A chromosome-scale assembly of the quinoa genome provides insights into the structure and dynamics of its subgenomes.

Quinoa (Chenopodium quinoa Willd.) is an allotetraploid seed crop with the potential to help address global food security concerns. Genomes have been assembled for four accessions of quinoa; however, all assemblies are fragmented and do not reflect known chromosome biology. Here, we use in vitro and in vivo Hi-C data to produce a chromosome-scale assembly of the Chilean accession PI 614886 (QQ74). The final assembly spans 1.326 Gb, of which 90.5% is assembled into 18 chromosome-scale scaffolds. The genome is annotated with 54,499 protein-coding genes, 96.9% of which are located on the 18 largest scaffolds. We also report an updated genome assembly for the B-genome diploid C. suecicum and use it, together with the A-genome diploid C. pallidicaule, to identify genomic rearrangements within the quinoa genome, including a large pericentromeric inversion representing 71.7% of chromosome Cq3B. Repetitive sequences comprise 65.2%, 48.6%, and 57.9% of the quinoa, C. pallidicaule, and C. suecicum genomes, respectively. Evidence suggests that the B subgenome is more dynamic and has expanded more than the A subgenome. These genomic resources will enable more accurate assessments of genome evolution within the Amaranthaceae and will facilitate future efforts to identify variation in genes underlying important agronomic traits in quinoa.

Inter-professional education of prospective speech-language therapists and primary school teachers through shared professional practice placements.

BACKGROUND: Preliminary studies of inter-professional education (IPE) among student speech-language therapists (SLTs) and student teachers suggest that workshop-based applications are beneficial in preparing participants for elements of collaborative practice. Situating IPE within the students' professional practice placements may provide another useful avenue to develop attitudes, knowledge and skills for inter-professional collaboration. Research examining the impact of different approaches to IPE is required to advance our understanding of effective design and evaluation of such initiatives. AIMS: To understand how student SLTs and student teachers develop competency for collaborative practice when co-working during professional practice placements to support children's speech and literacy development. METHODS & PROCEDURES: A case study design was used to monitor the impact of the IPE. Student SLTs (n = 4) were paired with student teachers (n = 4) to participate in shared professional practice placements in junior school classrooms. An inductive thematic analysis of interviews conducted with participants after the IPE was employed to explore the development of competencies in collaborative practice. Change in inter-disciplinary knowledge and perceptions over the IPE was evaluated via survey to further explore the development of collaborative competencies. OUTCOMES & RESULTS: Integration of qualitative and quantitative findings suggested that participants began to develop four broad areas of collaborative competency: understanding of professional roles and expertise, communication skills to support shared decision-making, inter-dependency in supporting children's learning, and flexibility to implement alternative instructional practices. Interview analysis also revealed factors related to the facilitators and learning contexts that supported and/or limited the collaboration between participants. CONCLUSIONS & IMPLICATIONS: Shared placement experiences between student SLTs and student teachers may be an effective method for building participants' competencies in multiple aspects of collaborative practice. Active facilitation by both SLT and classroom teacher supervisors alongside careful consideration of learning contexts (e.g., classroom structure) will help to ensure that learning is maximized for prospective professionals.

Thioflavin T fluorescence in human serum: correlations with vascular health and cardiovascular risk factors.

OBJECTIVES: Amyloid fibrils and amyloid-like structures are implicated in atherosclerosis via macrophage activation and inflammation. A common property of amyloid-like structures is their ability to induce thioflavin T (ThT) fluorescence. We measured ThT fluorescence in serum and related these levels to traditional cardiovascular risk factors and non-invasive measures of vascular dysfunction (elasticity). In addition, chemically modified serum components that contribute to serum ThT fluorescence were explored and identified. DESIGN, METHODS, AND RESULTS: Sera from 105 people, including 35 healthy subjects, and 70 high cardiovascular risk patients (36 with rheumatoid arthritis and 34 with systemic lupus erythrematosus) showed an 8.75-fold variation in induced ThT fluorescence. Although mean (+/-SD) ThT fluorescence did not differ significantly between groups (controls 0.97+/-0.26, RA 1.12+/-0.45, and SLE 0.74+/-0.23), the combined data set showed significant inverse correlation (p=0.046) between ThT fluorescence tertiles and small artery elasticity. Correlation was also found between ThT fluorescence tertiles and LDL-cholesterol, total-cholesterol, and C-reactive protein. Floatation fractionation of apoB containing lipoproteins showed that ThT reactivity in this fraction correlated with both serum oxidised-LDL and LDL-cholesterol levels. However, approximately 94% of ThT reactivity in serum was associated with the non-apoB containing serum fraction, with the majority of ThT fluorescence associated with albumin. Incubation of purified albumin with glucose or with methylglyoxal induced ThT fluorescence, suggesting that glycated or chemical adducts of albumin contribute to the variation in ThT fluorescence of human serum. CONCLUSIONS: We propose that the detection of these adducts in serum using ThT fluorescence measurements may provide a marker for chemically modified protein structures that could assist the assessment of cardiovascular disease risk.

Apolipoprotein C-II amyloid fibrils assemble via a reversible pathway that includes fibril breaking and rejoining.

Alzheimer's and several other diseases are characterized by the misfolding and assembly of protein subunits into amyloid fibrils. Current models propose that amyloid fibril formation proceeds via the self-association of several monomers to form a nucleus, which then elongates by the addition of monomer to form mature fibrils. We have examined the concentration-dependent kinetics of apolipoprotein C-II amyloid fibril formation and correlated this with the final size distribution of the fibrils determined by sedimentation velocity experiments. In contrast to predictions of the nucleation-elongation model, the final size distribution of the fibrils was found to be relatively independent of the starting monomer concentration. To explain these results, we extended the nucleation-elongation model to include fibril breaking and rejoining as integral parts of the amyloid fibril assembly mechanism. The system was examined under conditions that affected the stability of the mature fibrils including the effect of dilution on the free pool of monomeric apolipoprotein C-II and the time-dependent recovery of fibril size following sonication. Antibody-labelling transmission electron microscopy studies provided direct evidence for spontaneous fibril breaking and rejoining. These studies establish the importance of breaking and rejoining in amyloid fibril formation and identify prospective new therapeutic targets in the assembly pathway.

Phospholipid interaction induces molecular-level polymorphism in apolipoprotein C-II amyloid fibrils via alternative assembly pathways.

A common feature of many of the most important and prominent amyloid-forming proteins is their ability to bind lipids and lipid complexes. Lipids are ubiquitous components of disease-associated amyloid plaques and deposits in humans, yet the specific roles of lipid in the process of amyloid fibril formation are poorly understood. This study investigated the effect of phospholipids on amyloid fibril formation by human apolipoprotein (apo) C-II using phosphatidylcholine derivatives comprising acyl chains of up to 14 carbon atoms. Submicellar concentrations of short-chain phospholipids increase the rate of apoC-II fibril formation in an acyl-chain-length- and concentration-dependent fashion, while high micellar concentrations of phospholipids completely inhibited amyloid formation. At lower concentrations of soluble phospholipid complexes, fibril formation by apoC-II was only partially inhibited, and under these conditions, aggregation followed a two-phase process. Electron microscopy showed that the fibrils resulting from the second phase of aggregation were straight, cablelike, and about 13 nm wide, in contrast to the homogeneous twisted-ribbon morphology of apoC-II fibrils formed under lipid-free conditions. Seeding experiments showed that this alternative fibril structure could be templated both in the presence and in the absence of lipid complex, suggesting that the two morphologies result from distinct assembly pathways. Circular dichroism spectroscopy studies indicated that the secondary structural conformation within the straight-type and ribbon-type fibrils were distinct, further suggesting divergent assembly pathways. These studies show that phospholipid complexes can change the structural architecture of mature fibrils and generate new fibril morphologies with the potential to alter the in vivo behaviour of amyloid. Such lipid interactions may play a role in defining the structural features of fibrils formed by diverse amyloidogenic proteins.

Oxidized cholesterol metabolites found in human atherosclerotic lesions promote apolipoprotein C-II amyloid fibril formation.

Apolipoprotein amyloid deposits and lipid oxidation products are colocalized in human atherosclerotic tissue. In this study we show that the primary ozonolysis product of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (KA), rapidly promotes human apolipoprotein (apo) C-II amyloid fibril formation in vitro. Previous studies show that hydrophobic aldehydes, including KA, modify proteins by the formation of a Schiff base with the lysine epsilon-amino group or N-terminal amino group. High-performance liquid chromatography, mass spectrometry, and proteolysis of KA-modified apoC-II revealed that KA randomly modified six different lysine residues, with primarily one KA attached per apoC-II molecule. Competition experiments showed that an aldehyde scavenging compound partially inhibited the ability of KA to hasten apoC-II fibril formation. Conversely, the acid derivative of KA, lacking the ability to form a Schiff base, accelerated apoC-II fibril formation, albeit to a lesser extent, suggesting that amyloidogenesis triggered by KA involves both covalent and noncovalent mechanisms. The viability of a noncovalent mechanism mediated by KA has been observed previously with alpha-synuclein aggregation, implicated in Parkinson's disease. Electron microscopy demonstrated that fibrils formed in the presence of KA had a similar morphology to native fibrils; however, the isolated KA-apoC-II covalent adducts in the absence of unmodified apoC-II formed fibrillar structures with altered ropelike morphologies. KA-mediated fibril formation by apoC-II was inhibited by the addition of the amine-containing compound hydralazine and the lipid-binding protein apoA-I. These in vitro studies suggest that the oxidized small molecule pool could trigger or hasten the aggregation of apoC-II to form amyloid deposits.

A structural core within apolipoprotein C-II amyloid fibrils identified using hydrogen exchange and proteolysis.

Plasma apolipoproteins show alpha-helical structure in the lipid-bound state and limited conformational stability in the absence of lipid. This structural instability of lipid-free apolipoproteins may account for the high propensity of apolipoproteins to aggregate and accumulate in disease-related amyloid deposits. Here, we explore the properties of amyloid fibrils formed by apolipoproteins using human apolipoprotein (apo) C-II as a model system. Hydrogen-deuterium exchange and NMR spectroscopy of apoC-II fibrils revealed core regions between residues 19-37 and 57-74 with reduced amide proton exchange rates compared to monomeric apoC-II. The C-terminal core region was also identified by partial proteolysis of apoC-II amyloid fibrils using endoproteinase GluC and proteinase K. Complete tryptic hydrolysis of apoC-II fibrils followed by centrifugation yielded a single peptide in the pellet fraction identified using mass spectrometry as apoC-II(56-76). Synthetic apoC-II(56-76) readily formed fibrils, albeit with a different morphology and thioflavinT fluorescence yield compared to full-length apoC-II. Studies with smaller peptides narrowed this fibril-forming core to a region within residues 60-70. We postulate that the ability of apoC-II(60-70) to independently form amyloid fibrils drives fibril formation by apoC-II. These specific amyloid-forming regions within apolipoproteins may underlie the propensity of apolipoproteins and their peptide derivatives to accumulate in amyloid deposits in vivo.

Pyruvate induces mitochondrial biogenesis by a PGC-1 alpha-independent mechanism.

Oxidative cells increase mitochondrial mass in response to stimuli such as changes in energy demand or cellular differentiation. This plasticity enables the cell to adapt dynamically to achieve the necessary oxidative capacity. However, the pathways involved in triggering mitochondrial biogenesis are poorly defined. The present study examines the impact of altering energy provision on mitochondrial biogenesis in muscle cells. C2C12 myoblasts were chronically treated with supraphysiological levels of sodium pyruvate for 72 h. Treated cells exhibited increased mitochondrial protein expression, basal respiratory rate, and maximal oxidative capacity. The increase in mitochondrial biogenesis was independent of increases in peroxisomal proliferator activator receptor-gamma coactivator-1alpha (PGC-1alpha) and PGC-1beta mRNA expression. To further assess whether PGC-1alpha expression was necessary for pyruvate action, cells were infected with adenovirus containing shRNA for PGC-1alpha before treatment with pyruvate. Despite a 70% reduction in PGC-1alpha mRNA, the effect of pyruvate was preserved. Furthermore, pyruvate induced mitochondrial biogenesis in primary myoblasts from PGC-1alpha null mice. These data suggest that regulation of mitochondrial biogenesis by pyruvate in myoblasts is independent of PGC-1alpha, suggesting the existence of a novel energy-sensing pathway regulating oxidative capacity.

High density lipoproteins bind Abeta and apolipoprotein C-II amyloid fibrils.

Disease-associated amyloid deposits contain both fibrillar and nonfibrillar components. The majority of these amyloid components originate or coexist in the bloodstream. To understand the nature of the interaction between the nonfibrillar and fibrillar components, we have developed a centrifugation method to isolate fibril binding proteins from human serum. Amyloid fibrils composed of either Abeta peptide or apolipoprotein C-II (apoC-II) cosedimented with specific serum proteins. Gel electrophoresis, mass spectrometry peptide fingerprinting, and Western analysis identified the major binding species as proteins found in HDL particles, including apoA-I, apoA-II, apoE, clusterin, and serum amyloid A. Sedimentation analysis showed that purified human HDL and recombinant apoA-I lipid particles bound directly to Abeta and apoC-II amyloid fibrils. These studies reveal a novel function of HDL that may contribute to the well-established protective effect of this lipoprotein class in heart disease.

The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans.

Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two-module non-ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co-regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.

A randomized clinical trial of a tailored comprehensive care treatment program for temporomandibular disorders.

AIMS: To test the usefulness of tailoring cognitive-behavioral therapy (CBT) for patients with temporomandibular disorders (TMD) who demonstrated poor psychosocial adaptation to their TMD condition, independent of physical diagnosis. METHODS: A randomized clinical trial compared a 6-session CBT intervention delivered in conjunction with the usual TMD treatment to the usual conservative treatment by TMD specialist dentists. For study inclusion, Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD), Axis II criteria, were used to target patients with elevated levels of TMD pain-related interference with daily activities, independent of physical diagnosis (i.e., Axis I). RESULTS: At the post-treatment assessment, about 4 months after the baseline evaluations, the comprehensive care group, when compared to the usual treatment group, showed significantly lower levels of characteristic pain intensity, significantly higher self-reported ability to control their TMD pain, and a strong trend (P = .07) toward lower pain-related interference in daily activities. From post-intervention to 1-year follow-up, all subjects showed improvement. At the 1-year follow-up, the comprehensive care group, while not losing any of its early gains, was not significantly different from the usual care group with regard to reported levels of pain, ability to control pain, and levels of interference in activities. For many of these psychosocially disabled TMD patients, pain and interference 1 year after treatment remained at the same or higher levels than those observed at baseline among a group of patients selected for a separate randomized clinical trial on the basis of better psychosocial adaptation. CONCLUSION: The 6-session CBT intervention for patients with heightened psychologic and psychosocial disability was effective in improving pain-related variables over the course of the CBT in conjunction with usual treatment, but was too brief an intervention to result in further improvement after the sessions ended. Patient ratings of treatment satisfaction and helfulness were high for both groups, but they were significantly higher for the comprehensive care group.

A randomized clinical trial using research diagnostic criteria for temporomandibular disorders-axis II to target clinic cases for a tailored self-care TMD treatment program.

AIMS: To carry out a randomized clinical trial (RCT) contrasting usual conservative treatment of TMD by clinical TMD specialists with a structured self-care intervention, targeted to clinic cases independent of TMD physical diagnosis, who were reporting minimal levels of psychosocial dysfunction; the intervention was delivered by dental hygienists in lieu of usual treatment. METHODS: The Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) was used to target subjects who exhibited minimal TMD-related psychosocial interference. Criteria for study inclusion were: (1) self-report of facial and/or masticatory muscle pain discomfort for which usual care was prescribed by the clinic TMD specialist; (2) RDC/TMD Axis II graded scale of chronic pain (GCP) score of 0, I, or II-Low. (3) Age 18 to 70 years. RESULTS: On 1-year follow-up, while both groups showed improvement in all clinical and self-report categories measured, patients in the tailored self-care treatment program compared to usual TMD treatment showed significantly; (a) decreased TMD pain, (b) decreased pain-related interference in activity; (c) reduced number of masticatory muscles painful; (d) fewer additional visits for TMD treatment. Groups were comparable with regard to measures of vertical range of motion. The self-care program was associated with consistent, but non-statistically significant, trends towards lower levels of depression and somatization. Ability to cope with TMD, knowledge concerning TMD and patient satisfaction was significantly enhanced for the self-care group. No participating patients experienced physical or personal adverse effects during the 1-year post-treatment follow-up period. CONCLUSION: Use of RDC/TMD psychosocial assessment criteria can contribute to successful clinical decision-making for the management of TMD.

Characterization of a gene (sp1) encoding a secreted protein from Leptosphaeria maculans, the blackleg pathogen of Brassica napus.

SUMMARY A gene (sp1) encoding a 12.3 kDa protein with a predicted secretion signal has been characterized from Leptosphaeria maculans, the dothideomycete that causes blackleg disease of canola (Brassica napus). This protein (SP1) contains four cysteine residues and shows a high sequence similarity to proteins from other ascomycetes. L. maculans sp1 has been placed on genetic and physical maps. This gene is expressed during the infection of B. napus cotyledons 10 days post-inoculation, coinciding with detection of the constitutively expressed fungal gene, beta-tubulin. L. maculans sp1, along with opsin and glyceraldehyde phosphate dehydrogenase, is light regulated. A recombinant SP1 protein expressed in Escherichia coli and a crude protein fraction secreted by L. maculans induced an autofluorescence response on B. napus leaves. The sp1 gene was mutated by targeted gene disruption whereby a hygromycin resistance gene was inserted. Such mutants caused similar-sized lesions on B. napus cotyledons as those caused by the wild-type isolate, indicating that sp1 is not crucial for pathogenicity of L. maculans on B. napus. This is the first report of disruption of this gene in any fungus.

Brief group cognitive-behavioral intervention for temporomandibular disorders.

Temporomandibular disorders (TMD) are currently viewed as an interrelated set of clinical conditions presenting with signs and symptoms in masticatory and related muscles of the head and neck, and the soft tissue and bony components of the temporomandibular joint. Epidemiologic and clinical studies of TMD confirm its status as a chronic pain problem. In this report we present results from a randomized clinical trial which compared, at 3- and 12-month follow-ups, the effects of usual TMD treatment on TMD pain and related physical and psychological variables with the effects of a cognitive-behavioral (CB) intervention delivered to small groups of patients before usual TMD treatment began. The purpose of this study was to determine whether a minimal CB intervention followed by dental TMD treatment enhanced the effects of usual clinical dental treatment. A second purpose of the study was to determine whether patients classified as high in somatization and psychosocial dysfunction would respond less favorably to this minimal intervention than would those low in somatization and dysfunction. Patients who participated in the CB intervention followed by usual treatment showed greater long-term decreases in reported pain level and pain interference in daily activities than did patients who received only usual treatment. The benefits of CB intervention were not seen when the CB and UT groups were compared at 3-month follow-up. During the 3-12-month follow-up interval, however, the UT group maintained essentially the same level of improvement in characteristic pain while the CB group continued to improve, as hypothesized. During this same follow-up interval, the CB group also showed a strong trend toward continued improvement in pain interference. Such effects were not observed for depression, somatization, or clinical measures of jaw range of motion. Additionally, as hypothesized, dysfunctional chronic pain patients did not appear to benefit from the brief CB intervention. Intent to treat analyses were also performed to assess generalizability of the results.

Somatization and pain dispersion in chronic temporomandibular disorder pain.

We investigated the relationship between somatic and psychological symptoms and pain reported during a clinical examination for 220 patients with chronic temporomandibular disorder (TMD) pain. The clinical examination involved palpation of the muscles of the face and neck, as well as intraoral sites and non-TMD-related placebo sites. A distinction was drawn between somatization--the tendency to report numerous somatic symptoms--and psychological distress manifested by report of numerous affective and cognitive symptoms. Somatization was assessed with the Somatization scale of the SCL-90-R; cognitive/affective distress was assessed with the non-somatic items of the Anxiety and Depression scales. Heightened somatization and high-intensity pain were strong predictors of widely dispersed muscle palpation pain during the clinical examination. High-somatization patients were 3 times more likely than low-somatization subjects to report having a painful placebo site. Pain dispersion was more closely linked to report of number of somatic symptoms than to report of affective and cognitive symptoms of psychological distress.

Effect of temporomandibular disorder pain duration on facial expressions and verbal report of pain.

This study investigated how specific expressive behaviors (verbal report of pain level and the frequency of emitting specific non-verbal facial expressions of pain) may change over the course of a chronic pain condition. Based on the concept of chronic pain behaviors, we hypothesized that both verbal and non-verbal behavior would increase with duration of pain. Thirty-six women with chronic temporomandibular disorder (TMD) pain (duration over 6 months) were compared with 35 recent onset cases (first episode, duration < or = 2 months). Subjects completed questionnaires assessing depression, anxiety, somatization, daily hassles and pain coping strategies. They were videotaped during a resting baseline and 2 painful conditions: experimental cold pressor pain and the clinically relevant pain of palpation of the masticatory muscles and temporomandibular joint; tapes were coded for facial expression using the Facial Action Coding System. Visual analog scale (VAS) ratings of the aversiveness and intensity of ongoing TMD pain were collected at baseline, and similar ratings of cold pressor and clinical examination pain were gathered after the painful stimulus. Recent onset and chronic cases did not differ on self-report measures of anxiety, depression, somatization or daily stress. Coping strategies were also similar, although chronic cases showed a greater tendency to catastrophize. Self-report measures of ambient facial pain, as well as the pain of clinical examination and cold pressor stimulation, revealed no significant differences between the 2 groups. In contrast, rates of pain facial expression were significantly higher for chronic cases under all conditions of the experiment, including baseline.(ABSTRACT TRUNCATED AT 250 WORDS)