Conformational changes couple Na+ and glucose transport

The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na+/glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na+ and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady–state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in fluorescence closely followed the presteady–state charge movement. We conclude that conformational changes are responsible for the coupling of Na+ and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.

Allelic variation of a dehydrin gene cosegregates with chilling tolerance during seedling emergence

Dehydrins (DHNs, LEA D-11) are plant proteins present during environmental stresses associated with dehydration or low temperatures and during seed maturation. Functions of DHNs have not yet been defined. Earlier, we hypothesized that a ≈35-kDa DHN and membrane properties that reduce electrolyte leakage from seeds confer chilling tolerance during seedling emergence of cowpea (Vigna unguiculata L. Walp.) in an additive and independent manner. Evidence for this hypothesis was not rigorous because it was based on correlations of presence/absence of the DHN and slow electrolyte leakage with chilling tolerance in closely related cowpea lines that have some other genetic differences. Here, we provide more compelling genetic evidence for involvement of the DHN in chilling tolerance of cowpea. We developed near-isogenic lines by backcrossing. We isolated and determined the sequence of a cDNA corresponding to the ≈35-kDa DHN and used gene-specific oligonucleotides derived from it to test the genetic linkage between the DHN presence/absence trait and the DHN structural gene. We tested for association between the DHN presence/absence trait and both low-temperature seed emergence and electrolyte leakage. We show that allelic differences in the Dhn structural gene map to the same position as the DHN protein presence/absence trait and that the presence of the ≈35-kDa DHN is indeed associated with chilling tolerance during seedling emergence, independent of electrolyte leakage effects. Two types of allelic variation in the Dhn gene were identified in the protein-coding region, deletion of one Φ-segment from the DHN-negative lines and two single amino acid substitutions.

Declining brain activity in cognitively normal apolipoprotein E ɛ4 heterozygotes: A foundation for using positron emission tomography to efficiently test treatments to prevent Alzheimer's disease

Cross-sectional positron emission tomography (PET) studies find that cognitively normal carriers of the apolipoprotein E (APOE) ɛ4 allele, a common Alzheimer's susceptibility gene, have abnormally low measurements of the cerebral metabolic rate for glucose (CMRgl) in the same regions as patients with Alzheimer's dementia. In this article, we characterize longitudinal CMRgl declines in cognitively normal ɛ4 heterozygotes, estimate the power of PET to test the efficacy of treatments to attenuate these declines in 2 years, and consider how this paradigm could be used to efficiently test the potential of candidate therapies for the prevention of Alzheimer's disease. We studied 10 cognitively normal ɛ4 heterozygotes and 15 ɛ4 noncarriers 50–63 years of age with a reported family history of Alzheimer's dementia before and after an interval of approximately 2 years. The ɛ4 heterozygotes had significant CMRgl declines in the vicinity of temporal, posterior cingulate, and prefrontal cortex, basal forebrain, parahippocampal gyrus, and thalamus, and these declines were significantly greater than those in the ɛ4 noncarriers. In testing candidate primary prevention therapies, we estimate that between 50 and 115 cognitively normal ɛ4 heterozygotes are needed per active and placebo treatment group to detect a 25% attenuation in these CMRgl declines with 80% power and P = 0.005 in 2 years. Assuming these CMRgl declines are related to the predisposition to Alzheimer's dementia, this study provides a paradigm for testing the potential of treatments to prevent the disorder without having to study thousands of research subjects or wait many years to determine whether or when treated individuals develop symptoms.

Oral tolerance in myelin basic protein T-cell receptor transgenic mice: suppression of autoimmune encephalomyelitis and dose-dependent induction of regulatory cells.

Orally administered antigens induce a state of immunologic hyporesponsiveness termed oral tolerance. Different mechanisms are involved in mediating oral tolerance depending on the dose fed. Low doses of antigen generate cytokine-secreting regulatory cells, whereas high doses induce anergy or deletion. We used mice transgenic for a T-cell receptor (TCR) derived from an encephalitogenic T-cell clone specific for the acetylated N-terminal peptide of myelin basic protein (MBP) Ac-1-11 plus I-Au to test whether a regulatory T cell could be generated from the same precursor cell as that of an encephalitogenic Th1 cell and whether the induction was dose dependent. The MBP TCR transgenic mice primarily have T cells of a precursor phenotype that produce interleukin 2 (IL-2) with little interferon gamma (IFN-gamma), IL-4, or transforming growth factor beta (TGF-beta). We fed transgenic animals a low-dose (1 mg x 5) or high-dose (25 mg x 1) regimen of mouse MBP and without further immunization spleen cells were tested for cytokine production. Low-dose feeding induced prominent secretion of IL-4, IL-10, and TGF-beta, whereas minimal secretion of these cytokines was observed with high-dose feeding. Little or no change was seen in proliferation or IL-2/IFN-gamma secretion in fed animals irrespective of the dose. To demonstrate in vivo functional activity of the cytokine-secreting cells generated by oral antigen, spleen cells from low-dose-fed animals were adoptively transferred into naive (PLJ x SJL)F1 mice that were then immunized for the development of experimental autoimmune encephalomyelitis (EAE). Marked suppression of EAE was observed when T cells were transferred from MBP-fed transgenic animals but not from animals that were not fed. In contrast to oral tolerization, s.c. immunization of transgenic animals with MBP in complete Freund's adjuvant induced IFN-gamma-secreting Th1 cells in vitro and experimental encephalomyelitis in vivo. Despite the large number of cells reactive to MBP in the transgenic animals, EAE was also suppressed by low-dose feeding of MBP prior to immunization. These results demonstrate that MBP-specific T cells can differentiate in vivo into encephalitogenic or regulatory T cells depending upon the context by which they are exposed to antigen.

Functional anatomy of human eyeblink conditioning determined with regional cerebral glucose metabolism and positron-emission tomography.

Relative cerebral glucose metabolism was examined with positron-emission tomography (PET) as a measure of neuronal activation during performance of the classically conditioned eyeblink response in 12 young adult subjects. Each subject received three sessions: (i) a control session with PET scan in which unpaired presentations of the tone conditioned stimulus and corneal airpuff unconditioned stimulus were administered, (ii) a paired training session to allow associative learning to occur, and (iii) a paired test session with PET scan. Brain regions exhibiting learning-related activation were identified as those areas that showed significant differences in glucose metabolism between the unpaired control condition and well-trained state in the 9 subjects who met the learning criterion. Areas showing significant activation included bilateral sites in the inferior cerebellar cortex/deep nuclei, anterior cerebellar vermis, contralateral cerebellar cortex and pontine tegmentum, ipsilateral inferior thalamus/red nucleus, ipsilateral hippocampal formation, ipsilateral lateral temporal cortex, and bilateral ventral striatum. Among all subjects, including those who did not meet the learning criterion, metabolic changes in ipsilateral cerebellar nuclei, bilateral cerebellar cortex, anterior vermis, contralateral pontine tegmentum, ipsilateral hippocampal formation, and bilateral striatum correlated with degree of learning. The localization to cerebellum and its associated brainstem circuitry is consistent with neurobiological studies in the rabbit model of eyeblink classical conditioning and neuropsychological studies in brain-damaged humans. In addition, these data support a role for the hippocampus in conditioning and suggest that the ventral striatum may also be involved.

The association of feeding behavior with the resistance and tolerance to parasites in recently diverged sticklebacks

Divergent natural selection regimes can contribute to adaptive population divergence, but can be sensitive to human-mediated environmental change. Nutrient loading of aquatic ecosystems, for example, might modify selection pressures by altering the abundance and distribution of resources and the prevalence and infectivity of parasites. Here, we used a mesocosm experiment to test for interactive effects of nutrient loading and parasitism on host condition and feeding ecology. Specifically, we investigated whether the common fish parasite Gyrodactylus sp. differentially affected recently diverged lake and stream ecotypes of three-spined stickleback (Gasterosteus aculeatus). We found that the stream ecotype had a higher resistance to Gyrodactylus sp. infections than the lake ecotype, and that both ecotypes experienced a cost of parasitism, indicated by negative relationships between parasite load and both stomach fullness and body condition. Overall, our results suggest that in the early stages of adaptive population divergence of hosts, parasites can affect host resistance, body condition, and diet.

Development and field test of psychophysical tests for DWI arrest. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

Verification of test procedures for rulemaking - motorcycle headgear. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

The Effect of Fructose vs. Glucose Beverages on Low-Grade Systemic Inflammation

Thesis (Ph.D.)--University of Washington, 2016-04

Ototoxicity and tolerance assessment of a TrisEDTA and polyhexamethylene biguanide ear flush formulation in dogs

Clinically healthy mixed breed dogs (n = 20) were used to determine if a Tris (tromethamine)-buffered test solution, Otinide((R)) (Trademark of Dermcare-Vet Pty-Ltd, Australia), containing disodium ethylenediamine tetraacetic acid (EDTA; 1.21 g/L) and polyhexamethylene biguanide (PHMB; 0.22 g/L) caused ototoxicity or vestibular dysfunction. The dogs were randomly assigned to either a control group (group A, n = 10) receiving saline, or a treatment group (group B, n = 10) receiving the test solution. Phase 1 of the study consisted of applying 5.0 mL of saline to both ears of the control group (group A) and 5 mL of test solution to both ears of the test group (group B), for 21 days. A bilateral myringotomy was then performed on each dog under deep sedation. Phase 2 of the study then consisted of applying 2.0 mL of the saline to both ears of the control group (group A) and 2.0 mL of the test solution to both ears of the test group (group B), for 14 days. Throughout the study, dogs were examined for clinical health, and underwent otoscopic, vestibular and auditory examinations. The auditory examinations included brainstem auditory evoked potential (BAEP) threshold and supra-threshold assessments using both click and 8 kHz tone burst stimuli. The absence of vestibular signs and effects on the BAEP attributable to the test solution suggested the test solution could be applied safely to dogs, including those with a damaged tympanic membrane.

Extending a model of shift-work tolerance

The present study contributes to theory and practice through the development of a model of shift-work tolerance with the potential to indicate interventions that reduce nurses' intention toward turnover and increase job satisfaction in hospital-based settings. Survey data from 1257 nurses were used to conduct structural equation modeling that examine the direct and indirect effects of supervisor and colleague support, team identity, team climate, and control over working environment on time-based work/life conflict, psychological well-being, physical symptoms, job satisfaction, and turnover intention. The analysis of the proposed model revealed a good fit The chi-square difference test was non-significant (χ2(26)=338.56), the fit indices were high (CFI=.923, NFI=.918, and NNFI=.868), the distribution of residuals was symmetric and approached zero, the average standardized residual was low (AASR=.04), and the standardized RMR was .072. In terms of the predictor variable, the final model explained 48% of the variance in turnover intention. The data revealed considerable evidence of both direct effects on adjustment and complex indirect links between levels of adjustment and work-related social support, team identity, team climate, and control. Nurses with high supervisor and coworker support experienced more positive team climates, identified more strongly with their team, and increased their perceptions of control over their work environment. This in turn lowered their appraisals of their time-based work/life conflict, which consequently increased their psychological well-being and job satisfaction and reduced their physical health symptoms and turnover intention. The type of shift schedule worked by the nurses influenced levels of turnover intention, control over work environment, time-based work/life conflict, and physical symptoms.

Evaluating the effect of splitting cylindrical power on improving patient tolerance to toric lens misalignment

Purpose: Evaluating the impact of splitting toric power on patient tolerance to misorientation such as with intraocular lens rotation. Setting: University vision clinic. Methods: Healthy, non astigmats had +1.50D astigmatism induced with spectacle lenses at 90°, 135°, 180° and +3.00D at 90°. Two correcting cylindrical lenses of the opposite sign and half the power each were subsequently added to the trial frame misaligned by 0°, 5° or 10° in a random order and misorientated from the initial axis in a clockwise direction by up to 15° in 5° steps. A second group of adapted astigmats with between 1.00 and 3.00DC had their astigmatism corrected with two toric spectacle lenses of half the power separated by 0°, 5° or 10° and misorientated from the initial axis in both directions by up to 15° in 5° steps. Distance, high contrast visual acuity was measured using a computerised test chart at each lens misalignment and misorientation. Results: Misorientation of the split toric lenses caused a statistically significant drop in visual acuity (F= 70.341; p< 0.001). Comparatively better acuities were observed around 180°, as anticipated (F= 3.775; p= 0.035). Misaligning the split toric power produced no benefit in visual acuity retention with axis misorientation when subjects had astigmatism induced with a low (F= 2.190, p= 0.129) or high cylinder (F= 0.491, p= 0.617) or in the adapted astigmats (F= 0.120, p= 0.887). Conclusion: Misalignment of toric lens power split across the front and back lens surfaces had no beneficial effect on distance visual acuity, but also no negative effect. © 2013 British Contact Lens Association.

Laparoscopic greater curvature plication in morbidly obese women with type 2 diabetes:effects on glucose homeostasis, postprandial triglyceridemia and selected gut hormones

Background: Laparoscopic greater curvature plication (LGCP) is an emerging bariatric procedure that reduces the gastric volume without implantable devices or gastrectomy. The aim of this study was to explore changes in glucose homeostasis, postprandial triglyceridemia, and meal-stimulated secretion of selected gut hormones [glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), ghrelin, and obestatin] in patients with type 2 diabetes mellitus (T2DM) at 1 and 6 months after the procedure. Methods: Thirteen morbidly obese T2DM women (mean age, 53.2 ± 8.76 years; body mass index, 40.1 ± 4.59 kg/m2) were prospectively investigated before the LGCP and at 1- and 6-month follow-up. At these time points, all study patients underwent a standardized liquid mixed-meal test, and blood was sampled for assessment of plasma levels of glucose, insulin, C-peptide, triglycerides, GIP, GLP-1, ghrelin, and obestatin. Results: All patients had significant weight loss both at 1 and 6 months after the LGCP (p≤0.002), with mean percent excess weight loss (%EWL) reaching 29.7 ;plusmn2.9 % at the 6-month follow-up. Fasting hyperglycemia and hyperinsulinemia improved significantly at 6 months after the LGCP (p<0.05), with parallel improvement in insulin sensitivity and HbA1c levels (p<0.0001). Meal-induced glucose plasma levels were significantly lower at 6 months after the LGCP (p<0.0001), and postprandial triglyceridemia was also ameliorated at the 6-month follow-up (p<0.001). Postprandial GIP plasma levels were significantly increased both at 1 and 6 months after the LGCP (p<0.0001), whereas the overall meal-induced GLP-1 response was not significantly changed after the procedure (p ;gt0.05). Postprandial ghrelin plasma levels decreased at 1 and 6 months after the LGCP (p<0.0001) with no significant changes in circulating obestatin levels. Conclusion: During the initial 6-month postoperative period, LGCP induces significant weight loss and improves the metabolic profile of morbidly obese T2DM patients, while it also decreases circulating postprandial ghrelin levels and increases the meal-induced GIP response. © 2013 Springer Science+Business Media New York.

Development of surface flaw thresholds for pre-cured fiber reinforced polymer and groove size tolerance for near surface mounted fiber reinforced polymer retrofit systems

Since the introduction of fiber reinforced polymers (FRP) for the repair and retrofit of concrete structures in the 1980’s, considerable research has been devoted to the feasibility of their application and predictive modeling of their performance. However, the effects of flaws present in the constitutive components and the practices in substrate preparation and treatment have not yet been thoroughly studied. This research aims at investigating the effect of surface preparation and treatment for the pre-cured FRP systems and the groove size tolerance for near surface mounted (NSM) FRP systems; and to set thresholds for guaranteed system performance. This study was conducted as part of the National Cooperative Highway Research Program (NCHRP) Project 10-59B to develop construction specifications and process control manual for repair and retrofit of concrete structures using bonded FRP systems. The research included both analytical and experimental components. The experimental program for the pre-cured FRP systems consisted of a total of twenty-four (24) reinforced concrete (RC) T-beams with various surface preparation parameters and surface flaws, including roughness, flatness, voids and cracks (cuts). For the NSM FRP systems, a total of twelve (12) additional RC T-beams were tested with different grooves sizes for FRP bars and strips. The analytical program included developing an elaborate nonlinear finite element model using the general purpose software ANSYS. The bond interface between FRP and concrete was modeled by a series of nonlinear springs. The model was validated against test data from the present study as well as those available from the literature. The model was subsequently used to extend the experimental range of parameters for surface flatness in pre-cured FRP systems and for groove size study in the NSM FRP systems. Test results, confirmed by further analyses, indicated that contrary to the general belief in the industry, the impact of surface roughness on the global performance of pre-cured FRP systems was negligible. The study also verified that threshold limits set for wet lay-up FRP systems can be extended to pre-cured systems. The study showed that larger surface voids and cracks (cuts) can adversely impact both the strength and ductility of pre-cured FRP systems. On the other hand, frequency (or spacing) of surface cracks (cuts) may only affect system ductility rather than its strength. Finally, within the range studied, groove size tolerance of ±1/8 in. does not appear to have an adverse effect on the performance of NSM FRP systems.

Development of Surface Flaw Thresholds for Pre-Cured Fiber Reinforced Polymer and Groove Size Tolerance for Near Surface Mounted Fiber Reinforced Polymer Retrofit Systems

Since the introduction of fiber reinforced polymers (FRP) for the repair and retrofit of concrete structures in the 1980’s, considerable research has been devoted to the feasibility of their application and predictive modeling of their performance. However, the effects of flaws present in the constitutive components and the practices in substrate preparation and treatment have not yet been thoroughly studied. This research aims at investigating the effect of surface preparation and treatment for the pre-cured FRP systems and the groove size tolerance for near surface mounted (NSM) FRP systems; and to set thresholds for guaranteed system performance. The research included both analytical and experimental components. The experimental program for the pre-cured FRP systems consisted of a total of twenty-four (24) reinforced concrete (RC) T-beams with various surface preparation parameters and surface flaws, including roughness, flatness, voids and cracks (cuts). For the NSM FRP systems, a total of twelve (12) additional RC T-beams were tested with different grooves sizes for FRP bars and strips. The analytical program included developing an elaborate nonlinear finite element model using the general purpose software ANSYS. The model was subsequently used to extend the experimental range of parameters for surface flatness in pre-cured FRP systems, and for groove size study in the NSM FRP systems. Test results, confirmed by further analyses, indicated that contrary to the general belief in the industry, the impact of surface roughness on the global performance of pre-cured FRP systems was negligible. The study also verified that threshold limits set for wet lay-up FRP systems can be extended to pre-cured systems. The study showed that larger surface voids and cracks (cuts) can adversely impact both the strength and ductility of pre-cured FRP systems. On the other hand, frequency (or spacing) of surface cracks (cuts) may only affect system ductility rather than its strength. Finally, within the range studied, groove size tolerance of +1/8 in. does not appear to have an adverse effect on the performance of NSM FRP systems.