Membrane Structure of Aquaporin Observed with Combined Experimental and Theoretical Sum Frequency Generation Spectroscopy.

High-resolution structural information on membrane proteins is essential for understanding cell biology and for the structure-based design of new medical drugs and drug delivery strategies. X-ray diffraction (XRD) can provide angstrom-level information about the structure of membrane proteins, yet for XRD experiments, proteins are removed from their native membrane environment, chemically stabilized, and crystallized, all of which can compromise the conformation. Here, we describe how a combination of surface-sensitive vibrational spectroscopy and molecular dynamics simulations can account for the native membrane environment. We observe the structure of a glycerol facilitator channel (GlpF), an aquaporin membrane channel finely tuned to selectively transport water and glycerol molecules across the membrane barrier. We find subtle but significant differences between the XRD structure and the inferred in situ structure of GlpF.

Interaction of Amyloid-ß-(1-42) Peptide and Its Aggregates with Lipid/Water Interfaces Probed by Vibrational Sum-Frequency Generation Spectroscopy.

In this study, we use surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy to investigate the interaction between model lipid monolayers and Aß(1-42) in its monomeric and aggregated states. Combining VSFG with atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence measurements, we found that only small aggregates with probably a ß-hairpin-like structure adsorbed to the zwitterionic lipid monolayer (DOPC). In contrast, larger aggregates with an extended ß-sheet structure adsorbed to a negatively charged lipid monolayer (DOPG). The adsorption of small, initially formed aggregates strongly destabilized both monolayers, but only the DOPC monolayer was completely disrupted. We showed that the intensity of the amide-II' band in achiral (SSP) and chiral (SPP) polarization combinations increased in time when Aß(1-42) aggregates accumulated at the DOPG monolayer. Nevertheless, almost no adsorption of preformed mature fibrils to DOPG monolayers was detected. By performing spectral VSFG calculations, we revealed a clear correlation between the amide-II' signal and the degree of amyloid aggregates (e.g., oligomers or (proto)fibrils) of various Aß(1-42) structures. The calculations showed that only structures with a significant amyloid ß-sheet content have a strong amide-II' intensity, in line with previous Raman studies. The combination of the presented results substantiates the amide-II(') band as a legitimate amyloid marker.

Gram-negative bacterial toe web infection - a systematic review.

Gram-negative bacterial toe web infection (GNBTWI) is a frequent therapeutic challenge in clinical practice with high recurrence rates and frequent need of systemic drugs. The aim of this systematic review was to provide an updated overview and evidence-based data on pathogens, risk factors and treatment of GNBTWI along with promoting a consistent international terminology. This systematic review is based on a search in PubMed database for English and German articles published between 1980 and 2016. A total of seven articles were considered appropriate for inclusion in this review regarding to treatment and outcome. Throughout the medical literature, a variety of terms for bacterial toe web infections is used. Only few data on the incidence of GNBTWI were published. GNBTWI has been shown to have a significant male predominance. Pseudomonas aeruginosa is the most commonly identified organism beside a high mixed infection rate. We identified the following predisposing factors: interdigital tinea, occlusion and humidity, history of self-medication with antifungals, antibiotics and glucocorticosteroids. As for treatment, debridement of macerated skin lesions and the hyperkeratotic rim showed good response in three published cases. Bacteriological workup of swabs including an antibiogram is recommended for identification of the proper topical and systemic therapy. Autosensitization dermatitis and frequent recurrences are common complications of GNBTWI. Despite the fact, that GNBTWI is an accepted disease entity, scarce data on GNBTWI exist in the medical literature. Randomized controlled trials are missing although needed for evidence-based therapy. To facilitate communication and exchange of updates of GNBTWI, we promote the suggested terminology for bacterial toe web diseases.

The structure of insulin at the air/water interface: monomers or dimers?

The hydrophobic character of the air/water interface affects the oligomeric composition of insulin. By using interface-specific vibrational sum frequency spectroscopy and calculations of insulin monomer and dimer second-order nonlinear susceptibilities χ((2)), we show that insulin monomers segregate to the air/water interface.

Formation of lysozyme oligomers at model cell membranes monitored with sum frequency generation spectroscopy.

A growing number of studies suggest that the formation of toxic oligomers, precursors of amyloid fibrils, is initiated at the cell membrane and not in the cytosolic compartments of the cell. Studies of membrane-induced protein oligomerization are challenging due to the difficulties of probing small numbers of proteins present at membrane surfaces. Here, we employ surface-sensitive vibrational sum frequency generation (VSFG) to investigate the secondary structure of lysozyme at the surface of lipid monolayers. We investigate lysozyme aggregation at negatively charged 1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-1-glycerol) (DPPG) lipid monolayers under different pH conditions. The changes in the molecular vibrations of lipids, proteins, and water as a function of pH and surface pressure allow us to simultaneously monitor details of the conformation state of lysozyme, the organization of lipids, and the state of lipid-bound water. At pH = 6 lysozyme induces significant disordering of the lipid layer, and it exists in two states: a monomeric state with a predominantly α-helix content and an oligomeric (za-mer) state. At pH ≤ 3, all membrane-bound lysozyme self-associates into oligomers characterized by an antiparallel ß-sheet structure. This is different from the situation in bulk solution, for which circular dichroism (CD) shows that the protein maintains an α-helix conformation, under both neutral and acidic pH conditions. The transition from monomers to oligomers is also associated with a decreased hydration of the lipid monolayer resulting in an increase of the lipid acyl chains ordering. The results indicate that oligomerization requires cooperative action between lysozyme incorporated into the lipid membrane and peripherally adsorbed lysozyme and is associated with the membrane dehydration and lipid reorganization. Membrane-bound oligomers with antiparallel ß-sheet structure are found to destabilize lipid membranes.

Probing Albumin Adsorption onto Calcium Phosphates by XPS and ToF-SIMS.

In this study the binding and assembly of bovine serum albumin (BSA) onto three different calcium phosphate phases (hydroxyapatite, dibasic calcium phosphate dihydrate, and ß-tricalcium phosphate) was investigated using a combination of X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). XPS was used to record adsorption isotherms and to quantify the amount of BSA adsorbed onto the different CaP surfaces. On all three surfaces a monolayer of adsorbed BSA was formed. ToF-SIMS was then used to investigate how the structure of BSA changes upon surface binding. ToF-SIMS data from BSA films on the three CaP surfaces showed intensity differences of secondary ions originating from both hydrophobic and hydrophilic amino acids. For a more quantitative examination of structural changes, we developed a ratio comparing the sum of intensities of secondary ions from hydrophobic and hydrophilic residues. A small, but statistically significant, increase in the value of this ratio (7%) was observed between a BSA film on hydroxyapatite versus dibasic calcium phosphate dihydrate. From this ratio we can make some initial hypotheses about what specific changes in BSA structure relate to these differences observed in the ToF-SIMS data.

Multi-technique Characterization of Adsorbed Peptide and Protein Orientation: LK310 and Protein G B1.

The ability to orient biologically active proteins on surfaces is a major challenge in the design, construction, and successful deployment of many medical technologies. As methods to orient biomolecules are developed, it is also essential to develop techniques that can an accurately determine the orientation and structure of these materials. In this study, two model protein and peptide systems are presented to highlight the strengths of three surface analysis techniques for characterizing protein films: time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum-frequency generation (SFG) vibrational spectroscopy, and near-edge x-ray absorption fine structure (NEXAFS) spectroscopy. First, the orientation of Protein G B1, a rigid 6 kDa domain covalently attached to a maleimide-functionalized self-assembled monolayer, was examined using ToF-SIMS. Although the thickness of the Protein G layer was similar to the ToF-SIMS sampling depth, orientation of Protein G was successfully determined by analyzing the C2H5S+ intensity, a secondary ion derived from a methionine residue located at one end of the protein. Next, the secondary structure of a 13-mer leucine-lysine peptide (LK310) adsorbed onto hydrophilic quartz and hydrophobic fluorocarbon surfaces was examined. SFG spectra indicated that the peptide's lysine side chains were ordered on the quartz surface, while the peptide's leucine side chains were ordered on the fluorocarbon surface. NEXAFS results provided complementary information about the structure of the LK310 film and the orientations of amide bonds within the LK310 peptide.

Amine Terminated SAMs: Investigating Why Oxygen is Present in these Films.

Self-assembled monolayers (SAMs) on gold prepared from amine-terminated alkanethiols have long been employed as model positively charged surfaces. Yet in previous studies significant amounts of unexpected oxygen containing species are always detected in amine terminated SAMs. Thus, the goal of this investigation was to determine the source of these oxygen species and minimize their presence in the SAM. The surface composition, structure, and order of amine-terminated SAMs on Au were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), sum frequency generation (SFG) and near edge X-ray absorption fine structure (NEXAFS) spectroscopy. XPS determined compositions of amine-terminated SAMs in the current study exhibited oxygen concentrations of 2.4 ± 0.4 atomic %, a substantially lower amount of oxygen than reported in previously published studies. High-resolution XPS results from the S(2p), C(1s) and N(1s) regions did not detect any oxidized species. Angle-resolved XPS indicated that the small amount of oxygen detected was located at or near the amine head group. Small amounts of oxidized nitrogen, carbon and sulfur secondary ions, as well as ions attributed to water, were detected in the ToF-SIMS data due to the higher sensitivity of ToF-SIMS. The lack of N-O, S-O, and C-O stretches in the SFG spectra are consistent with the XPS and ToF-SIMS results and together show that oxidation of the amine-terminated thiols alone can only account for, at most, a small fraction of the oxygen detected by XPS. Both the SFG and angle-dependent NEXAFS indicated the presence of gauche defects in the amine SAMs. However, the SFG spectral features near 2865 cm(-1), assigned to the stretch of the methylene group next to the terminal amine unit, demonstrate the SAM is reasonably ordered. The SFG results also show another broad feature near 3200 cm(-1) related to hydrogen-bonded water. From this multi-technique investigation it is clear that the majority of the oxygen detected within these amine-terminated SAMs arises from the presence of oxygen containing adsorbates such as tightly bound water.

Effect of exercise on upper respiratory tract infection in sedentary subjects.

OBJECTIVE: To determine if exercise training affects the severity and duration of a naturally acquired upper respiratory tract infection (URTI) in sedentary subjects. METHODS: Subjects were sedentary volunteers (two or fewer days a week of exercise for less than 30 minutes a day for the previous three months), 18-29 years of age, with a naturally acquired URTI (three to four days of onset). All subjects were screened-for example, asthma, hay fever-by a doctor and were afebrile. Volunteers were alternately assigned to an exercise (EX) group (four men, seven women) or a non-exercise (NEX) group (three men, eight women). Subjects in the EX group completed 30 minutes of supervised exercise at 70% of target heart rate range for five days of a seven day period. For the initial screening, and every 12 hours, all subjects completed a 13 item symptom severity checklist and a physical activity log. Cold symptom scores were obtained until the subjects were asymptomatic. Significance was set at p

Cloning and functional expression of the D-beta-hydroxybutyrate dehydrogenase gene of Rhodobacter sp. DSMZ 12077.

Nucleotide sequence and biochemical analysis of D-beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30), isolated from Rhodobacter sp., indicate functional oligomers composed of subunits of 257 amino acids with a calculated M(r) of 26,800 and a pI of 5.90. Compared to mammalian short-chain alcohol dehydrogenases, the bacterial enzyme lacks a C-terminal lipid anchor domain and was found to be highly active upon expression in Escherichia coli even without lipid supplement. The recombinant enzyme could be highly enriched using a single chromatography step and was shown to be stable over a broad range of pH and temperature.

The effect of exercise training on the severity and duration of a viral upper respiratory illness.

PURPOSE: The purpose of this investigation was to determine whether exercise training affects the severity and duration of a rhinovirus-caused upper respiratory illness (URI). METHODS: Subjects who were rhinovirus 16 (RV 16) antibody-free completed a graded exercise test. Thirty-four individuals (ages 18-29 yr) of moderate fitness (32 mL.kg-1.min-1 to 60 mL.kg-1.min-1) were randomly assigned to the exercise group (EX) while 16 additional individuals of similar age and fitness served as a nonexercise (NEX) control group. All EX and NEX subjects were inoculated with RV 16 on 2 consecutive days. EX subjects completed 40 min of supervised exercise every other day at 70% of heart rate (HR) reserve for a 10-d period. Every 12 h, all subjects completed a 13-item symptom severity checklist and a physical activity log. Used facial tissues were collected and weighed (symptom severity measure) during these same reporting periods. RESULTS: A two group by nine measure (2 x 9) repeated measures ANOVA procedure showed no difference in symptom questionnaire mean scores and the mucous weights of the EX and NEX groups for days 2-10 of the experiment. A two measure by five measure (2 x 5) repeated measures ANOVA procedure indicated no differences between the pre- and post-exercise questionnaire means for the five sessions that EX subjects exercised. Statistical significance was set at P < 0.05. CONCLUSION: These results suggest that moderate exercise training during a rhinovirus-caused URI under the conditions of this study design do not alter the severity and duration of the illness.

Effect of a rhinovirus-caused upper respiratory illness on pulmonary function test and exercise responses.

Upper respiratory illness (URI) may cause more frequent acute disability among athletes than all other diseases combined. The purposes of this study were to determine the impact of a rhinovirus-caused URI on resting pulmonary function submaximal exercise responses and on maximal exercise functional capacity. Twenty-four men and 21 women (18-29 yr) of varying fitness levels were assigned to the experimental group (URI), and 10 additional individuals served as a control group (CRL). An initial serological screening was performed on all URI group subjects to exclude those with the rhinovirus 16 (HRV16) antibody. All subjects completed both a baseline pulmonary function test and a graded exercise test to volitional fatigue. URI subjects were inoculated with HRV 16 on two consecutive days within 10 d of completing these tests. The day following the second inoculation (peak of illness), post-inoculation pulmonary function and graded exercise tests were performed. A noninfected control group completed these same pulmonary and exercise tests 1 wk apart. ANOVA identified no significant differences (P < 0.05) at minutes 2, 5, and 8 for the physiological responses measured between the pre- and post-exercise tests for both the URI and CRL, groups. Furthermore, there were no significant differences between maximal exercise performance between running trials for either group. There was also no significant interaction between treatment (pre/post URI) and group for any of the pulmonary function measures obtained. In conclusion, physiological responses to pulmonary function testing and submaximal and maximal exercise do not appear to be altered by an URI.

Effects of viral upper respiratory illness on running gait.

OBJECTIVE: To determine the kinematic changes that may occur during running with a cold of known etiology and to assess the impact of select accompanying upper respiratory illness symptoms. DESIGN AND SETTING: In this nonrandomized study, subjects with colds and subjects without colds were videotaped while exercising on a treadmill. Three weeks later, the trials were repeated. SUBJECTS: Eighteen young adults (5 females, 13 males; mean age = 20.4+/- 2.4 yr) with naturally acquired moderate to severe (total symptom score) colds were screened and selected for inclusion in the illness group (ILL). A control group (CRL) of 20 subjects (2 females, 18 males) was also examined. Virologic confirmation of specific viral infections, unprecedented in this line of research, revealed that 12 of the 18 subjects in the ILL group (67%) were infected with human rhinoviruses. None of the subjects had a fever. MEASUREMENTS: All subjects exercised on a treadmill for 5 minutes at a heart rate of approximately 85% of their age-predicted maximum. Both groups were videotaped kinematically during two running trials 3 weeks apart. All subjects in the ILL group displayed upper respiratory illness symptoms for the first running trial and were asymptomatic by the second. RESULTS: We identified significant differences in mean changes between the ILL and CRL group stride lengths (p <.01), stride frequencies (p <.05), and ankle maximum angle displacement (p <.01). Mean changes in stride length (p <.03) and in stride frequency (p <.04) were larger for ILL subjects who felt feverish. CONCLUSIONS: Alterations in running gait during a rhinovirus-caused upper respiratory illness, and possibly increases in injury incidence, may be associated with feeling feverish. Gait alterations may increase injury incidence or decrease athletic performance, or both.

Sport, exercise, and the common cold.

Upper respiratory illness may cause more disability among athletes than all other diseases combined. This paper presents the essential epidemiology, risks of infection, and transmission features of upper respiratory illness. Those who provide health care for athletes must understand the subsequent implications of an upper respiratory illness on sport performance and should be familiar with participation and clinical management guidelines for athletes with an upper respiratory illness. The literature suggests that regular, rigorous exercise increases both the incidence and severity of upper respiratory illness, yet the immune system appears to have a distinct level at which moderate exercise promotes optimum health. Although research indicates that upper respiratory illness infections are surprisingly reluctant transmitters, upper respiratory illness transmission may escalate during winter sports seasons. The impact of upper respiratory illness on selected pulmonary, cardiac, and skeletal muscle functions may lead to illness complications in athletes, and sport performance during illness may also decline. Athletes should monitor symptoms, adjust training schedules, and rest during an upper respiratory illness.

Athletic training continuing education needs assessment: pilot study.

Continuing education for certified athletic trainers is both required and essential. The purpose of this study was to determine the need, including solutions and priorities, for continuing education for athletic trainers in various employment settings. Focus group sessions were conducted during the Spring 1992 District 4 meeting of the National Athletic Trainers Association. Representatives for each of the following employment settings were selected from the preregistration list and invited to participate: high school (7 participated), college/university (8), corporate/industrial (6), professional (3), and clinical (8). Data were compiled from written lists and audio cassette recordings of group discussions. Focus group participants across all employment settings felt that their primary continuing education needs were not being addressed at district meetings. Less traditional topics (eg, ergonomics, budgeting, public relations, and functional capacity evaluations) were identified as the more essential education needs. Focus group opinions varied somewhat regarding specific topics and methods of presentation. Recommendations included a more thematic approach to topics and presentations and ample opportunities for work or discussions in small groups. Results of the focus groups could impact the professional preparation of athletic trainers.

Reporting behaviors and activity levels of intercollegiate athletes with an URI.

Some contend that upper respiratory illness (URI) causes more disability among athletes than all other diseases combined. The purpose of this study was to describe the reporting behaviors, activity levels, and perceived physical performance levels of intercollegiate athletes with an URI. Respondents rated the severity of 14 cold symptoms and indicated to whom they reported their cold, and within how many days. Additionally, respondents were to indicate whether they self-treated their illness, whether they missed a practice or game due to the cold, and whether the cold affected their performance. The subjects for this study were 290 intercollegiate athletes (165 males, 125 females) in a large Midwestern conference. The instrument was administered at three regular intervals during the sport seasons. There was a total of 118 illness episodes. Respondents reported their colds to the athletic trainer (50.4%, N = 61), doctor/nurse (33%, N = 41), and/or coach (28.3%, N = 32). Athletes reported their cold symptoms earliest to the coach (2.45 d) or athletic trainer (2.71 d). The number of days prior to reporting to a doctor or nurse was longer (3.64 d). Of the illness episodes reported, 17.8% (N = 21) caused the athlete to miss a practice and 5.1% (N = 6) caused the athlete to miss a game. Athletes with an URI felt in some instances that their illness episode affected their performance. Cold symptoms of cough, fever, laryngitis, aching muscles/joints, and nasal discharge were significantly (P < 0.05) correlated with reporting behaviors, activity levels, and/or perceived physical performance. Possible implications for athlete participation and education are offered.

Literature review: upper respiratory illness and sport and exercise.

Upper respiratory illness (URI) may cause more acute disability among athletes than all other diseases combined. This paper presents the essential epidemiological, infectious, and transmission features of URI. Those who provide health care for athletes must also understand the subsequent implications of URI on sport and exercise participation. While there are reports suggesting that regular, vigorous exercise reduces both the incidence and severity of URI, other research suggests that the immune system may have a distinct level of moderate exercise for optimum health. During winter sports season, URI transmission may escalate. The impact of URI on selected pulmonary, cardiac and skeletal muscle functions may lead to illness complications and protracted courses of URI in athletes. Sport performance during illness may also decline. Athletes should monitor symptoms and training schedules and should rest during a URI.

Evaluation of professional preparation in athletic training by employed, entry-level athletic trainers.

The purpose of this study was to determine the perceived adequacy of professional preparation in athletic training among employed, entry-level certified athletic trainers. Data were gathered by means of a self-reporting questionnaire designed specifically for the study. There were 277 questionnaires mailed, and 183 entry-level certified athletic trainers (66%) responded to the survey. Demographic and employment characteristics of the respondents were examined. Respondents rated their perceptions of adequacy of professional preparation and growth in several academic and clinical task areas. Entry-level athletic trainers felt more prepared in the areas of prevention of athletic injuries/illnesses, evaluation and recognition of athletic injuries/illnesses, and first aid/emergency care. They felt less prepared in the areas of rehabilitation and reconditioning, organization and administration of athletic training programs, counseling and guidance of athletes, and education of athletes. Many entry-level certified athletic trainers were not strongly impressed with the adequacy of the number of clinical hours required or the areas of clinical experiences encountered during their professional preparation programs. Respondents felt that their mentors/certified athletic trainers could have provided more leadership, guidance, and evaluation. According to this study, professional preparation programs in athletic training are adequately preparing entry-level professionals, but certain task areas need increased emphasis.

Gramicidin-D-stimulated influx of monovalent cations into plant roots.

Gramicidin D and nigericin were found to stimulate K(+) influx into oat roots. Valinomycin and nonactin had little effect on K(+) influx. The region of the root most sensitive to gramicidin was the elongation zone. Monocot roots were more sensitive to gramicidin than dicot roots. At 0.2 mM KCl, gramicidin stimulated K(+) influx by 4- to 8fold over a 30-min absorption period. Although a gramicidin response is detectable within one minute, maximum stimulation occurred after a slight (approximately 2-min) lag period. The gramicidin effect was much greater at 0.2 mM KCl than at 20 mM KCl. Respiratory inhibitors reduced the gramicidin-stimulated K(+) influx by 50-80%. The results are discussed in terms of possible mechanisms of action of the various ionophores on ion transport in roots.