Function and anatomy of plant siRNA pools derived from hairpin transgenes

<p>Abstract</p> <p>Background</p> <p>RNA interference results in specific gene silencing by small-interfering RNAs (siRNAs). Synthetic siRNAs provide a powerful tool for manipulating gene expression but high cost suggests that novel siRNA production methods are desirable. Strong evolutionary conservation of siRNA structure suggested that siRNAs will retain cross-species function and that transgenic plants expressing heterologous siRNAs might serve as useful siRNA bioreactors. Here we report a detailed evaluation of the above proposition and present evidence regarding structural features of siRNAs extracted from plants.</p> <p>Results</p> <p>Testing the gene silencing capacity of plant-derived siRNAs in mammalian cells proved to be very challenging and required partial siRNA purification and design of a highly sensitive assay. Using the above assay we found that plant-derived siRNAs are ineffective for gene silencing in mammalian cells. Plant-derived siRNAs are almost exclusively double-stranded and most likely comprise a mixture of bona fide siRNAs and aberrant partially complementary duplexes. We also provide indirect evidence that plant-derived siRNAs may contain a hitherto undetected physiological modification, distinct from 3' terminal 2-O-methylation.</p> <p>Conclusion</p> <p>siRNAs produced from plant hairpin transgenes and extracted from plants are ineffective for gene silencing in mammalian cells. Thus our findings establish that a previous claim that transgenic plants offer a cost-effective, scalable and sustainable source of siRNAs is unwarranted. Our results also indicate that the presence of aberrant siRNA duplexes and possibly a plant-specific siRNA modification, compromises the gene silencing capacity of plant-derived siRNAs in mammalian cells.</p

Synthetic control of a fitness tradeoff in yeast nitrogen metabolism

Background: Microbial communities are involved in many processes relevant to industrial and medical biotechnology, such as the formation of biofilms, lignocellulosic degradation, and hydrogen production. The manipulation of synthetic and natural microbial communities and their underlying ecological parameters, such as fitness, evolvability, and variation, is an increasingly important area of research for synthetic biology. Results: Here, we explored how synthetic control of an endogenous circuit can be used to regulate a tradeoff between fitness in resource abundant and resource limited environments in a population of Saccharomyces cerevisiae. We found that noise in the expression of a key enzyme in ammonia assimilation, Gdh1p, mediated a tradeoff between growth in low nitrogen environments and stress resistance in high ammonia environments. We implemented synthetic control of an endogenous Gdh1p regulatory network to construct an engineered strain in which the fitness of the population was tunable in response to an exogenously-added small molecule across a range of ammonia environments. Conclusion: The ability to tune fitness and biological tradeoffs will be important components of future efforts to engineer microbial communities

An Innovative Approach for Community Engagement: Using an Audience Response System

Community-based participatory research methods allow for community engagement in the effort to reduce cancer health disparities. Community engagement involves health professionals becoming a part of the community in order to build trust, learn from the community and empower them to reduce disparities through their own initiatives and ideas. Audience Response Systems (ARS) are an innovative and engaging way to involve the community and obtain data for research purposes using keypads to report results via power point. The use of ARS within communities is very limited and serves to widen the disparity gap by not delivering new advances in medical knowledge and technology among all population groups. ARS was implemented at a community town hall event sponsored by a National Institute on Minority Health and Health Disparities Exploratory Center of Excellence, the Center for Equal Health. Participants appreciated being able to see how everyone else answered and felt included in the research process. ARS is beneficial because the community can answer truthfully and provides instant research results. Additionally, researchers can collect large amounts of data quickly, in a non-threatening way while tracking individual responses anonymously. Audience Response Systems proved to be an effective tool for successfully accomplishing community-based participatory research

A Focus Group Study of Child Nutrition Professionals' Attitudes about Food Allergies and Current Training Practices

Citation: Lee, Y. M., Kwon, J., & Sauer, K. (2014). A focus group study of child nutrition professionals’ attitudes about food allergies and current training practices. Journal of Child Nutrition and Management, 38(1).Purpose/Objectives: The purpose of this study was to explore child nutrition professionals' (CNPs) attitudes about food allergies, current practices of food allergy training, and operational issues related to food allergy training in school foodservice operations. Methods: Three focus groups were conducted with 21 CNPs with managerial responsibilities from a midwestern state. Responses to guided, open-ended questions were audio-recorded, transcribed verbatim, and analyzed to identify common themes. Results: Participants felt that the prevalence and types of food allergies affecting school nutrition programs have increased in recent years. They also felt that communicating with other stakeholders and verifying physicians' recommendations regarding food allergies can be difficult. Participants agreed that training could improve food allergy knowledge and awareness of their employees and improve safety of children with food allergies. However, only a few reported providing specific food allergy training for employees. Cost, scheduling difficulties, and time constraints were identified as barriers to providing food allergy training. Participants preferred having credentialed professionals to conduct employee food allergy training. Support from school administrators and witnessing a food allergic reaction in the cafeteria would trigger a decision to initiate food allergy training. Applications to Child Nutrition Professionals: Improved communication between school foodservice staff and other stakeholders is crucial to better serve students with food allergies. A well-planned, structured training program could better prepare school foodservice employees serving students with food allergies. CNPs might need continuous guidance and assistance regarding employee food allergy training. State agencies and professional organizations should work with CNPs to develop and communicate best practices for prevention and management of food allergic reactions

Development of a composite model derived from cardiopulmonary exercise tests to predict mortality risk in patients with mild-to-moderate heart failure

Objective: Cardiopulmonary exercise testing (CPET) is used to predict outcome in patients with mild-to-moderate heart failure (HF). Single CPET-derived variables are often used, but we wanted to see if a composite score achieved better predictive power. Methods: Retrospective analysis of patient records at the Department of Cardiology, Castle Hill Hospital, Kingston-upon-Hull. 387 patients [median (25th-75th percentile)] [age 65 (56-72) years; 79% males; LVEF 34 (31-37) %] were included. Patients underwent a symptomlimited, maximal CPET on a treadmill. During a median follow up of 8.6 ± 2.1 years in survivors, 107 patients died. Survival models were built and validated using a hybrid approach between the bootstrap and Cox regression. Nine CPET-derived variables were included. Z-score defined each variable's predictive strength. Model coefficients were converted to a risk score. Results: Four CPET-related variables were independent predictors of all-cause mortality in the survival model: the presence of exertional oscillatory ventilation (EOV), increasing slope of the relation between ventilation and carbon dioxide production (VE/VCO2 slope), decreasing oxygen uptake efficiency slope (OUES), and an increase in the lowest ventilatory equivalent for carbon dioxide (VEqCO2 nadir). Individual predictors of mortality ranged from 0.60 to 0.71 using Harrell’s C-statistic, but the optimal combination of EOV + VE/VCO2 slope + OUES + VEqCO2 nadir reached 0.75. The Hull CPET risk score had a significantly higher area under the curve (0.78) when compared to the Heart Failure Survival Score (AUC=0.70;

Hydrostatic machine tool spindles

Includes bibliographical references (p. 300-301).Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1996.This work investigates the application of hydrostatic bearings, particularly those of the self-compensating type, to precision machine tool spindles. First, relations are developed that can be used to calculate the hydraulic resistances of various bearing land flows in tilted and untilted orientations. These relations are then applied to predicting the load carrying characteristics of several different types of hydrostatic bearings. The bearing calculations are then integrated with shaft bending calculations to predict static spindle stiffness. Relations are also presented to calculate the frictional and thermal characteristics of the bearings at high speeds. The relations developed are compared to experimental data collected on three prototype test spindles. These spindles were found to have excellent qualities that represent a significant advance in machine tool spindle technology. Finally, a design case study is presented that illustrates how the methods developed in this work may be applied to practical spindle design.by Kevin L. Wasson.Ph.D

On the perception of pitch accents

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Architecture, 1988.Includes bibliographical references.Work was supported by the Defense Advanced Research Projects Agency (DARPA). N00039-86-C-0406by Kevin L. Landel.M.S

The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase

Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H→C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs) that are sensitive to the physical nature of the chemical step, and protein mass-modulation that slows down fast dynamics (femto- to picosecond timescale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5–45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction’s transition state (or tunneling ready state – TRS). Mass modulation of these enzymes through isotopic labeling with 13C, 15N, and 2H at nonexchangeable hydrogens yield an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass-modulation of the human DHFR affects neither DAD distribution nor the DAD’s conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H→C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically-modulated heavy enzymes in general