produces allergens with protease activity. ► Growth on protein based substrates induces the secretion of proteases. ► Mainly serine proteases secreted in response pig lung homogenate or mucin media. ► Predominately MMP secreted in response to casein media. ► Aspergillus fumigatus can sense and respond to changes in growth environment.

The chondrodysplasias are a group of >450 different phenotypes (1) that occur when endochondral bone growth is disrupted (2). The clinical presentation of chondrodysplasia varies from mild to lethal but is generally distinguished by disproportionate short stature. Multiple epiphyseal dysplasia (MED) is characterized by delayed and irregular endochondral ossification and early-onset osteoarthritis (3, 4). MED is genetically heterogeneous, and autosomal-dominant forms of the disease arise from mutations in the genes encoding matrilin 3, cartilage oligomeric matrix protein (COMP), and type IX collagen (3–5). Although present in other musculoskeletal tissues, matrilin 3, COMP, and type IX collagen are expressed primarily by chondrocytes and have been shown to interact with each other, both in vitro and in vivo (6–13). The matrilins are a family of 4 noncollagenous extracellular matrix (ECM) proteins. Each matrilin monomer comprises a C-terminal α-helical coiled-coil oligomerization domain, a varying number of epidermal growth factor–like domains, and 1 (matrilin 3) or 2 (matrilins 1, 2, and 4) von Willebrand factor A domains (6, 14). Matrilin proteins bind to numerous ECM components, including type II collagen, type IX collagen, aggrecan, COMP, biglycan, and decorin, and are believed to act as adaptor proteins in the ECM (6). More than 20 different missense mutations within the matrilin 3 gene have been shown to cause autosomal-dominant MED, and MATN3 mutations are believed to account for ∼20% of all cases of autosomal-dominant MED (5). The majority of MATN3 mutations affect residues that are located within the central β-sheet of the single A domain of matrilin 3 (5, 15–17), while a smaller proportion of mutations that affect residues in the α-helices of the same domain have recently been described (5, 18, 19). Several different cell culture model systems have been used in the past to study the effect of matrilin 3 mutations in vitro (15, 19, 20). The most striking effect of MATN3 mutations was the intracellular retention of mutant protein in cells in culture and also in patient cartilage. This distinctive finding was recently verified in a murine model of MED caused by a Matn3 V194D mutation, which replicated the human phenotype by exhibiting mild short-limbed dwarfism (21, 22). In this murine model, mutant matrilin 3 was retained within the endoplasmic reticulum (ER) of chondrocytes from the growth plates of endochondral bones. The retention of mutant matrilin 3 elicited a conventional unfolded protein response characterized by the up-regulation of classic chaperone proteins such as BiP/glucose-regulated protein 78-kd, calnexin, and calreticulin, as well as less-characterized ER stress–associated genes such as Creld2 and Armet/Manf (22). Despite intracellular retention of the majority of mutant matrilin 3 in vivo, a small proportion of mutant protein was secreted into the ECM, even in mice that were homozygous for the V194D mutation (21). The structural and/or functional effect of this mutant matrilin 3 in the ECM is not known, nor is the extent to which its presence in the ECM might contribute to the chondrodysplasia phenotype. Given the widespread literature describing the hetero-oligomerization of matrilin 3 with matrilin 1 (23–26), we considered the possibility that the secretion of mutant matrilin 3 may be dependent on its oligomerization into heterotetramers containing wild-type matrilin 1. We hypothesized that deletion of matrilin 1, which would abolish the formation of matrilin 1/matrilin 3 heterotetramers, would also eliminate the secretion of mutant matrilin 3 into the ECM and increase the severity of chondrodysplasia in mice with the Matn3 V194D mutation. A study testing this hypothesis would provide new insight into the disease mechanisms of MED and the role of hetero-oligomerization on the trafficking and secretion of matrilin 1/matrilin 3 proteins. We therefore crossed mice with the Matn3 V194D (Matn3m/m) mutation with Matn1−/− mice and generated a novel mouse line that was homozygous for the Matn3 V194D mutation and null for matrilin 1 (Matn1−/−Matn3m/m). We investigated the phenotypic, morphologic, and biochemical characterization of this novel murine model to determine the interdependency of matrilin 1 and matrilin 3 oligomerization on the trafficking and secretion of these proteins and their effect on disease severity.

Objectives.Asymptomatic bacteriuria/candidiuria (ASB) and culture-negative pyuria (CNP) are common and often result in inappropriate antibiotic use. We aimed to evaluate whether a standardized educational memorandum could reduce antimicrobial utilization for ASB/CNP.Design, Setting, and Patients.Quasi-experimental study with a control group, from a convenience sample of inpatients with abnormal urinalysis or urine culture results in a Veterans Affairs hospital.Intervention.An educational memorandum outlining guidelines for diagnosis and treatment of ASB was placed in the chart of patients with ASB/CNP who were receiving antimicrobials.Methods.The records of patients meeting inclusion criteria were abstracted for demographics, comorbidities, antimicrobials, and symptoms suggestive of possible urinary tract infection (UTI). Patients were categorized as having ASB, CNP, or UTI. The number of antimicrobial-days attributed to ASB/CNP was compared between the control group and the intervention group.Results.Charts of 301 patients with abnormal urine results were reviewed. Thirty of 117 (26%) patients in the control group received antimicrobials for ASB/CNP for an average of 6.3 days. In the intervention group, 24 of 92 (26%) patients received antimicrobials for ASB/CNP for an average of 2.2 days (t-test: PConclusions.ASB and CNP resulted in antimicrobial exposure in more than one-quarter of our study patients. Placing a standardized memorandum in the electronic record was associated with a 65% relative reduction in antimicrobial-days for ASB and CNP.

In cystic fibrosis, abnormal mucus viscosity and mucociliary clearance are due to decreased water and more solids, and not to changes in mucus pHhttp://ow.ly/NvBS30myLwk

The intestinal mucosal barrier, part of the innate immune defence, is responsive to the external environment and changes in response to infection. There is disparate evidence for the epithelial and goblet cell products within the intrinsic barrier being part of a response to resolve infection. We comprehensively analysed the changes of mucosal glycoconjugates during acute and chronic infection by utilising the Trichuris muris (T. muris) model. Transcription factors, atonal homolog 1 (Math-1) and SAM pointed domain containing ETS transcription factor (Spdef) were activated during acute infection, which promoted stem cell fate towards a secretory cell phenotype. The thickness of the intermediate barrier, the carbohydrate-rich glycocalyx, composed of cell surface mucins increased with exposure to T. muris, with an increase in Muc4, Muc13 and Muc17. Overall, hypersecretion of glycoproteins into the extrinsic barrier (mediated by IL-13) via the gamma amino-butyric acid-α3 receptor (GABA-α3), was observed during acute infection. Furthermore, altered glycosylation was observed during acute and chronic infection; mucins were more highly charged during acute infection than during chronic infection. This study readdresses the changes within the mucosal barrier, in particular in the cell surface and secreted mucins during acute and chronic nematode infection.

Airway mucus plugs, composed of mucin glycoproteins mixed with plasma proteins, are an important cause of airway obstruction in acute severe asthma, and they are poorly treated with current therapies.To investigate mechanisms of airway mucus clearance in health and in acute severe asthma.We collected airway mucus from patients with asthma and nonasthmatic control subjects, using sputum induction or tracheal aspiration. We used rheological methods complemented by centrifugation-based mucin size profiling and immunoblotting to characterize the physical properties of the mucus gel, the size profiles of mucins, and the degradation products of albumin in airway mucus.Repeated ex vivo measures of size and entanglement of mucin polymers in airway mucus from nonasthmatic control subjects showed that the mucus gel is normally degraded by proteases and that albumin inhibits this degradation. In airway mucus collected from patients with asthma at various time points during acute asthma exacerbation, protease-driven mucus degradation was inhibited at the height of exacerbation but was restored during recovery. In immunoblots of human serum albumin digested by neutrophil elastase and in immunoblots of airway mucus, we found that albumin was a substrate of neutrophil elastase and that products of albumin degradation were abundant in airway mucus during acute asthma exacerbation.Rheological methods complemented by centrifugation-based mucin size profiling of airway mucins in health and acute asthma reveal that mucin degradation is inhibited in acute asthma, and that an excess of plasma proteins present in acute asthma inhibits the degradation of mucins in a protease-dependent manner. These findings identify a novel mechanism whereby plasma exudation may impair airway mucus clearance.

Previous studies described the characteristics of individuals committed under sexual offender civil commitment laws in Arizona, Florida, and Washington. This study describes the diagnostic and risk profiles of 331 sexual offenders held under Wisconsin’s sexual offender statute and compares them to the people held in those other states. Persons detained under Wisconsin’s law substantially differ from those in the other states. This variability is attributed to differences in both diagnostic procedures and statutory risk thresholds. The authors believe this is the first study to directly compare diagnoses and risk measures of sex offenders referred for commitment in different states, thereby offering a more national view of who is detained under these laws.

The airways mucus gel performs a critical function in defending the respiratory tract against pathogenic and environmental challenges. In normal physiology, the secreted mucins, in particular the polymeric mucins MUC5AC and MUC5B, provide the organizing framework of the airways mucus gel and are major contributors to its rheological properties. However, overproduction of mucins is an important factor in the morbidity and mortality of chronic airways disease (e.g., asthma, cystic fibrosis, and chronic obstructive pulmonary disease). The roles of these enormous, multifunctional, O-linked glycoproteins in health and disease are discussed.

MUC5B is a major polymeric mucin in the airway mucus gel and is an essential component of innate defense of the respiratory epithelium. Knowledge of the synthesis and intracellular processing of MUC5B is incomplete. We investigated the molecular details of MUC5B assembly in primary human bronchial epithelial cells (HBECs) grown at an air-liquid interface (ALI). Electrophoretic and centrifugal separations of intracellular forms of MUC5B probed with antibodies specific for non-O-glycosylated and O-glycosylated forms of the mucin identified three major intracellular populations of MUC5B (non-O-glycosylated monomer and dimer, and O-glycosylated polymers). Biophysical analysis of recombinant MUC5B COOH-terminus (CT5B; D4-B-C-CK) expressed in 293-EBNA cells showed that MUC5B dimerizes by disulfide linkage. Pulse-chase studies in the HBEC ALI cultures showed that non-O-glycosylated MUC5B was synthesized within 20 min of metabolic labeling and O-glycosylated, polymeric mucin within 2 h. Radiolabeled O-glycosylated mucin polymers were secreted within 2 h and the majority were released by 48 h. These data indicate that MUC5B follows a similar assembly to the related glycoprotein, von Willebrand factor (vWF); however, unlike vWF the MUC5B polypeptide shows no evidence of major proteolytic processing of D-domains during the production of the mature secreted polymeric mucin in normal and cystic fibrosis (CF) primary bronchial epithelial cells. In contrast, MUC5B D-domains were modified by neutrophil elastase, a protease commonly found in CF sputum, demonstrating that proteolytic degradation of MUC5B is an extracellular event in CF sputum. These results define the pathway for synthesis of MUC5B in primary human goblet cells.

Mucins secreted by intestinal goblet cells are considered an important component of innate defense in a number of enteric infections, including many parasitic infections, but also likely provide protection against the gut microbiota. Nod proteins are intracellular receptors that play key roles in innate immune response and inflammation. Here, we investigated the role of Nod proteins in regulation of intestinal goblet cell response in naive mice and mice infected with the enteric parasite Trichuris muris . We observed significantly fewer periodic acid-Schiff (PAS)-stained intestinal goblet cells and less mucin (Muc2) in Nod1 and Nod2 double-knockout (Nod DKO) mice after T. muris infection than in wild-type (WT) mice. Expulsion of parasites from the intestine was significantly delayed in Nod DKO mice. Treatment of naive WT mice with Nod1 and Nod2 agonists simultaneously increased numbers of PAS-stained goblet cells and Muc2-expressing cells, whereas treatment with Nod1 or Nod2 separately had no significant effect. Stimulation of mucin-secreting LS174T cells with Nod1 and Nod2 agonists upregulated core 3 β1,3- N -acetylglucosaminyltransferase (C3GnT; an important enzyme in mucin synthesis) and MUC2. We also observed lower numbers of PAS-stained goblet cells and less Muc2 in germfree mice. Treatment with Nod1 and Nod2 agonists enhanced the production of PAS-stained goblet cells and Muc2 in germfree mice. These data provide novel information on the role of Nod proteins in goblet cell response and Muc2 production in relation to intestinal innate defense.

Mutations in matrilin-3 result in multiple epiphyseal dysplasia, which is characterized by delayed and irregular bone growth and early onset osteoarthritis. The majority of disease-causing mutations are located within the beta-sheet of the single A-domain of matrilin-3, suggesting that they disrupt the structure and/or function of this important domain. Indeed, the expression of mutant matrilin-3 results in its intracellular retention within the rough endoplasmic reticulum of cells, where it elicits an unfolded protein response. To understand the folding characteristics of the matrilin-3 A-domain we determined its structure using CD, analytical ultracentrifugation, and dual polarization interferometry. This study defined novel structural features of the matrilin-3 A-domain and identified a conformational change induced by the presence or the absence of Zn(2+). In the presence of Zn(2+) the A-domain adopts a more stable "tighter" conformation. However, after the removal of Zn(2+) a potential structural rearrangement of the metal ion-dependent adhesion site motif occurs, which leads to a more "relaxed" conformation. Finally, to characterize the interactions of the matrilin-3 A-domain we performed binding studies on a BIAcore using type II and IX collagen and cartilage oligomeric matrix protein. We were able to demonstrate that it binds to type II and IX collagen and cartilage oligomeric matrix protein in a Zn(2+)-dependent manner. Furthermore, we have also determined that the matrilin-3 A-domain appears to bind exclusively to the COL3 domain of type IX collagen and that this binding is abolished in the presence of a disease causing mutation in type IX collagen.

Horses frequently suffer from respiratory diseases, which, irrespective of etiology, are often associated with airway mucus accumulation. Studies on human airways have shown that the key structural components of the mucus layer are oligomeric mucins, which can undergo changes of expression and properties in disease. However, there is little information on these gel-forming glycoproteins in horse airways mucus. Therefore, the aims of this study were to isolate equine airways oligomeric mucins, characterize their macromolecular properties, and identify their gene products. To this end, pooled tracheal washes, collected from healthy horses and horses suffering from respiratory diseases, were solubilized with 6 M guanidinium chloride (GdmCl). The oligomeric mucins were purified by density gradient centrifugation followed by size exclusion chromatography. Biochemical and biophysical analyses showed the mucins were stiffened random coils in solution that were polydisperse in size ( Mr= 6–20 MDa, average Mr= 14 MDa) and comprised of disulfide-linked subunits (average Mr= 7 MDa). Agarose gel electrophoresis showed that the pooled mucus sample contained at least two populations of oligomeric mucins. Electrospray ionization tandem mass spectrometry of tryptic digests of the unfractionated mucin preparation showed that the oligomeric mucins Muc5b and Muc5ac were present. In summary, we have shown that equine airways mucus is a mixture of Muc5b and Muc5ac mucins that have a similar macromolecular organization to their human counterparts. This study will form the basis for future studies to analyze the contribution of these two mucins to equine airways pathology associated with mucus accumulation.

Disruption to endochondral ossification leads to delayed and irregular bone formation and can result in a heterogeneous group of genetic disorders known as the chondrodysplasias. One such disorder, multiple epiphyseal dysplasia (MED), is characterized by mild dwarfism and early-onset osteoarthritis and can result from mutations in the gene encoding matrilin-3 (MATN3). To determine the disease mechanisms that underpin the pathophysiology of MED we generated a murine model of epiphyseal dysplasia by knocking-in a matn3 mutation. Mice that are homozygous for the mutation develop a progressive dysplasia and have short-limbed dwarfism that is consistent in severity with the relevant human phenotype. Mutant matrilin-3 is retained within the rough endoplasmic reticulum of chondrocytes and is associated with an unfolded protein response. Eventually, there is reduced proliferation and spatially dysregulated apoptosis of chondrocytes in the cartilage growth plate, which is likely to be the cause of disrupted linear bone growth and the resulting short-limbed dwarfism in the mutant mice.

We have generated an antiserum to the variable domain of mouse collagen XXVII, a recently discovered novel member of the fibrillar collagen family. Collagen XXVII protein was first detectable in the mouse at embryonic day 12.5 (E12.5). By E14.5, the protein localized to cartilage, developing dermis, cornea, the inner limiting membrane of the retina, and major arteries of the heart. However, at E18.5, collagen XXVII protein was no longer apparent in most tissues and appeared restricted mainly to cartilage where expression continued into adulthood. Type XXVII collagen immunolocalized to 10-nm-thick nonstriated fibrils that were distinct from fibrils formed by the classical fibrillar collagens. The transient nature of its expression and unusual fibrillar structure suggest that collagen XXVII plays a developmental role distinct from those of the classical fibrillar collagens.

This study sought to determine delta granule normal ranges for children and to validate methodology for the appropriate diagnosis of delta granule deficiency (storage pool disease) by using the whole-mount technique in electron microscopy. Specimens obtained from 40 healthy volunteers (2 months of age through 21 years old, 21 females and 19 males) were tested. Results showed dense granules/platelet (DG/Plt) ranged from 1.78 to 5.25. The 5th percentile was 1.96 DG/Plt with an overall mean ± SEM 3.07 ± 0.12 DG/Plt. In comparison, a previously published lower cutoff value, 3.68 DG/Plt, was significantly higher than the mean from our volunteers ( P < 0.0001). We found no variability in dense granules/platelet based on race or sex and no significant variation by age subgroup. Pending wider studies, the value of 2 DG/Plt is a more appropriate lower limit of normal. In the absence of wider studies (in healthy volunteers and patients), laboratories should consider establishing their own reference ranges.

Sensorimotor integration (SMI) across the dorsal stream enables online monitoring of speech. Jenson et al. (2014) used independent component analysis (ICA) and event related spectral perturbation (ERSP) analysis of electroencephalography (EEG) data to describe anterior sensorimotor (e.g., premotor cortex, PMC) activity during speech perception and production. The purpose of the current study was to identify and temporally map neural activity from posterior (i.e., auditory) regions of the dorsal stream in the same tasks. Perception tasks required “active” discrimination of syllable pairs (/ba/ and /da/) in quiet and noisy conditions. Production conditions required overt production of syllable pairs and nouns. ICA performed on concatenated raw 68 channel EEG data from all tasks identified bilateral “auditory” alpha (α) components in 15 of 29 participants localized to pSTG (left) and pMTG (right). ERSP analyses were performed to reveal fluctuations in the spectral power of the α rhythm clusters across time. Production conditions were characterized by significant α event related synchronization (ERS; pFDR < 0.05) concurrent with EMG activity from speech production, consistent with speech-induced auditory inhibition. Discrimination conditions were also characterized by α ERS following stimulus offset. Auditory α ERS in all conditions temporally aligned with PMC activity reported in Jenson et al. (2014). These findings are indicative of speech-induced suppression of auditory regions, possibly via efference copy. The presence of the same pattern following stimulus offset in discrimination conditions suggests that sensorimotor contributions following speech perception reflect covert replay, and that covert replay provides one source of the motor activity previously observed in some speech perception tasks. To our knowledge, this is the first time that inhibition of auditory regions by speech has been observed in real-time with the ICA/ERSP technique.

Cartilage is a highly specialized load-bearing tissue with a small number of cells and a high proportion of extracellular matrix (ECM). The abundance of heavily sulfated proteoglycans and a poorly soluble collagenous ECM presents a major technical challenge to 2-DE. Here we report proteomic analysis of mouse growth plate cartilage using novel methodology for tissue dissection and sample prefractionation. We have successfully resolved cartilage tissue extracts by 2-DE for the first time and identified cartilage ECM proteins by Western blotting and MS/MS.

The relationship between self-esteem assessed prior to treatment using the Self-Esteem Scale and sexual recidivism was explored in two samples of adult male sex offenders, 53 of whom were beginning treatment in the community and 172 of whom were beginning treatment in prison. Sexual reconviction rates were obtained for both samples using a 6-year followup for the community sample and an average follow-up of just less than 4 years for the prison sample. Lower levels of self-esteem were associated with higher sexual recidivism rates with similar trends being apparent in both samples. The linear main effect of self-esteem was significant at beyond the .01 level in a logistic regression analysis. Receiver operating characteristics analysis was used to assess the strength of this association and an area-under-the-curve coefficient of .69 was obtained. Results are discussed in terms of their meaning for the relevance of self-esteem as a predictor of recidivism and as a target for treatment.

Mucociliary transport in the airways significantly depends on the liquid and mucin components of the airway surface liquid (ASL). The regulation of ASL water and mucin content during pathological conditions is not well understood. We hypothesized that airway epithelial mucin production and liquid transport are regulated in response to inflammatory stimuli and tested this hypothesis by investigating the effects of the pleiotropic, early-response cytokine, IL-1β, on cultured primary human bronchial epithelial and second-passage, normal human tracheo-bronchial epithelial (NHTBE) cell cultures. Fully differentiated NHTBE cultures secreted two major airway mucins, MUC5AC and MUC5B. IL-1β, in a dose- and time-dependent manner, increased the secretion of MUC5AC, but not MUC5B. MUC5AC mRNA levels were only transiently increased at 1 and 4 h after the start of IL-1β treatment and returned to control levels thereafter, even though MUC5AC mucin production remained elevated for at least 72 h. Synchronous with elevated MUC5AC secretion, ASL volume increased, its percentage of solid was reduced, and the pH/[[Formula: see text]] of the ASL was elevated. ASL volume changes reflected altered ion transport, including an upregulation of Cl-secretory currents (via CFTR and Ca2+-activated Cl-conductance) and an inhibition of epithelial sodium channel (ENaC)-mediated absorptive Na+currents. IL-1β increased CFTR mRNA levels without affecting those for ENaC subunits. The synchronous regulation of ASL mucin and liquid metabolism triggered by IL-1β may be an important defense mechanism of the airway epithelium to enhance mucociliary clearance during airway inflammation.

Mucus is essential for protection of the airways; however, in chronic airway disease mucus hypersecretion is an important factor in morbidity and mortality. The properties of the mucus gel are dictated in large part by the oligomeric mucins and, over the past decade, we have gained a better understanding of the molecular nature of these complex O-linked glycoproteins. We know now that MUC5AC mucins, as well as different glycoforms of the MUC5B mucin, are the predominant gel-forming glycoproteins in airways mucus. Furthermore, the amount, molecular size, and morphology of these glycoproteins can be altered in disease. From more recent data, it has become clear that oligomeric mucins alone do not constitute mucus, and other mucin and nonmucin components must be important contributors to mucus organization and hence airways defense. Therefore, the challenge over the coming decade will be to investigate how the oligomeric mucins are organized to yield "functional" mucus. Such studies will provide a clearer perception of airways mucosal protection and may highlight specific components as potential targets for therapeutic strategies for the treatment of hypersecretory disease.

The TIM10 complex, composed of the homologous proteins Tim10 and Tim9, chaperones hydrophobic proteins inserted at the mitochondrial inner membrane. A salient feature of the TIM10 complex subunits is their conserved "twin CX3C" motif. Systematic mutational analysis of all cysteines of Tim10 showed that their underlying molecular defect is impaired folding (demonstrated by circular dichroism, aberrant homo-oligomer formation, and thiol trapping assays). As a result of defective folding, clear functional consequences were manifested in (i) complex formation with Tim9, (ii) chaperone activity, and (iii) import into tim9ts mitochondria lacking both endogenous Tim9 and Tim10. The organization of the four cysteines in intrachain disulfides was determined by trypsin digestion and mass spectrometry. The two distal CX3C motifs are juxtaposed in the folded structure and disulfide-bonded to each other rather than within each other, with an inner cysteine pair connecting Cys44 with Cys61 and an outer pair between Cys40 and Cys65. These cysteine pairs are not equally important for folding and assembly; mutations of the inner Cys are severely affected and form wrong, non-native disulfides, in contrast to mutations of the outer Cys that can still maintain the native inner disulfide pair and display weaker functional defects. Taken together these data reveal this specific intramolecular disulfide bonding as the crucial mechanism for Tim10 folding and show that the inner cysteine pair has a more prominent role in this process.

The macromolecular organization within saliva was investigated by tracer diffusion measurements of fluorescent polystyrene microspheres by fluorescence recovery after photobleaching using a confocal microscope (confocal-FRAP). There was a concentration-dependent reduction in microsphere diffusion; this was much greater in the presence of calcium (10 mm) and was reduced by the addition of EGTA (10 mm). These effects on tracer diffusion showed that native saliva contained a macromolecular organization that was sensitive to free calcium concentrations. This was supported by a major increase in the weight average molecular weight of the high molecular weight mucin fraction in saliva (10-62 x 106) and an increase in intrinsic viscosity of saliva (733 to 1203 ml/g) both caused by calcium. Analysis of the change in tracer diffusion in saliva showed a 20-fold increase in the apparent pore size (from 130 nm in 10 mm CaCl2 to 2600 nm in 10 mm EGTA at physiological concentration). The effect was specific for calcium and was unaffected by up to 2 m NaCl. The calcium binding activity was contained in a high buoyant density fraction of saliva excluded from Sepharose CL-2B. Calcium binding to this fraction gave an approximate Kd of 7 x 10-6 m, and the binding was irreversibly destroyed by treatment with 6 m guanidinium chloride and by mild reduction, suggesting it to be to a protein site. This fraction of saliva was shown to contain MUC5B as the single major protein species by positive ion electrospray ionization-tandem mass spectrometry analysis. The results suggested that oligomeric MUC5B in saliva is assembled into much larger linear or branched assemblies through calcium-mediated protein cross-links.

Mucosal surfaces are exposed to the external environment and pathogens and are therefore protected by a secreted layer of mucus rich in mucin glycoproteins, which are the main components of mucus. Mucus provides physical protection and hydration, excludes pathogens, and is a reservoir for antimicrobial molecules. Underlying mucus further protection is provided by epithelial cell surface mucins, which limit microbial adherence and regulate growth and apoptosis. Differentiation of the cells that produce mucins, and expression of mucins and proteins involved in mucin biosynthesis, is regulated by innate and adaptive immunity. Experimental deficiencies in mucins lead to infectious and inflammatory diseases, and mucin gene polymorphisms are associated with disease. Many chronic mucosal inflammatory diseases are characterized by mucus hypersecretion driven by immune and microbial factors, which contributes to pathology and is a target for therapy. In this chapter we describe the nature of this mucosal barrier, its regulation and function, and its involvement in human disease, with particular emphasis on the mucin component of the barrier.

The TIM10 complex is localized in the mitochondrial intermembrane space and mediates insertion of hydrophobic proteins at the inner membrane. We have characterized TIM10 assembly and analyzed the structural properties of its subunits, Tim9 and Tim10. Both proteins are alpha-helical with a protease-resistant central domain, and each self-associates to form mainly dimers and trimers in solution. Tim9 and Tim10 bound to one another with submicromolar affinity in equimolar amounts and assembled in a stable, significantly extended complex that was indistinguishable from the native mitochondrial TIM10 complex. Importantly, the reconstituted TIM10 complex is functional because it bound to the physiological substrate ADP/ATP carrier and displayed chaperone activity in refolding the model substrate firefly luciferase. These data demonstrate that the individual subunits can exist as independent, dynamically self-associating proteins. Assembly into the thermodynamically stable hexameric complex is necessary for the TIM10 chaperone function.

We have developed a new approach to study the molecular organization of salivary mucus and salivary mucins using confocal fluorescence recovery after photobleaching (confocal-FRAP). MUC5B mucin, its reduced subunit and T-domains were prepared from saliva and fluorescently labelled. The translational self-diffusion coefficients were determined up to 3.6mg/ml by confocal-FRAP. The results suggest that, in solutions of purified MUC5B mucin, at concentrations at which the hydrodynamic domains overlap, the intermolecular interactions are predominantly due to dynamic entanglements, and there was no evidence of specific self-association of MUC5B mucin, or of its subunits, or T-domains. The analysis of the salivary mucus gel also showed no specific interactions with the purified MUC5B components, but it was much less permeable than expected from its MUC5B content. The saliva was completely permeable to microspheres of 207nm diameter, but showed size-dependent effects on the diffusion of larger microspheres (499nm and 711nm diameter). From these analyses the salivary mucus was shown to be both permeable and dynamic, and with the characteristics of a semi-dilute transient network at physiological concentration. Comparison of the results from saliva and purified MUC5B mucin solutions showed that the network properties of saliva were equivalent to a solution of purified MUC5B mucin of 10–20 times higher concentration. This showed that saliva has additional structure and organization not present in the purified MUC5B mucin and suggests there are other interactions and/or components within saliva that combine with MUC5B to produce its complete properties.

Activity in premotor and sensorimotor cortices is found in speech production and some perception tasks. Yet, how sensorimotor integration supports these functions is unclear due to a lack of data examining the timing of activity from these regions. Beta (~20Hz) and alpha (~10Hz) spectral power within the EEG µ rhythm are considered indices of motor and somatosensory activity, respectively. In the current study, perception conditions required discrimination (same/different) of syllables pairs (/ba/ and /da/) in quiet and noisy conditions. Production conditions required covert and overt syllable productions and overt word production. Independent component analysis was performed on EEG data obtained during these conditions to 1) identify clusters of µ components common to all conditions and 2) examine real-time event-related spectral perturbations (ERSP) within alpha and beta bands. 17 and 15 out of 20 participants produced left and right µ-components, respectively, localized to precentral gyri. Discrimination conditions were characterized by significant (pFDR

We have isolated the high-Mr mucins from growth medium of the early stage of an HT-29 cell culture by gel chromatography and isopycnic density gradient centrifugation. The mucins (buoyant density 1.34-1.44 g/ml) were reactive with an anti-peptide antiserum (MAN-5ACI) raised against a sequence from within the MUC5AC mucin. Similar antisera raised against the MUC2 and MUC5B mucins were not reactive. The MUC5AC reduced-mucin subunits exhibited a homogeneous charge distribution on anion-exchange chromatography, but appeared as two bands, one major and one more minor, after agarose gel electrophoresis. The unreduced mucins had an average Mr in excess of 40 MDa and were visualized in the electron microscope as large, fine filamentous threads (many microns in length) that after reduction were greatly reduced in size (number average length 570 nm). Agarose gel electrophoresis of unreduced MUC5AC mucins identified a major band just entering the gel with evidence of a ‘ladder’ of faster-migrating minor bands. Partial reduction of the mucins increased the proportion of the faster bands and at least 16 could be discriminated. Mr measurements showed that these bands differed by single monomer units. The mucins behaved as very stiff extended structures in solution and this characteristic might explain the poor separation of different-sized oligomers in sedimentation-rate experiments. The cell-culture mucin preparation had similar characteristics of charge and buoyant density to MUC5AC mucins from respiratory secretions in vivo. In addition the MUC5AC mucin from respiratory tract secretions exhibited similar behaviour, reduced and unreduced on agarose gel electrophoresis, indicating that the mucin has a similar molecular phenotype in vivo and in vitro.