Single-molecule force spectroscopy of membrane proteins from membranes freely spanning across nanoscopic pores.

Single-molecule force spectroscopy (SMFS) provides detailed insight into the mechanical (un)folding pathways and structural stability of membrane proteins. So far, SMFS could only be applied to membrane proteins embedded in native or synthetic membranes adsorbed to solid supports. This adsorption causes experimental limitations and raises the question to what extent the support influences the results obtained by SMFS. Therefore, we introduce here SMFS from native purple membrane freely spanning across nanopores. We show that correct analysis of the SMFS data requires extending the worm-like chain model, which describes the mechanical stretching of a polypeptide, by the cubic extension model, which describes the bending of a purple membrane exposed to mechanical stress. This new experimental and theoretical approach allows to characterize the stepwise (un)folding of the membrane protein bacteriorhodopsin and to assign the stability of single and grouped secondary structures. The (un)folding and stability of bacteriorhodopsin shows no significant difference between freely spanning and directly supported purple membranes. Importantly, the novel experimental SMFS setup opens an avenue to characterize any protein from freely spanning cellular or synthetic membranes.

High-resolution atomic force microscopy imaging of rhodopsin in rod outer segment disk membranes.

Atomic force microscopy (AFM) is a powerful imaging technique that allows recording topographical information of membrane proteins under near-physiological conditions. Remarkable results have been obtained on membrane proteins that were reconstituted into lipid bilayers. High-resolution AFM imaging of native disk membranes from vertebrate rod outer segments has unveiled the higher-order oligomeric state of the G protein-coupled receptor rhodopsin, which is highly expressed in disk membranes. Based on AFM imaging, it has been demonstrated that rhodopsin assembles in rows of dimers and paracrystals and that the rhodopsin dimer is the fundamental building block of higher-order structures.

Diagnosis and therapy of retained fetal membranes, puerperal metritis and clinical endometritis in cattle: Results of the Online-survey among Swiss practitioners. II. Puerperal metritis and clinical endometritis

The aim of the study was to obtain the diagnostic and therapeutic approach among Swiss practitioners in cows with puerperal metritis and clinical endometritis (part 2). All members of the Association for ruminant health were contacted per email via the newsletter. The survey was completed by 128 veterinarians, partially responded by 140 veterinarians. The following main symptoms of puerperal metritis were stated by the practitioners: purulent vaginal discharge, fever and reduced appetite. A vaginal and rectal examination was performed to diagnose the disease. Usually, an intrauterine treatment with tetracycline or cefapirin was done. Parenteral administration of tetracycline or penicillin was often combined with PGF(2α), NSAIDS or cortisone. Clinical endometritis was also diagnosed by vaginal and rectal examination and the main symptom indicated was purulent vaginal discharge. The therapy consisted of the administration of PGF(2α), uterine infusions predominantly with cefapirin, and rarely with parenteral administration of antibiotics. Further diagnostic tools were not used and normally cows were not rechecked. The success of the therapy of puerperal metritis and clinical endometritis was judged to be satisfactory to excellent.

Diagnosis and therapy of retained fetal membranes, puerperal metritis and clinical endometritis in cattle: Results of the Online-survey among Swiss practitioners. I Retained fetal membranes

The aim of this study was to obtain the diagnostic and therapeutic approach among Swiss practitioners in cows with retained fetal membranes (RFM) (part 1). All members of the Association for ruminant health were contacted per email via the newsletter. The survey was completed by 128 veterinarians, partially responded by 140 veterinarians. The manual removal of the fetal membranes is practiced by 129 of the responding veterinarians. Cows with/without fever are treated usually with intrauterine antibiotics. Cows with RFM with/without fever are most commonly treated parenterally with tetracycline or penicillin. The use of cephalosporins and quinolones in cows with fever is more common than in cows without fever. With the present results of the survey veterinarians should critically question the supposed benefits of the manual removal of the placenta and the use of antibiotics in cows with RFM.

Flagellar membranes are rich in raft-forming phospholipids.

The observation that the membranes of flagella are enriched in sterols and sphingolipids has led to the hypothesis that flagella might be enriched in raft-forming lipids. However, a detailed lipidomic analysis of flagellar membranes is not available. Novel protocols to detach and isolate intact flagella from Trypanosoma brucei procyclic forms in combination with reverse-phase liquid chromatography high-resolution tandem mass spectrometry allowed us to determine the phospholipid composition of flagellar membranes relative to whole cells. Our analyses revealed that phosphatidylethanolamine, phosphatidylserine, ceramide and the sphingolipids inositol phosphorylceramide and sphingomyelin are enriched in flagella relative to whole cells. In contrast, phosphatidylcholine and phosphatidylinositol are strongly depleted in flagella. Within individual glycerophospholipid classes, we observed a preference for ether-type over diacyl-type molecular species in membranes of flagella. Our study provides direct evidence for a preferential presence of raft-forming phospholipids in flagellar membranes of T. brucei.

Cessation of facial growth in subjects with short, average, and long facial types - Implications for the timing of implant placement

Placement of a single-tooth implant should be performed when a patient's facial growth has ceased. In this retrospective observational study, we evaluated if there was a difference in the timing of cessation of craniofacial growth in short, average, and long facial types. Based on the value of the angle between cranial base and mandibular plane (SN/MP angle), three groups comprising 48 subjects with short facial type (SF; SN/MP ≤28°), 77 with average facial type (AF; SN/MP ≥31.5° and ≤34.5°), and 44 with long facial type (LF; SN/MP ≥38°) were selected. Facial growth was assessed on lateral cephalograms taken at 15.4 years of age, and 2, 5, and 10 years later. Variables were considered to be stable when the difference between two successive measurements was less than 1 mm or 1°. We found no difference between facial types in the timing of cessation of facial growth. Depending on the variable, the mean age when variables became stable ranged from 18.0 years (Is-Pal in LF group) to 22.0 years (SN/MP in LF group). However, facial growth continued at the last follow-up in approximately 20% subjects. This study demonstrates that facial type is not associated with the timing of cessation of facial growth.

Nasolabial symmetry and esthetics in cleft lip and palate: analysis of 3D facial images

OBJECTIVES To determine the relationship between nasolabial symmetry and esthetics in subjects with orofacial clefts. MATERIAL AND METHODS Eighty-four subjects (mean age 10 years, standard deviation 1.5) with various types of nonsyndromic clefts were included: 11 had unilateral cleft lip (UCL); 30 had unilateral cleft lip and alveolus (UCLA); and 43 had unilateral cleft lip, alveolus, and palate (UCLAP). A 3D stereophotogrammetric image of the face was taken for each subject. Symmetry and esthetics were evaluated on cropped 3D facial images. The degree of asymmetry of the nasolabial area was calculated based on all 3D data points using a surface registration algorithm. Esthetic ratings of various elements of nasal morphology were performed by eight lay raters on a 100 mm visual analog scale. Statistical analysis included ANOVA tests and regression models. RESULTS Nasolabial asymmetry increased with growing severity of the cleft (p = 0.029). Overall, nasolabial appearance was affected by nasolabial asymmetry; subjects with more nasolabial asymmetry were judged as having a less esthetically pleasing nasolabial area (p < 0.001). However, the relationship between nasolabial symmetry and esthetics was relatively weak in subjects with UCLAP, in whom only vermilion border esthetics was associated with asymmetry. CONCLUSIONS Nasolabial symmetry assessed with 3D facial imaging can be used as an objective measure of treatment outcome in subjects with less severe cleft deformity. In subjects with more severe cleft types, other factors may play a decisive role. CLINICAL SIGNIFICANCE Assessment of nasolabial symmetry is a useful measure of treatment success in less severe cleft types.

Regional facial asymmetries in unilateral orofacial clefts

OBJECTIVES Assess facial asymmetry in subjects with unilateral cleft lip (UCL), unilateral cleft lip and alveolus (UCLA), and unilateral cleft lip, alveolus, and palate (UCLP), and to evaluate which area of the face is most asymmetrical. METHODS Standardized three-dimensional facial images of 58 patients (9 UCL, 21 UCLA, and 28 UCLP; age range: 8.6-12.3 years) and 121 controls (age range 9-12 years) were mirrored and distance maps were created. Absolute mean asymmetry values were calculated for the whole face, cheek, nose, lips, and chin. One-way analysis of variance, Kruskal-Wallis, and t-test were used to assess the differences between clefts and controls for the whole face and separate areas. RESULTS Clefts and controls differ significantly for the whole face as well as in all areas. Asymmetry is distributed differently over the face for all groups. In UCLA, the nose was significantly more asymmetric compared with chin and cheek (P = 0.038 and 0.024, respectively). For UCL, significant differences in asymmetry between nose and chin and chin and cheek were present (P = 0.038 and 0.046, respectively). In the control group, the chin was the most asymmetric area compared to lip and nose (P = 0.002 and P = 0.001, respectively) followed by the nose (P = 0.004). In UCLP, the nose, followed by the lips, was the most asymmetric area compared to chin, cheek (P < 0.001 and P = 0.016, respectively). LIMITATIONS Despite division into regional areas, the method may still exclude or underrate smaller local areas in the face, which are better visualized in a facial colour coded distance map than quantified by distance numbers. The UCL subsample is small. CONCLUSION Each type of cleft has its own distinct asymmetry pattern. Children with unilateral clefts show more facial asymmetry than children without clefts.

Oral-facial-digital syndrome type VI: is C5orf42 really the major gene?

Oral-facial-digital type VI syndrome (OFDVI) is a rare phenotype of Joubert syndrome (JS). Recently, C5orf42 was suggested as the major OFDVI gene, being mutated in 9 of 11 families (82 %). We sequenced C5orf42 in 313 JS probands and identified mutations in 28 (8.9 %), most with a phenotype of pure JS. Only 2 out of 17 OFDVI patients (11.7 %) were mutated. A comparison of mutated vs. non-mutated OFDVI patients showed that preaxial and mesoaxial polydactyly, hypothalamic hamartoma and other congenital defects may predict C5orf42 mutations, while tongue hamartomas are more common in negative patients.

Oxygen Supplementing, Biocompatible Outer Membranes for Enhanced Performance of Implantable Glucose Sensors

Lack of linearity and sensitivity, oxygen dependence, biofouling and tissue inflammation hinder the development of implantable biosensors for continuous monitoring of glucose. Herein, we report the development of stacked outer membranes based on LBL/PVA hydrogels that improve sensor sensitivity, linearity, oxygen independence and counter biofouling and inflammation. While the inner LBL membrane affords tunable diffusivity, the outer PVA is capable of releasing anti-inflammatory drugs/tissue response modifying agents to counter acute and chronic inflammation, and to induce neo-angiogenesis at the implant site. Sensors were fabricated by immobilizing GOx enzyme on top of 50 μm platinum wires, followed by deposition of stacked LBL/PVA hydrogel membranes. The response of the sensors at 0.7V to various glucose concentrations was studied. Michelis-Menten analysis was performed to quantify sensor performance in terms of linearity and oxygen dependence. The interplay between sensor performance and inward glucose diffusivity was elucidated using (i) various LBL membranes and (ii) various freeze-thaw (FT) cycles of PVA. Incorporation of LBL/PVA stacked membranes resulted in an 8 fold increase in sensor linearity and a 9 fold decrease in oxygen dependence compared to controls. The enhancement in the sensor performance is attributed to (i) the oxygen storing capability of PVA hydrogel due to the formation of hydrophobic domains during its freezing/ thawing employed for its physical crosslinking and (ii) regulation of glucose flux by the inner LBL membrane. Such membranes offer significant advantages over presently available outer membranes in lieu of (i) their ability to control inflammation, (ii) their modulus that closely matches that of subcutaneous human tissue, (iii) non-necessity of reactive chemical crosslinking agents, (iv) tunable sensitivity and (v) supplemental storage of oxygen.

Relationship of Turfgrass Growth and Quality to Soil Nitrate Desorbed from Anion Exchange Membranes

Anion exchange membranes (AEMs) are a potential method for determining the plant available N status of soils; however, their capacity for use with turfgrass has not been researched extensively. The main objective of this experiment was to determine the relationship between soil nitrate desorbed from AEMs and growth response and quality of turfgrass managed as a residential lawn. Two field experiments were conducted with a bluegrass-ryegrass-fescue mixture receiving four rates of N fertilizer (0, 98, 196, and 392 kg N ha(-1) yr(-1)) with clippings returned or removed. The soils at the two sites were a Paxton fine sandy loam (coarse-loamy, mixed, active, mesic Oxyaquic Dystrudepts) and a variant of a Hinckley gravelly sandy loam (sandy-skeletal, mixed, mesic Typic Udorthents). Anion exchange membranes were inserted into plots and exchanged weekly during the growing seasons of 1998 and 1999. Nitrate-N was desorbed from AEMs and quantified. As N fertilization rates increased, desorbed NO3-N increased. The relationship of desorbed NO3-N from AEMs to clipping yield and turfgrass quality was characterized using quadratic response plateau (QRP) and Cate-Nelson models (C-Ns). Critical levels of desorbed NO3-N ranged from 0.86 to 8.0 microgram cm(-2) d(-1) for relative dry matter yield (DMY) and from 2.3 to 12 microgram cm(-2) d(-1) for turfgrass quality depending upon experimental treatment. Anion exchange membranes show promise of indicating the critical levels of soil NO3-N desorbed from AEMs necessary to achieve maximum turfgrass quality and yield without overapplication of N.

Relationships between soil nitrate desorbed from anion-exchange membranes, canopy reflectance and nitrate leaching losses from cool-season lawn turf.

Nutrient leaching studies are expensive and require expertise in water collection and analyses. Less expensive or easier methods that estimate leaching losses would be desirable. The objective of this study was to determine if anion-exchange membranes (AEMs) and reflectance meters could predict nitrate (NO3-N) leaching losses from a cool-season lawn turf. A two-year field study used an established 90% Kentucky bluegrass (Poa pratensis L.)-10% creeping red fescue (Festuca rubra L.) turf that received 0 to 98 kg N ha-1 month-1, from May through November. Soil monolith lysimeters collected leachate that was analyzed for NO3-N concentration. Soil NO3-N was estimated with AEMs. Spectral reflectance measurements of the turf were obtained with chlorophyll and chroma meters. No significant (p > 0.05) increase in percolate flow-weighted NO3-N concentration (FWC) or mass loss occurred when AEM desorbed soil NO3-N was below 0.84 µg cm-2 d-1. A linear increase in FWC and mass loss (p < 0.0001) occurred, however, when AEM soil NO3-N was above this value. The maximum contaminant level (MCL) for drinking water (10 mg L-1 NO3-N) was reached with an AEM soil NO3-N value of 1.6 µg cm-2 d-1. Maximum meter readings were obtained when AEM soil NO3 N reached or exceeded 2.3 µg cm-2 d-1. As chlorophyll index and hue angle (greenness) increased, there was an increased probability of exceeding the NO3-N MCL. These data suggest that AEMs and reflectance meters can serve as tools to predict NO3-N leaching losses from cool-season lawn turf, and to provide objective guides for N fertilization.

Evaluation of a 3-Dimensional Measurement System: A Method for Assessing Facial Asymmetry

Submitted in partial fulfillment of the requirements for a Certificate in Orthodontics, Dept. of Orthodontics, University of Connecticut Health Center, 1992