Production, purification and characterization of recombinant, full-length human claudin-1

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-ß-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

Experimental and theoretical studies on a distributed temperature sensor based on Brillouin scattering

Results are reported from recent research on the use of the Brillouin gain/loss mechanism for distributed sensing. A theoretical model of the interaction of the pulsed and CW beams is described and compared with experiments. Results from a system with a 51 km sensing length are presented. We finally investigate issues related to the variation within the sensing fiber of the polarizations of the two beams.

Experimental and theoretical studies on a distributed temperature sensor based on Brillouin scattering

Results are reported from recent research on the use of the Brillouin gain/loss mechanism for distributed sensing. A theoretical model of the interaction of the pulsed and CW beams is described and compared with experiments. Results from a system with a 51 km sensing length are presented. We finally investigate issues related to the variation within the sensing fiber of the polarizations of the two beams.

Short-term stability in refractive status despite large fluctuations in glucose levels in diabetes mellitus type 1 and 2

Purpose: This work investigates how short-term changes in blood glucose concentration affect the refractive components of the diabetic eye in patients with long-term Type 1 and Type 2 diabetes. Methods: Blood glucose concentration, refractive error components (mean spherical equivalent MSE, J0, J45), central corneal thickness (CCT), anterior chamber depth (ACD), crystalline lens thickness (LT), axial length (AL) and ocular aberrations were monitored at two-hourly intervals over a 12-hour period in: 20 T1DM patients (mean age ± SD) 38±14 years, baseline HbA1c 8.6±1.9%; 21 T2DM patients (mean age ± SD) 56±11 years, HbA1c 7.5±1.8%; and in 20 control subjects (mean age ± SD) 49±23 years, HbA1c 5.5±0.5%. The refractive and biometric results were compared with the corresponding changes in blood glucose concentration. Results: Blood glucose concentration at different times was found to vary significantly within (p<0.0005) and between groups (p<0.0005). However, the refractive error components and ocular aberrations were not found to alter significantly over the day in either the diabetic patients or the control subjects (p>0.05). Minor changes of marginal statistical or optical significance were observed in some biometric parameters. Similarly there were some marginally significant differences between the baseline biometric parameters of well-controlled and poorly-controlled diabetic subjects. Conclusion: This work suggests that normal, short-term fluctuations (of up to about 6 mM/l on a timescale of a few hours) in the blood glucose levels of diabetics are not usually associated with acute changes in refractive error or ocular wavefront aberrations. It is therefore possible that factors other than refractive error fluctuations are sometimes responsible for the transient visual problems often reported by diabetic patients. © 2012 Huntjens et al.

Insights into the influence of solvent polarity on the crystallization of poly(ethylene oxide) spin-coated thin films via in situ grazing incidence wide-angle X-ray scattering

Controlling polymer thin-film morphology and crystallinity is crucial for a wide range of applications, particularly in thin-film organic electronic devices. In this work, the crystallization behavior of a model polymer, poly(ethylene oxide) (PEO), during spin-coating is studied. PEO films were spun-cast from solvents possessing different polarities (chloroform, THF, and methanol) and probed via in situ grazing incidence wide-angle X-ray scattering. The crystallization behavior was found to follow the solvent polarity order (where chloroform < THF < methanol) rather than the solubility order (where THF > chloroform > methanol). When spun-cast from nonpolar chloroform, crystallization largely followed Avrami kinetics, resulting in the formation of morphologies comprising large spherulites. PEO solutions cast from more polar solvents (THF and methanol) do not form well-defined highly crystalline morphologies and are largely amorphous with the presence of small crystalline regions. The difference in morphological development of PEO spun-cast from polar solvents is attributed to clustering phenomena that inhibit polymer crystallization. This work highlights the importance of considering individual components of polymer solubility, rather than simple total solubility, when designing processing routes for the generation of morphologies with optimum crystallinities or morphologies.

Raman fiber lasers with a random distributed feedback based on Rayleigh scattering

We demonstrate lasing based on a random distributed feedback due to the Raman amplified Rayleigh backscattering in different types of cavities with and without conventional point-action reflectors. Quasistationary generation of a narrowband spectrum is achieved despite the random nature of the feedback. The generated spectrum is localized at the reflection or gain spectral maxima in schemes with and without point reflectors, respectively. The length limit for a conventional cavity and the minimal pump power required for the lasing based purely on a random distributed feedback are determined. © 2010 The American Physical Society.

Measurement of single spin asymmetry and fifth structure function for the p( e&ar; ,e'K +)Λ reaction with CEBAF Large Acceptance Spectrometer (CLAS)

The single spin asymmetry, ALT ′, and the polarized structure function, σ LT′, for the p( e&ar; , e′K +)Λ reaction in the resonance region have been measured and extracted using the CEBAF Large Acceptance Spectrometer (CLAS) at Jefferson Lab. Data were taken at an electron beam energy of 2.567 GeV. The large acceptance of CLAS allows for full azimuthal angle coverage over a large range of center-of-mass scattering angles. Results were obtained that span a range in Q 2 from 0.5 to 1.3 GeV2 and W from threshold up to 2.1 GeV and were compared to existing theoretical calculations. The polarized structure function is sensitive to the interferences between various resonant amplitudes, as well as to resonant and non-resonant amplitudes. This measurement is essential for understanding the structure of nucleons and searching for previously undetected nucleon excited states (resonances) predicted by quark models. The W dependence of the σ LT′ in the kinematic regions dominated by s and u channel exchange (cos qcmk = −0.50, −0.167, 0.167) indicated possible resonance structures not predicted by theoretical calculations. The σLT ′ behavior around W = 1.875 GeV could be the signature of a resonance predicted by the quark models and possibly seen in photoproduction. In the very forward angles where the reaction is dominated by the t-channel, the average σLT ′ was zero. There was no indication of the interference between resonances or resonant and non-resonant amplitudes. This might be indicating the dominance of a single t-channel exchange. Study of the sensitivity of the fifth structure function data to the resonance around 1900 MeV showed that these data were highly sensitive to the various assumptions of the models for the quantum number of this resonance. This project was part of a larger CLAS program to measure cross sections and polarization observables for kaon electroproduction in the nucleon resonance region. ^

Coherent rho meson electroproduction off deuteron

QCD predicts Color Transparency (CT), which refers to nuclear medium becoming transparent to a small color neutral object produced in high momentum transfer reactions, due to reduced strong interaction. Despite several studies at BNL, SLAC, FNAL, DESY and Jefferson Lab, a definitive signal for CT still remains elusive. In this dissertation, we present the results of a new study at Jefferson Lab motivated by theoretical calculations that suggest fully exclusive measurement of coherent rho meson electroproduction off the deuteron is a favorable channel for studying CT. Vector meson production has a large cross section at high energies, and the deuteron is the best understood and simplest nuclear system. Exclusivity allows the production and propagation to be controlled separately by controlling Q 2, lf (formation length), lc (coherence length) and t. This control is important as the rapid expansion of small objects increases their interaction probability and masks CT. The CT signal is investigated in a ratio of cross sections at high t (where re-scattering is significant) to low t (where single nucleon reactions dominate). The results are presented over a Q2 range of 1 to 3 GeV2 based on the data taken with beam energy of 6 GeV.

Risk -taking propensity of faculty, administrators, and academic department heads at a large, urban, multi -campus community college

This research was undertaken to explore dimensions of the risk construct, identify factors related to risk-taking in education, and study risk propensity among employees at a community college. Risk-taking propensity (RTP) was measured by the 12-item BCDQ, which consisted of personal and professional risk-related situations balanced for the money, reputation, and satisfaction dimensions of the risk construct. Scoring ranged from 1.00 (most cautious) to 6.00 (most risky).^ Surveys including the BCDQ and seven demographic questions relating to age, gender, professional status, length of service, academic discipline, highest degree, and campus location were sent to faculty, administrators, and academic department heads. A total of 325 surveys were returned, resulting in a 66.7% response rate. Subjects were relatively homogeneous for age, length of service, and highest degree.^ Subjects were also homogeneous for risk-taking propensity: no substantive differences in RTP scores were noted within and among demographic groups, with the possible exception of academic discipline. The mean RTP score for all subjects was 3.77, for faculty was 3.76, for administrators was 3.83, and for department heads was 3.64.^ The relationship between propensity to take personal risks and propensity to take professional risks was tested by computing Pearson r correlation coefficients. The relationships for the total sample, faculty, and administrator groups were statistically significant, but of limited practical significance. Subjects were placed into risk categories by dividing the response scale into thirds. A 3 x 3 factorial ANOVA revealed no interaction effects between professional status and risk category with regard to RTP score. A discriminant analysis showed that a seven-factor model was not effective in predicting risk category.^ The homogeneity of the study sample and the effect of a risk-encouraging environment were discussed in the context of the community college. Since very little data on risk-taking in education is available, risk propensity data from this study could serve as a basis for comparison to future research. Results could be used by institutions to plan professional development activities, designed to increase risk-taking and encourage active acceptance of change. ^

Development of effective approaches to the large-scale aerodynamic testing of low-rise buildings

Low-rise buildings are often subjected to high wind loads during hurricanes that lead to severe damage and cause water intrusion. It is therefore important to estimate accurate wind pressures for design purposes to reduce losses. Wind loads on low-rise buildings can differ significantly depending upon the laboratory in which they were measured. The differences are due in large part to inadequate simulations of the low-frequency content of atmospheric velocity fluctuations in the laboratory and to the small scale of the models used for the measurements. A new partial turbulence simulation methodology was developed for simulating the effect of low-frequency flow fluctuations on low-rise buildings more effectively from the point of view of testing accuracy and repeatability than is currently the case. The methodology was validated by comparing aerodynamic pressure data for building models obtained in the open-jet 12-Fan Wall of Wind (WOW) facility against their counterparts in a boundary-layer wind tunnel. Field measurements of pressures on Texas Tech University building and Silsoe building were also used for validation purposes. The tests in partial simulation are freed of integral length scale constraints, meaning that model length scales in such testing are only limited by blockage considerations. Thus the partial simulation methodology can be used to produce aerodynamic data for low-rise buildings by using large-scale models in wind tunnels and WOW-like facilities. This is a major advantage, because large-scale models allow for accurate modeling of architectural details, testing at higher Reynolds number, using greater spatial resolution of the pressure taps in high pressure zones, and assessing the performance of aerodynamic devices to reduce wind effects. The technique eliminates a major cause of discrepancies among measurements conducted in different laboratories and can help to standardize flow simulations for testing residential homes as well as significantly improving testing accuracy and repeatability. Partial turbulence simulation was used in the WOW to determine the performance of discontinuous perforated parapets in mitigating roof pressures. The comparisons of pressures with and without parapets showed significant reductions in pressure coefficients in the zones with high suctions. This demonstrated the potential of such aerodynamic add-on devices to reduce uplift forces.

Proton Form Factor Puzzle and the CEBAF Large Acceptance Spectrometer (CLAS) two-photon exchange experiment

The electromagnetic form factors are the most fundamental observables that encode information about the internal structure of the nucleon. The electric (GE) and the magnetic ( GM) form factors contain information about the spatial distribution of the charge and magnetization inside the nucleon. A significant discrepancy exists between the Rosenbluth and the polarization transfer measurements of the electromagnetic form factors of the proton. One possible explanation for the discrepancy is the contributions of two-photon exchange (TPE) effects. Theoretical calculations estimating the magnitude of the TPE effect are highly model dependent, and limited experimental evidence for such effects exists. Experimentally, the TPE effect can be measured by comparing the ratio of positron-proton elastic scattering cross section to that of the electron-proton [R = σ(e +p)/σ(e+p)]. The ratio R was measured over a wide range of kinematics, utilizing a 5.6 GeV primary electron beam produced by the Continuous Electron Beam Accelerator Facility (CEBAF) at Jefferson Lab. This dissertation explored dependence of R on kinematic variables such as squared four-momentum transfer (Q2) and the virtual photon polarization parameter (&epsis;). A mixed electron-positron beam was produced from the primary electron beam in experimental Hall B. The mixed beam was scattered from a liquid hydrogen (LH2) target. Both the scattered lepton and the recoil proton were detected by the CEBAF Large Acceptance Spectrometer (CLAS). The elastic events were then identified by using elastic scattering kinematics. This work extracted the Q2 dependence of R at high &epsis;(&epsis; > 0.8) and the $&epsis; dependence of R at ⟨Q 2⟩ approx 0.85 GeV2. In these kinematics, our data confirm the validity of the hadronic calculations of the TPE effect by Blunden, Melnitchouk, and Tjon. This hadronic TPE effect, with additional corrections contributed by higher excitations of the intermediate state nucleon, largely reconciles the Rosenbluth and the polarization transfer measurements of the electromagnetic form factors.

Nutrient cycling in Alaskan tundra in response to experimental manipulation of growing season length and soil temperature : a climate change scenario

Climate change in the Arctic is predicted to increase plant productivity through decomposition-related enhanced nutrient availability. However, the extent of the increase will depend on whether the increased nutrient availability can be sustained. To address this uncertainty, I assessed the response of plant tissue nutrients, litter decomposition rates, and soil nutrient availability to experimental climate warming manipulations, extended growing season and soil warming, over a 7 year period. Overall, the most consistent effect was the year-to-year variability in measured parameters, probably a result of large differences in weather and time of snowmelt. The results of this study emphasize that although plants of arctic environments are specifically adapted to low nutrient availability, they also posses a suite of traits that help to reduce nutrient losses such as slow growth, low tissue concentrations, and low tissue turnover that result in subtle responses to environmental changes.

Weak electron scattering for the 3He-3H transition and the weak nuclear form factors

We calculate the differential cross section for weak electron scattering reaction, e + 3He-' 3H + ve, for energies from 100 MeV to 6 GeV as a function of outgoing nucleus angle from 0 to n/2 radians. We find that the differential cross section at low [q2] increases with electron energy from 0.1 GeV to 6.0 GeV, such that the peak value at 6.0 GeV is approximately 3.2 x 10-40 cm 2 / ster, a factor of 10 larger than the peak value at 0.1 GeV. We also find that the width of the peak falls very rapidly with increasing electron energy. At high [q2] we find that the differential cross section falls by approximately three orders of magnitude making experimental observation at this time unlikely. The contributions of the individual form factors are obtained for electron energies of 0.5GeV and 2.0 GeV. It is found that at low [q2] the form factors, FA(q2) and Fv(q2), make contributions of similar size to the differential cross section and might be simultaneously determined , but for the case of FM(q2) we find that the contribution is too small to determine. It is also found that at large [q2] values, the contribution of FM(q2) is substantially enhanced , but that the cross section is probably too small to enable a direct determination of FM(q2).

Quantitative Comparison of Plant Community Hydrology Using Large-Extent, Long-Term Data

Large-extent vegetation datasets that co-occur with long-term hydrology data provide new ways to develop biologically meaningful hydrologic variables and to determine plant community responses to hydrology. We analyzed the suitability of different hydrological variables to predict vegetation in two water conservation areas (WCAs) in the Florida Everglades, USA, and developed metrics to define realized hydrologic optima and tolerances. Using vegetation data spatially co-located with long-term hydrological records, we evaluated seven variables describing water depth, hydroperiod length, and number of wet/dry events; each variable was tested for 2-, 4- and 10-year intervals for Julian annual averages and environmentally-defined hydrologic intervals. Maximum length and maximum water depth during the wet period calculated for environmentally-defined hydrologic intervals over a 4-year period were the best predictors of vegetation type. Proportional abundance of vegetation types along hydrological gradients indicated that communities had different realized optima and tolerances across WCAs. Although in both WCAs, the trees/shrubs class was on the drier/shallower end of hydrological gradients, while slough communities occupied the wetter/deeper end, the distribution ofCladium, Typha, wet prairie and Salix communities, which were intermediate for most hydrological variables, varied in proportional abundance along hydrologic gradients between WCAs, indicating that realized optima and tolerances are context-dependent.

The relationship between Common Language and Length of Treatment in Occupational Therapy

The diversity of ethnic and cultural groups and the effects of language in the therapeutic relationship are timely professional issues of concern to occupational therapy practitioners. The tri-ethnic, tri-cultural South Florida area offers a natural environment where one can study how patient-therapist interactions are influenced by language barriers in a diverse society. This study examines the effects of language on the adequacy of occupational therapy services, specifically how language affects the length of the treatment program. The nature of diagnosis therapists' ethnicity, and how they impact treatment outcomes are also addressed. A sample was drawn from the occupational therapy outpatient department of a large county hospital. Data taken from patients' charts examined race, sex, age, diagnosis, and language. Number of treatment sessions and length of treatment were viewed as proxy measures for adequacy. Findings indicate that the effect of language cannot be understood aside from ethnicity. Implications for occupational therapy practice are discussed.