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Description
Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins.
ContributorsHansen, Debra (Author) / Robida, Mark (Author) / Craciunescu, Felicia (Author) / Loskutov, Andrey (Author) / Dorner, Katerina (Author) / Rodenberry, John-Charles (Author) / Wang, Xiao (Author) / Olson, Tien (Author) / Patel, Hetal (Author) / Fromme, Petra (Author) / Sykes, Kathryn (Author) / Biodesign Institute (Contributor) / Innovations in Medicine (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor)
Created2016-02-24
Description
Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.
ContributorsNogly, Przemyslaw (Author) / Panneels, Valerie (Author) / Nelson, Garrett (Author) / Gati, Cornelius (Author) / Kimura, Tetsunari (Author) / Milne, Christopher (Author) / Milathianaki, Despina (Author) / Kubo, Minoru (Author) / Wu, Wenting (Author) / Conrad, Chelsie (Author) / Coe, Jesse (Author) / Bean, Richard (Author) / Zhao, Yun (Author) / Bath, Petra (Author) / Dods, Robert (Author) / Harimoorthy, Rajiv (Author) / Beyerlein, Kenneth R. (Author) / Rheinberger, Jan (Author) / James, Daniel (Author) / Deponte, Daniel (Author) / Li, Chufeng (Author) / Sala, Leonardo (Author) / Williams, Garth J. (Author) / Hunter, Mark S. (Author) / Koglin, Jason E. (Author) / Berntsen, Peter (Author) / Nango, Eriko (Author) / Iwata, So (Author) / Chapman, Henry N. (Author) / Fromme, Petra (Author) / Frank, Matthias (Author) / Abela, Rafael (Author) / Boutet, Sebastien (Author) / Barty, Anton (Author) / White, Thomas A. (Author) / Weierstall, Uwe (Author) / Spence, John (Author) / Neutze, Richard (Author) / Schertler, Gebhard (Author) / Standfuss, Jorg (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / School of Molecular Sciences (Contributor)
Created2016-08-22
Description
Chloroform and methanol are superior solvents for lipid extraction from photosynthetic microorganisms, because they can overcome the resistance offered by the cell walls and membranes, but they are too toxic and expensive to use for large-scale fuel production. Biomass from the photosynthetic microalga Scenedesmus, subjected to a commercially available pre-treatment technology called Focused-Pulsed® (FP), yielded 3.1-fold more crude lipid and fatty acid methyl ester (FAME) after extraction with a range of solvents. FP treatment increased the FAME-to-crude-lipid ratio for all solvents, which means that the extraction of non-lipid materials was minimized, while the FAME profile itself was unchanged compared to the control. FP treatment also made it possible to use only a small proportion of chloroform and methanol, along with isopropanol, to obtain equivalent yields of lipid and FAME as with 100% chloroform plus methanol.
ContributorsLai, Yenjung Sean (Author) / Parameswaran, Prathap (Author) / Li, Ang (Author) / Baez, Maria (Author) / Rittmann, Bruce (Author) / Biodesign Institute (Contributor) / Swette Center for Environmental Biotechnology (Contributor)
Created2014-12-01
Description
Sulfadiazine (SD), one of broad-spectrum antibiotics, exhibits limited biodegradation in wastewater treatment due to its chemical structure, which requires initial mono-oxygenation reactions to initiate its biodegradation. Intimately coupling UV photolysis with biodegradation, realized with the internal loop photobiodegradation reactor, accelerated SD biodegradation and mineralization by 35 and 71 %, respectively. The main organic products from photolysis were 2-aminopyrimidine (2-AP), p-aminobenzenesulfonic acid (ABS), and aniline (An), and an SD-photolysis pathway could be identified using C, N, and S balances. Adding An or ABS (but not 2-AP) into the SD solution during biodegradation experiments (no UV photolysis) gave SD removal and mineralization rates similar to intimately coupled photolysis and biodegradation. An SD biodegradation pathway, based on a diverse set of the experimental results, explains how the mineralization of ABS and An (but not 2-AP) provided internal electron carriers that accelerated the initial mono-oxygenation reactions of SD biodegradation. Thus, multiple lines of evidence support that the mechanism by which intimately coupled photolysis and biodegradation accelerated SD removal and mineralization was through producing co-substrates whose oxidation produced electron equivalents that stimulated the initial mono-oxygenation reactions for SD biodegradation.
ContributorsPan, Shihui (Author) / Yan, Ning (Author) / Liu, Xinyue (Author) / Wang, Wenbing (Author) / Zhang, Yongming (Author) / Liu, Rui (Author) / Rittmann, Bruce (Author) / Biodesign Institute (Contributor) / Swette Center for Environmental Biotechnology (Contributor)
Created2014-11-01
Description
We studied the microbial community structure of pilot two-stage membrane biofilm reactors (MBfRs) designed to reduce nitrate (NO[subscript 3]–) and perchlorate (ClO[subscript 4]–) in contaminated groundwater. The groundwater also contained oxygen (O[subscript 2]) and sulfate (SO[2 over 4]–), which became important electron sinks that affected the NO[subscript 3]– and ClO[subscript 4]– removal rates. Using pyrosequencing, we elucidated how important phylotypes of each “primary” microbial group, i.e., denitrifying bacteria (DB), perchlorate-reducing bacteria (PRB), and sulfate-reducing bacteria (SRB), responded to changes in electron-acceptor loading. UniFrac, principal coordinate analysis (PCoA), and diversity analyses documented that the microbial community of biofilms sampled when the MBfRs had a high acceptor loading were phylogenetically distant from and less diverse than the microbial community of biofilm samples with lower acceptor loadings. Diminished acceptor loading led to SO[2 over 4]– reduction in the lag MBfR, which allowed Desulfovibrionales (an SRB) and Thiothrichales (sulfur-oxidizers) to thrive through S cycling. As a result of this cooperative relationship, they competed effectively with DB/PRB phylotypes such as Xanthomonadales and Rhodobacterales. Thus, pyrosequencing illustrated that while DB, PRB, and SRB responded predictably to changes in acceptor loading, a decrease in total acceptor loading led to important shifts within the “primary” groups, the onset of other members (e.g., Thiothrichales), and overall greater diversity.
ContributorsOntiveros-Valencia, Aura (Author) / Tang, Youneng (Author) / Zhao, Heping (Author) / Friese, David (Author) / Overstreet, Ryan (Author) / Smith, Jennifer (Author) / Evans, Patrick (Author) / Rittmann, Bruce (Author) / Krajmalnik-Brown, Rosa (Author) / Biodesign Institute (Contributor) / Swette Center for Environmental Biotechnology (Contributor) / Julie Ann Wrigley Global Institute of Sustainability (Contributor) / School of Sustainability (Contributor)
Created2014-07-01
Description
Introduction: There is currently a lack of industry-wide gold standardization in accelerometer study
protocols, including within sleep-focused studies. This study seeks to address accuracy of
accelerometer data in detection of the beginnings and ends of sleep bouts in young adults with
polysomnography (PSG) corroboration. An existing algorithm used to differentiate valid/invalid wear
time and detect bouts of sleep has been modified with the goal of maximizing accuracy of sleep bout
detection. Methods: Three key decisions and thresholds of the algorithm have been modified with three
experimental values each being tested. The main experimental variable Sleepwindow controls the
amount of time before and after a determined bout of sleep that is searched for additional sedentary
time to incorporate and consider part of the same sleep bout. Results were compared to PSG and sleep
diary data for absolute agreement of sleep bout start time (START), end time (END) and time in bed
(TIB). Adjustments were made for outliers as well as sleep latency, snooze time, and the sum of both.
Results: Only adjustments made to a sleep window variable yielded altered results. Between a 5-, 15-,
and 30-minute window, a 15-minute window incurred the least error and most agreement to
comparisons for START, while a 5-minute window was best for END and TIB. Discussion: Contrary
to expectation, corrections for snooze, latency, and both did not substantially improve agreement to
PSG. Algorithm-derived estimates of START and END always fell after sleep diary and PSG both,
suggesting either participants’ sedentary behavior beginning and ends were at a delay from sleep and
wake times, or the algorithm estimates consistently later times than appropriate. The inclusion of a
sleep window variable yields substantial variety in results. A 15-minute window appears best at
determining START while a 5-minute window appears best for END and TIB. Further investigation on
the optimal window length per demographic and condition is required.
protocols, including within sleep-focused studies. This study seeks to address accuracy of
accelerometer data in detection of the beginnings and ends of sleep bouts in young adults with
polysomnography (PSG) corroboration. An existing algorithm used to differentiate valid/invalid wear
time and detect bouts of sleep has been modified with the goal of maximizing accuracy of sleep bout
detection. Methods: Three key decisions and thresholds of the algorithm have been modified with three
experimental values each being tested. The main experimental variable Sleepwindow controls the
amount of time before and after a determined bout of sleep that is searched for additional sedentary
time to incorporate and consider part of the same sleep bout. Results were compared to PSG and sleep
diary data for absolute agreement of sleep bout start time (START), end time (END) and time in bed
(TIB). Adjustments were made for outliers as well as sleep latency, snooze time, and the sum of both.
Results: Only adjustments made to a sleep window variable yielded altered results. Between a 5-, 15-,
and 30-minute window, a 15-minute window incurred the least error and most agreement to
comparisons for START, while a 5-minute window was best for END and TIB. Discussion: Contrary
to expectation, corrections for snooze, latency, and both did not substantially improve agreement to
PSG. Algorithm-derived estimates of START and END always fell after sleep diary and PSG both,
suggesting either participants’ sedentary behavior beginning and ends were at a delay from sleep and
wake times, or the algorithm estimates consistently later times than appropriate. The inclusion of a
sleep window variable yields substantial variety in results. A 15-minute window appears best at
determining START while a 5-minute window appears best for END and TIB. Further investigation on
the optimal window length per demographic and condition is required.
ContributorsMartin, Logan Rhett (Author) / Buman, Matthew (Thesis director) / Toledo, Meynard John (Committee member) / Kurka, Jonathan (Committee member) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2019-12
Description
This thesis paper examines the effects of increased standing and light physical activity in the workplace on postprandial glucose. Sedentary behavior is detrimental to our health, affecting metabolic risk factors. An easy way to implement change is by decreasing sedentary time in workplaces where sitting is common, such as office workspaces. To consider how postprandial glucose is affected by decreasing sedentary time, participants ate a standardized meal for lunch and were asked to decrease their sitting time by replacing it with standing and light physical activity.
ContributorsChilders, Autumn Skye (Author) / Buman, Matthew (Thesis director) / Sears, Dorothy (Committee member) / Hasanaj, Kristina (Committee member) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2020-12
Description
The purpose of this study was to examine the overall maintenance of behavior during the 12 to 24 month period of the Stand&Move@Work study and the impact of implementation factors (i.e., facilitators, advocate activity, and the amount of strategies used) on behavior change. The design of the study was a cluster randomized trial which was facilitated by researchers for the first 12 months of the study. The primary aim of the study was to examine the maintenance of behavior change (i.e., sitting time) at the 12 month and 24 month marks using objectively measured sedentary behavior (activPAL micro). The secondary aim of the study was to examine the impact of implementation factors (i.e., facilitators, advocate activity, and the amount of strategies used) on behavior change during the 12 through 24 months maintenance period. Participants (N=630) included full-time, caucasian, middle-aged office workers. For the primary aim, descriptive means were used to cluster for observations within-persons and were adjusted for age, gender, race, job-type, and ordering effects.. For the secondary aim, descriptive means adjusted for workplace culture and environment were computed. At the 24 month mark, participants spent 280.67 ± 87.67 min/8hr workday sitting and 161.94 ± 85.87 min/8hr workday standing. The top performing worksites displayed reductions in sitting time which largely translated into standing time by about 30 minutes per 8 hour workday at 24 months. Feasibility findings indicated that implementation strategies do not show differences between the top 25% and bottom 25% performing worksites. This study provides insight to implementation strategies for interventions in the workplace.
ContributorsTong, Alyssa Taylor (Author) / Buman, Matthew (Thesis director) / Larouche, Miranda (Committee member) / Estabrooks, Paul (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Cardiovascular disease attributed to about 800,000 deaths per year and is the leading cause of all-cause mortality in the U.S. Previous studies indicate that reducing sedentary time or increasing physical activity (PA) can independently reduce cardiometabolic risk (CMR). Further, studies have shown that higher levels of moderate-to-vigorous PA can attenuate the negative effects of sedentary behavior on CMR.
In this study, we evaluated the association between sedentary time, light-intensity PA (LPA), and moderate-vigorous PA (MVPA) and CMR biomarkers (high density lipoprotein level, low density lipoprotein level, triglycerides, fasting glucose, total cholesterol, blood pressure, and body mass index). Additionally, we examined if the detrimental association between sedentary time and CMR biomarkers is partially or fully attenuated by MVPA. Baseline objective physical activity and cardiometabolic risk data from a two-arm-cluster randomized trial (Stand&Move@work) were used in this study. Multilevel models clustered by worksite evaluated the fixed effects and interaction between MVPA and sedentary time on CMR. Data from 630 sedentary working adults (from 24 worksites) were included in the analysis. The sample was mainly middle aged (44.6±11.2) females (74%) with race distributions as follows; 70.5% white, 13.8% hispanic, 4.1% black, 5.1% asian, and 2.1% other. Our study showed detrimental trends consistent with previous studies between sedentary behavior and cardiometabolic outcomes including HDL, LDL, and total cholesterol. MVPA demonstrated beneficial associations with lipoproteins including HDL, LDL, total cholesterol, and triglycerides. We observed that high levels of MVPA may be able to partially attenuate the negative effects of highly sedentary behavior on fasting glucose, total cholesterol, and LDL levels. Overall, sedentary behavior indicated deleterious associations with cardiometabolic outcomes. Future directions for this study could examine a more at-risk population or a highly active population for further assessment of CMR biomarkers and the effects of behavior.
In this study, we evaluated the association between sedentary time, light-intensity PA (LPA), and moderate-vigorous PA (MVPA) and CMR biomarkers (high density lipoprotein level, low density lipoprotein level, triglycerides, fasting glucose, total cholesterol, blood pressure, and body mass index). Additionally, we examined if the detrimental association between sedentary time and CMR biomarkers is partially or fully attenuated by MVPA. Baseline objective physical activity and cardiometabolic risk data from a two-arm-cluster randomized trial (Stand&Move@work) were used in this study. Multilevel models clustered by worksite evaluated the fixed effects and interaction between MVPA and sedentary time on CMR. Data from 630 sedentary working adults (from 24 worksites) were included in the analysis. The sample was mainly middle aged (44.6±11.2) females (74%) with race distributions as follows; 70.5% white, 13.8% hispanic, 4.1% black, 5.1% asian, and 2.1% other. Our study showed detrimental trends consistent with previous studies between sedentary behavior and cardiometabolic outcomes including HDL, LDL, and total cholesterol. MVPA demonstrated beneficial associations with lipoproteins including HDL, LDL, total cholesterol, and triglycerides. We observed that high levels of MVPA may be able to partially attenuate the negative effects of highly sedentary behavior on fasting glucose, total cholesterol, and LDL levels. Overall, sedentary behavior indicated deleterious associations with cardiometabolic outcomes. Future directions for this study could examine a more at-risk population or a highly active population for further assessment of CMR biomarkers and the effects of behavior.
ContributorsMeyer, Emily Camille (Author) / Buman, Matthew (Thesis director) / Toledo, Meynard (Committee member) / Pereira, Mark (Committee member) / School of Life Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
While type 2 diabetes (T2D) rates have soared, the number of Americans classified as ‘prediabetic’ has also increased. Despite this, current preventative approaches are costly and often not without undue side-effects. Instead, behavioral lifestyle approaches hold promise in reducing conversion rates of T2D as the latest treatment option that could mitigate and transform disease management. However, present interventions do not possess the scope necessary for implementation in a realistic, scalable way that can target the large at-risk population.
The application (app) “BeWell24” mitigates this diabetes risk through targeting sleep, physical activity, sedentary behavior, and diet, and is being delivered through mHealth technology to attenuate the higher-risk of the prediabetic Veteran population. In order for full scale dissemination, this thesis examines a provider perspective of the ‘Post-intervention interview guide’, performed with a Phoenix Veterans Affairs Health Care System (PVAHCS) provider. It then suggests revisions to the interview guide based on the provider’s interview and existing literature. This thesis also emphasizes the rationale behind these proposed changes to be organized in line with the iPARIHS framework (integrated Promoting Action on Research Implementation in Health Services).
Overall, the provider responded positively to BeWell24 and the ‘Post-intervention interview guide’, with constructive suggestions for each question in the interview guide. The main theme of the provider’s answers and comments were to prioritize efficiency and preserve standard clinical flow. A revised interview guide is provided, which prospectively presents as a more brief and focused interview organized by the iPARIHS framework. This revised interview guide could aid in the clarity of provider responses, specifically for the prospective interviews of the ongoing larger BeWell24 study and future studies.
The application (app) “BeWell24” mitigates this diabetes risk through targeting sleep, physical activity, sedentary behavior, and diet, and is being delivered through mHealth technology to attenuate the higher-risk of the prediabetic Veteran population. In order for full scale dissemination, this thesis examines a provider perspective of the ‘Post-intervention interview guide’, performed with a Phoenix Veterans Affairs Health Care System (PVAHCS) provider. It then suggests revisions to the interview guide based on the provider’s interview and existing literature. This thesis also emphasizes the rationale behind these proposed changes to be organized in line with the iPARIHS framework (integrated Promoting Action on Research Implementation in Health Services).
Overall, the provider responded positively to BeWell24 and the ‘Post-intervention interview guide’, with constructive suggestions for each question in the interview guide. The main theme of the provider’s answers and comments were to prioritize efficiency and preserve standard clinical flow. A revised interview guide is provided, which prospectively presents as a more brief and focused interview organized by the iPARIHS framework. This revised interview guide could aid in the clarity of provider responses, specifically for the prospective interviews of the ongoing larger BeWell24 study and future studies.
ContributorsWojtas, Abby Ann (Author) / Buman, Matthew (Thesis director) / Larouche, Miranda (Committee member) / Epstein, Dana (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05