Validation of the Fibromyalgia Survey Questionnaire within a cross-sectional survey

The Fibromyalgia Survey Questionnaire (FSQ) assesses the key symptoms of fibromyalgia syndrome. The FSQ can be administrated in survey research and settings where the use of interviews to evaluate the number of pain sites and extent of somatic symptom intensity and tender point examination would be difficult. We validated the FSQ in a cross-sectional survey with FMS patients. In a cross-sectional survey, participants with physician diagnosis of FMS were recruited by FMS-self help organisations and nine clinical institutions of different levels of care. Participants answered the FSQ (composed by the Widespread Pain Index [WPI] and the Somatic Severity Score [SSS]) assessing the Fibromyalgia Survey Diagnostic Criteria (FSDC) and the Patient Health Questionnaire PHQ 4. American College of Rheumatology 1990 classification criteria were assessed in a subgroup of participants. 1,651 persons diagnosed with FMS were included into analysis. The acceptance of the FSQ-items ranged between 78.9 to 98.1% completed items. The internal consistency of the items of the SSS ranged between 0.75-0.82. 85.5% of the study participants met the FSDC. The concordance rate of the FSDC and ACR 1990 criteria was 72.7% in a subsample of 128 patients. The Pearson correlation of the SSS with the PHQ 4 depression score was 0.52 (p<0.0001) and with the PHQ anxiety score was 0.51 (p<0.0001) (convergent validity). 64/202 (31.7%) of the participants not meeting the FSDC criteria and 152/1283 (11.8%) of the participants meeting the FSDC criteria reported an improvement (slightly too very much better) in their health status since FMS-diagnosis (Chi(2)?=?55, p<0.0001) (discriminant validity). The study demonstrated the feasibility of the FSQ in a cross-sectional survey with FMS-patients. The reliability, convergent and discriminant validity of the FSQ were good. Further validation studies of the FSQ in clinical and general population settings are necessary.     

Medication Adherence in the General Population

Background

Adherence to medication is low in specific populations who need chronic medication. However, adherence to medication is also of interest in a more general fashion, independent of specific populations or side effects of particular drugs. If clinicians and researchers expect patients to show close to full adherence, it is relevant to know how likely the achievement of this goal is. Population based rates can provide an estimate of efforts needed to achieve near complete adherence in patient populations. The objective of the study was to collect normative data for medication nonadherence in the general population.

Methods and Findings

We assessed 2,512 persons (a representative sample of German population). Adherence was measured by Rief Adherence Index. We also assessed current medication intake and side effects. We found that at least 33% of Germans repeatedly fail to follow their doctor''s recommendations regarding pharmacological treatments and only 25% of Germans describe themselves as fully adherent. Nonadherence to medication occurs more often in younger patients with higher socioeconomic status taking short-term medications than in older patients with chronic conditions. Experience with medication side effects was the most prominent predictor of nonadherence.

Conclusions

The major strengths of our study are a representative sample and a novel approach to assess adherence. Nonadherece seems to be commonplace in the general population. Therefore adherence cannot be expected per se but needs special efforts on behalf of prescribers and public health initiatives. Nonadherence to medication should not only be considered as a drug-specific behaviour problem, but as a behaviour pattern that is independent of the prescribed medication.     

Characterization of HLA class II/peptide-TCR interactions of the immunodominant T cell epitope in Art v 1, the major mugwort pollen allergen

More than 95% of mugwort pollen-allergic individuals are sensitized to Art v 1, the major allergen in mugwort pollen. Interestingly, the CD4 T cell response to Art v 1 involves only one single immunodominant peptide, Art v 1(25-36) (KCIEWEKAQHGA), and is highly associated with the expression of HLA-DR1. Therefore, we investigated the molecular basis of this unusual immunodominance among allergens. Using artificial APC expressing exclusively HLA-DRB1*0101 and HLA-DRA*0101, we formally showed that DR1 acts as restriction element for Art v 1(25-36)-specific T cell responses. Further assessment of binding of Art v 1(25-36) to artificial HLA-DR molecules revealed that its affinity was high for HLA-DR1. Amino acid I27 was identified as anchor residue interacting with DR molecules in pocket P1. Additionally, Art v 1(25-36) bound with high affinity to HLA-DRB1*0301 and *0401, moderately to HLA-DRB1*1301 and HLA-DRB5*0101, and weakly to HLA-DRB1*1101 and *1501. T cell activation was also inducible by Art v 1(25-36)-loaded, APC-expressing HLA molecules other than DR1, indicating degeneracy of peptide binding and promiscuity of TCR recognition. Specific binding of HLA-DRB1*0101 tetramers containing Art v 1(19-36) allowed the identification of Art v 1(25-36)-specific T cells by flow cytometry. In summary, the immunodominance of Art v 1(25-36) relies on its affinity to DR1, but is not dictated by it. Future investigations at the molecular HLA/peptide/TCR and cellular level using mugwort pollen allergy as a disease model may allow new insights into tolerance and pathomechanisms operative in type I allergy, which may instigate new, T cell-directed strategies in specific immunotherapy.     

Projection structure of yidC: a conserved mediator of membrane protein assembly

Bacteria, mitochondria and chloroplasts harbour factors that facilitate the insertion, folding and assembly of membrane proteins. In Escherichia coli, yidC is required for membrane insertion, acting in both a Sec-dependent and a Sec-independent manner. There is an expanding volume of biochemical work on its role in this process, but none so far on its structure. We present the first of this class of membrane proteins determined by electron cryomicroscopy in the near-nativelike state of the membrane. yidC forms dimers in the membrane and each monomer has an area of low density that may be part of the path transmembrane segments follow during their insertion. Upon consideration of the structures of yidC and SecYEG, we speculate on the nature of the interfaces that facilitate the alternative pathways (Sec-dependent and -independent) of membrane protein insertion.     

Evaluation of the effects of dietary particle fractions on fermentation profile and concentration of microbiota in the rumen of dairy cows fed grass silage-based diets

The study evaluated the effects of three different theoretical particle lengths (TPL) of grass silage on the distribution of particle fractions of the diet and the resulting effects on fermentation profile and concentrations of protozoa and mixed bacterial mass in the rumen of three lactating Holstein cows fed total mixed rations (45% grass silage, 5% grass hay and 50% concentrate) ad libitum. Decreasing TPL of grass silage (long, medium, short) reduced particles retained on the 19-mm sieve of the Penn State Particle Separator, while particle fractions from 8 mm to 19 mm and smaller than 8 mm were increased. Different TPL did not affect pH and the concentration of volatile fatty acids in the rumen. However, lowering the TPL from long to medium increased significantly the bicarbonate concentration, acetate proportion and protozoal number in the rumen, whereas the proportion of bacterial protein in ruminal digesta and its amino acid concentration were significantly increased by the short TPL. For the current feeding conditions, it can be concluded that increasing the fraction of particles between 8 and 19 mm and probably even the fraction below 8 mm by decreasing TPL of grass silage do not adversely affect rumen conditions and can be beneficial in terms of optimising concentration and activity of ruminal microbiota in high-yielding dairy cows.     

SAM domain-based protein oligomerization observed by live-cell fluorescence fluctuation spectroscopy

Sterile-alpha-motif (SAM) domains are common protein interaction motifs observed in organisms as diverse as yeast and human. They play a role in protein homo- and hetero-interactions in processes ranging from signal transduction to RNA binding. In addition, mutations in SAM domain and SAM-mediated oligomers have been linked to several diseases. To date, the observation of heterogeneous SAM-mediated oligomers in vivo has been elusive, which represents a common challenge in dissecting cellular biochemistry in live-cell systems. In this study, we report the oligomerization and binding stoichiometry of high-order, multi-component complexes of (SAM) domain proteins Ste11 and Ste50 in live yeast cells using fluorescence fluctuation methods. Fluorescence cross-correlation spectroscopy (FCCS) and 1-dimensional photon counting histogram (1dPCH) confirm the SAM-mediated interaction and oligomerization of Ste11 and Ste50. Two-dimensional PCH (2dPCH), with endogenously expressed proteins tagged with GFP or mCherry, uniquely indicates that Ste11 and Ste50 form a heterogeneous complex in the yeast cytosol comprised of a dimer of Ste11 and a monomer of Ste50. In addition, Ste50 also exists as a high order oligomer that does not interact with Ste11, and the size of this oligomer decreases in response to signals that activate the MAP kinase cascade. Surprisingly, a SAM domain mutant of Ste50 disrupted not only the Ste50 oligomers but also Ste11 dimerization. These results establish an in vivo model of Ste50 and Ste11 homo- and hetero-oligomerization and highlight the usefulness of 2dPCH for quantitative dissection of complex molecular interactions in genetic model organisms such as yeast.     

Peptide specificity and lipid activation of the lysosomal transport complex ABCB9 (TAPL)

The lysosomal ABC transporter associated with antigen processing-like (TAPL, ABCB9) acts as an ATP-dependent polypeptide transporter with broad length selectivity. To characterize in detail its substrate specificity, a procedure for functional reconstitution of human TAPL was developed. By intensive screening of detergents, ideal solubilization conditions were evolved with respect to efficiency, long term stability, and functionality of TAPL. TAPL was isolated in a two-step procedure with high purity and, subsequently, reconstituted into proteoliposomes. The peptide transport activity of reconstituted TAPL strongly depends on the lipid composition. With the help of combinatorial peptide libraries, the key positions of the peptides were localized to the N- and C-terminal residues with respect to peptide transport. At both ends, TAPL favors positively charged, aromatic, or hydrophobic residues and disfavors negatively charged residues as well as asparagine and methionine. Besides specific interactions of both terminal residues, electrostatic interactions are important, since peptides with positive net charge are more efficiently transported than negatively charged ones.     

Comparative analysis of meiotic progression in female mice bearing mutations in genes of the DNA mismatch repair pathway

The DNA mismatch repair (MMR) family functions in a variety of contexts to preserve genome integrity in most eukaryotes. In particular, members of the MMR family are involved in the process of meiotic recombination in germ cells. MMR gene mutations in mice result in meiotic disruption during prophase I, but the extent of this disruption often differs between male and female meiocytes. To address the role of MMR proteins specifically in female meiosis, we explored the progression of oocytes through prophase I and the meiotic divisions in mice harboring deletions in members of the MMR pathway (Mlh1, Mlh3, Exo1, and an ATPase-deficient variant of Mlh1, Mlh1(G67R)). The colocalization of MLH1 and MLH3, key proteins involved in stabilization of nascent crossovers, was dependent on intact heterodimer formation and was highly correlated with the ability of oocytes to progress through to metaphase II. The exception was Exo1(-/-) oocytes, in which normal MLH1/MLH3 localization was observed followed by failure to proceed to metaphase II. All mutant oocytes were able to resume meiosis after dictyate arrest, but they showed a dramatic decline in chiasmata (to less than 25% of normal), accompanied by varied progression through metaphase I. Taken together, these results demonstrate that MMR function is required for the formation and stabilization of crossovers in mammalian oocytes and that, in the absence of a functional MMR system, the failure to maintain chiasmata results in a reduced ability to proceed normally through the first and second meiotic divisions, despite near-normal levels of meiotic resumption after dictyate arrest.     

Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes

Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.