Mechanisms of chloroperoxidase-catalyzed enantioselective reactions as probed by site-directed mutagenesis and isotopic labeling

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in k cat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide. Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins. For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.

Perceiving Spanish in Miami: The Interaction of Dialect and National Labeling

The current study implements a speech perception experiment that interrogates local perceptions of Spanish varieties in Miami. Participants (N=292) listened to recordings of three Spanish varieties (Peninsular, Highland Colombian, and Post-Castro Cuban) and were given background information about the speakers, including the parents’ country of origin. In certain cases, the parents’ national-origin label matched the country of origin of the speaker, but otherwise the background information and voices were mismatched. The manipulation distinguishes perceptions determined by bottom-up cues (dialect) from top-down ones (social information). Participants then rated each voice for a range of personal characteristics and answered hypothetical questions about the speakers’ employment, family, and income. Results show clear top-down effects of the social information that often drive perceptions up or down depending on the traits themselves. Additionally, the data suggest differences in perceptions between Hispanic/non-Hispanic and Cuban/non-Cuban participants, although the Cuban participants do not drive the Hispanic participants’ perceptions.

Mechanisms of Chloroperoxidase-catalyzed Enantioselective Reactions as Probed by Site-directed Mutagenesis and Isotopic Labeling

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in kcat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide. Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins. For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.

The construction and optimization of a surface plasmon resonance sensor for the studies of bio-molecular interactions

Bio-molecular interactions exist ubiquitously in all biological systems. This dissertation project was to construct a powerful surface plasmon resonance (SPR) sensor. The SPR system is used to study bio-molecular interactions in real time and without labeling. Surface plasmon is the oscillation of free electrons in metals coupled with surface electromagnetic waves. These surface electromagnetic waves provide a sensitive probe to study bio-molecular interactions on metal surfaces. This project resulted in the successful construction and optimization of a homemade SPR sensor and the development of several new powerful protocols to study bio-molecular interactions. It was discovered through this project that the limitations of earlier SPR sensors are related not only to the instrumentation design and operating procedures, but also to the complex behaviors of bio-molecules on sensor surfaces that were very different from that in solution. Based on these discoveries the instrumentation design and operating procedures were fully optimized. A set of existing sensor surface treatment protocols were tested and evaluated and new protocols were developed in this project. The new protocols have demonstrated excellent performance to study biomolecular interactions. The optimized home-made SPR sensor was used to study protein-surface interactions. These protein-surface interactions are responsible for many complex organic cell activities. The co-existence of different driving forces and their correlation with the structure of the protein and the surface make the understanding of the fundamental mechanism of protein-surface interactions a very challenging task. Using the improved SPR sensor, the electrostatic interaction and hydrophobic interaction were studied separately. The results of this project directly confirmed the theoretical predictions for electrostatic force between the protein and surface. In addition, this project demonstrated that the strength of the protein-surface hydrophobic interaction does not solely depend on the hydrophobicity as reported earlier. Surface structure also plays a significant role.

Conjugated Polymer Nanoparticles for Biological Labeling and Delivery

Cancer remains one of the world’s most devastating diseases, with more than 10 million new cases every year. However, traditional treatments have proven insufficient for successful medical management of cancer due to the chemotherapeutics’ difficulty in achieving therapeutic concentrations at the target site, non-specific cytotoxicity to normal tissues, and limited systemic circulation lifetime. Although, a concerted effort has been placed in developing and successfully employing nanoparticle(NP)-based drug delivery vehicles successfully mitigate the physiochemical and pharmacological limitations of chemotherapeutics, work towards controlling the subcellular fate of the carrier, and ultimately its payload, has been limited. Because efficient therapeutic action requires drug delivery to specific organelles, the subcellular barrier remains critical obstacle to maximize the full potential of NP-based delivery vehicles. The aim of my dissertation work is to better understand how NP-delivery vehicles’ structural, chemical, and physical properties affect the internalization method and subcellular localization of the nanocarrier. In this work we explored how side-chain and backbone modifications affect the conjugated polymer nanoparticle (CPN) toxicity and subcellular localization. We discovered how subtle chemical modifications had profound consequences on the polymer’s accumulation inside the cell and cellular retention. We also examined how complexation of CPN with polysaccharides affects uptake efficiency and subcellular localization. This work also presents how changes to CPN backbone biodegradability can significantly affect the subcellular localization of the material. A series of triphenyl phosphonium-containing CPNs were synthesized and the effect of backbone modifications have on the cellular toxicity and intracellular fate of the material. A mitochondrial-specific polymer exhibiting time-dependent release is reported. Finally, we present a novel polymerization technique which allows for the controlled incorporation of electron-accepting benzothiadiazole units onto the polymer chain. This facilitates tuning CPN emission towards red emission. The work presented here, specifically, the effect that side-chain and structure, polysaccharide formulation and CPN degradability have on material’s uptake behavior, can help maximize the full potential of NP-based delivery vehicles for improved chemotherapeutic drug delivery.

Conjugated polymer nanoparticles for biological labeling and delivery

Cancer remains one of the world’s most devastating diseases, with more than 10 million new cases every year. However, traditional treatments have proven insufficient for successful medical management of cancer due to the chemotherapeutics’ difficulty in achieving therapeutic concentrations at the target site, non-specific cytotoxicity to normal tissues, and limited systemic circulation lifetime. Although, a concerted effort has been placed in developing and successfully employing nanoparticle(NP)-based drug delivery vehicles successfully mitigate the physiochemical and pharmacological limitations of chemotherapeutics, work towards controlling the subcellular fate of the carrier, and ultimately its payload, has been limited. Because efficient therapeutic action requires drug delivery to specific organelles, the subcellular barrier remains critical obstacle to maximize the full potential of NP-based delivery vehicles. The aim of my dissertation work is to better understand how NP-delivery vehicles’ structural, chemical, and physical properties affect the internalization method and subcellular localization of the nanocarrier. ^ In this work we explored how side-chain and backbone modifications affect the conjugated polymer nanoparticle (CPN) toxicity and subcellular localization. We discovered how subtle chemical modifications had profound consequences on the polymer’s accumulation inside the cell and cellular retention. We also examined how complexation of CPN with polysaccharides affects uptake efficiency and subcellular localization. ^ This work also presents how changes to CPN backbone biodegradability can significantly affect the subcellular localization of the material. A series of triphenyl phosphonium-containing CPNs were synthesized and the effect of backbone modifications have on the cellular toxicity and intracellular fate of the material. A mitochondrial-specific polymer exhibiting time-dependent release is reported. Finally, we present a novel polymerization technique which allows for the controlled incorporation of electron-accepting benzothiadiazole units onto the polymer chain. This facilitates tuning CPN emission towards red emission. ^ The work presented here, specifically, the effect that side-chain and structure, polysaccharide formulation and CPN degradability have on material’s uptake behavior, can help maximize the full potential of NP-based delivery vehicles for improved chemotherapeutic drug delivery.^

The costs and controls of flowering and fruiting in {\it Spondias purpurea\/}

Small potted trees of Spondias purpurea were monitored to determine the costs and controls of flowering and fruiting. The effect of photoperiod, extremes in moisture and temperature, and defoliation were examined. The carbon exchange rates of the leaves, shoots and fruits were determined. Light response curves and diurnal levels were also investigated. $\sp{13}$Carbon labeling was used to determine which plant parts are carbon sinks. Photoperiod induces dormancy and bud activity. Extremes in soil moisture and temperature induce leaf fall. Flowers, fruits, and roots are carbon sinks. The results were used to develop a phenological model with latitude, soil moisture, and air temperature as variables. ^

Improved estimation of postmortem interval with Cardiac Troponin T and improved analytical methods

Accurate knowledge of the time since death, or postmortem interval (PMI), has enormous legal, criminological, and psychological impact. In this study, an investigation was made to determine whether the relationship between the degradation of the human cardiac structure protein Cardiac Troponin T and PMI could be used as an indicator of time since death, thus providing a rapid, high resolution, sensitive, and automated methodology for the determination of PMI. ^ The use of Cardiac Troponin T (cTnT), a protein found in heart tissue, as a selective marker for cardiac muscle damage has shown great promise in the determination of PMI. An optimized conventional immunoassay method was developed to quantify intact and fragmented cTnT. A small sample of cardiac tissue, which is less affected than other tissues by external factors, was taken, homogenized, extracted with magnetic microparticles, separated by SDS-PAGE, and visualized with Western blot by probing with monoclonal antibody against cTnT. This step was followed by labeling and available scanners. This conventional immunoassay provides a proper detection and quantitation of cTnT protein in cardiac tissue as a complex matrix; however, this method does not provide the analyst with immediate results. Therefore, a competitive separation method using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was developed to study the interaction between human cTnT protein and monoclonal anti-TroponinT antibody. ^ Analysis of the results revealed a linear relationship between the percent of degraded cTnT and the log of the PMI, indicating that intact cTnT could be detected in human heart tissue up to 10 days postmortem at room temperature and beyond two weeks at 4C. The data presented demonstrates that this technique can provide an extended time range during which PMI can be more accurately estimated as compared to currently used methods. The data demonstrates that this technique represents a major advance in time of death determination through a fast and reliable, semi-quantitative measurement of a biochemical marker from an organ protected from outside factors. ^

Think before you eat: Calories and exercise equivalents presented on menus at point-of-choice

Although calorie information at the point-of-purchase at fast food restaurants is proposed as a method to decrease calorie choices and combat obesity, research results have been mixed. Much of the supportive research has weak methodology, and is limited. There is a demonstrated need to develop better techniques to assist consumers to make lower calorie food choices. Eating at fast food restaurants has been positively associated with weight gain. The current study explored the possibility of adding exercise equivalents (EE) (physical activity required to burn off the calories in the food), along with calorie information as a possible way to facilitate lower calorie choice at the point-of-choice in fast food restaurants. This three-group experimental study, in 18-34 year old, overweight and obese women, examines whether presenting caloric information in the form of EE at the point-of-choice at fast food restaurants, will lead to lower calorie food choices compared to presenting simple caloric information or no information at all. Methods. A randomized repeated measures experiment was conducted. Participants ordered a fast food meal from Burger King with menus that contained only the names of the food choices (Lunch 1). One week later (Lunch 2), study participants were given one of three menus that varied: no information, calorie information, or calorie information and EE. Study participants included 62 college aged students. Additionally, the study controlled for dietary restraint by blocking participants, before randomization, to the three groups. Results. A repeated measures analysis of variance was conducted. The study was not sufficiently powered, and while the study was designed to determine large effect sizes, a small effect size of .026, was determined. No significant differences were found in the foods ordered among the various menu conditions. Conclusion. Menu labeling alone might not be enough to reduce calories at the point-of-choice at restaurants. Additional research is necessary to determine if calorie information and EE at the point-of-choice would lead to fewer calories chosen at a meal. Studies should also look at long-term, repeated exposure to determine the effectiveness of calories and or EE at the point-of-choice at fast food restaurants.

Professionalism and Ethics in Hospitality

In their discussion - Professionalism and Ethics in Hospitality - by James R. Keiser, Associate Professor and John Swinton, Instructor, Hotel, Restaurant and Institutional Management, The Pennsylvania State University, Keiser and Swinton initially offer: “Referring to “the hospitality profession” necessitates thinking of the ethics of that profession and how ethics can be taught. The authors discuss what it means for the hospitality industry to be a profession.” The authors will have you know, a cursory nod to the term or description, profession and/or professional, is awarded to the hospitality industry at large; at least in an academic sense. Keiser and Swinton also want you to know that ethics, and professionalism are distinctly unique concepts, however, they are related. Their intangible nature does make them difficult, at best, to define, but ethics in contemporary hospitality has, to some degree, been charted and quantified. “We have left the caveat emptor era, and the common law, the Uniform Commercial Code, and a variety of local ordinances now dictate that the goods and services hospitality offers carry an implied warranty of merchantability,” the authors inform you. About the symbiotic relationship between ethics and professionalism, the authors say this: The less precise a code of ethics goes, the general rule, the fewer claims the group has to professional status.” The statement above may be considered a cornerstone principle. “However, the mere existence of an ethical code (or of professional status, for that matter) does not ensure ethical behavior in any group,” caution Keiser and Swinton. “Codes of ethics do not really define professionalism except as they adopt a group's special, arcane, exclusionary jargon. Worse, they can define the minimum, agreed-upon standards of conduct and thereby encourage ethical corner-cutting,” they further qualify the thought. And, in bridging academia, Keiser and Swinton say, “Equipped now with a sense of the ironies and ambiguities inherent in labeling any work "professional," we can turn to the problem of instilling in students a sense of what is professionally ethical. Students appear to welcome this kind of instruction, and while we would like to think their interest comes welling up from altruism and intellectual curiosity rather than drifting down as Watergate and malpractice fallout, our job is to teach, not to weigh the motives that bring us our students, and to provide a climate conducive to ethical behavior, not supply a separate answer for every contingency.” Keiser and Swinton illustrate their treatise on ethics via the hypothetical tale [stylized case study] of Cosmo Cuisiner, who manages the Phoenix, a large suburban restaurant. Cosmo is “…a typical restaurant manager faced with a series of stylized, over-simplified, but illustrative decisions, each with its own ethical skew for the students to analyze.” A shortened version of that case study is presented. Figure 1 outlines the State Restaurant Association Code of Ethics.

Microspheres for liver radiomicrospheres therapy and planning

Liver cancer accounts for nearly 10% of all cancers in the US. Intrahepatic Arterial Radiomicrosphere Therapy (RMT), also known as Selective Internal Radiation Treatment (SIRT), is one of the evolving treatment modalities. Successful patient clinical outcomes require suitable treatment planning followed by delivery of the microspheres for therapy. The production and in vitro evaluation of various polymers (PGCD, CHS and CHSg) microspheres for a RMT and RMT planning are described. Microparticles with a 30±10 µm size distribution were prepared by emulsion method. The in vitro half-life of the particles was determined in PBS buffer and porcine plasma and their potential application (treatment or treatment planning) established. Further, the fast degrading microspheres (≤ 48 hours in vitro half-life) were labeled with 68Ga and/or 99mTc as they are suitable for the imaging component of treatment planning, which is the primary emphasis of this dissertation. Labeling kinetics demonstrated that 68Ga-PGCD, 68Ga-CHSg and 68Ga-NOTA-CHSg can be labeled with more than 95% yield in 15 minutes; 99mTc-PGCD and 99mTc-CHSg can also be labeled with high yield within 15-30 minutes. In vitro stability after four hours was more than 90% in saline and PBS buffer for all of them. Experiments in reconstituted hemoglobin lysate were also performed. Two successful imaging (RMT planning) agents were found: 99mTc-CHSg and 68Ga-NOTA-CHSg. For the 99mTc-PGCD a successful perfusion image was obtained after 10 minutes, however the in vivo degradation was very fast (half-life), releasing the 99mTc from the lungs. Slow degrading CHS microparticles (> 21 days half-life) were modified with p-SCN-b-DOTA and labeled with 90 Y for production of 90Y-DOTA-CHS. Radiochemical purity was evaluated in vitro and in vivo showing more than 90% stability after 72 and 24 hours respectively. All agents were compared to their respective gold standards (99mTc-MAA for 68Ga-NOTA-CHSg and 99m Tc-CHSg; 90Y-SirTEX for 90Y-DOTA-CHS) showing superior in vivo stability. RMT and RMT planning agents (Therapy, PET and SPECT imaging) were designed and successfully evaluated in vitro and in vivo.

An Evaluation of Three Nutrition Labeling Formats for Restaurant Menus

This study evaluated three menu nutrition labeling formats: calorie only information, a healthy symbol, and a nutrient list. Daily sales data for a table-service restaurant located on a university campus were recorded during a four-week period from January to February 2013 to examine changes in average nutritional content of the entrees purchased by customers when different nutrition labels were provided. A survey was conducted to assess the customers’ use of nutrition labels, their preferences among the three labeling formats, their entree selections, their cognitive beliefs with regard to healthy eating, and their demographic characteristics. A total of 173 questionnaires were returned and included in data analysis. Analysis of Variance (ANOVA) and regression analyses were performed using SAS. The results showed that favorable attitudes toward healthy eating and the use of nutrition labels were both significantly associated with healthier entrée selections. Age and diet status had some effects on the respondent’s use of nutrition labels. The calorie only information format was the most effective in reducing calories contained in the entrees sold, and the nutrient list was most effective in reducing fat and saturated fat content of the entrees sold. The healthy symbol was the least effective format, but interestingly enough, was most preferred by respondents. The findings provide support for future research and offer implications for policy makers, public health professionals, and foodservice operations.

Text analytics of social media: Sentiment analysis, event detection and summarization

In the last decade, large numbers of social media services have emerged and been widely used in people's daily life as important information sharing and acquisition tools. With a substantial amount of user-contributed text data on social media, it becomes a necessity to develop methods and tools for text analysis for this emerging data, in order to better utilize it to deliver meaningful information to users. ^ Previous work on text analytics in last several decades is mainly focused on traditional types of text like emails, news and academic literatures, and several critical issues to text data on social media have not been well explored: 1) how to detect sentiment from text on social media; 2) how to make use of social media's real-time nature; 3) how to address information overload for flexible information needs. ^ In this dissertation, we focus on these three problems. First, to detect sentiment of text on social media, we propose a non-negative matrix tri-factorization (tri-NMF) based dual active supervision method to minimize human labeling efforts for the new type of data. Second, to make use of social media's real-time nature, we propose approaches to detect events from text streams on social media. Third, to address information overload for flexible information needs, we propose two summarization framework, dominating set based summarization framework and learning-to-rank based summarization framework. The dominating set based summarization framework can be applied for different types of summarization problems, while the learning-to-rank based summarization framework helps utilize the existing training data to guild the new summarization tasks. In addition, we integrate these techneques in an application study of event summarization for sports games as an example of how to better utilize social media data. ^

The effect of sexist attitudes on the perception of visual artists by community college and university students

This study compared the effects of sexist labeling on the perceptions of visual artists by the community college and university students and determined their sex role orientation. The 370 students were shown five slides of an artist's works and were given six versions of an artist's biography. It contained embedded sexual labeling (woman, girl, person/ she, man, guy, person/he). The Artist Evaluation Questionnaire was administered to the female and male community college and university students that required the students to evaluate the female and male artists on several aspects of affective and cognitive measures. The questionnaire consisted of 9 items that had to be rated by the participants. In addition, the students filled out the Demographic Questionnaire and the BEM Sex Role Inventory, titled the Attitude Questionnaire. The Analysis of Variance testing procedures were administered to analyze the responses. The results disclosed gender differences in students' ratings. The female artist's work, when the artist was referred to by the neutral sexual label, "person", received significantly higher ratings from the female students. The male students gave the female artist her highest ratings when she was referred to by the low status sexual label, "girl". Both sexes did not express statistically significant preferences for any of the male sexual labels. Gender difference became apparent when it was found that female students rated both sexes equally, and their ratings were lower than those of the male students. The male students rated the female artist's work higher than the work of the male artist. The analysis of the sex role inventory questionnaire revealed the absence of the feminine (expressive) and masculine (instrumental) personalities among the students. The personalities of almost all the students were androgynous, with a few within the range of the near feminine, and a few within the range of the near masculine. The study reveals that there are differences in perception of sexual labels among the community college and university students.

Think Before You Eat: Calories and Exercise Equivalents Presented on Menus at Point-of-Choice

Although calorie information at the point-of-purchase at fast food restaurants is proposed as a method to decrease calorie choices and combat obesity, research results have been mixed. Much of the supportive research has weak methodology, and is limited. There is a demonstrated need to develop better techniques to assist consumers to make lower calorie food choices. Eating at fast food restaurants has been positively associated with weight gain. The current study explored the possibility of adding exercise equivalents (EE) (physical activity required to burn off the calories in the food), along with calorie information as a possible way to facilitate lower calorie choice at the point-of-choice in fast food restaurants. This three-group experimental study, in 18-34 year old, overweight and obese women, examines whether presenting caloric information in the form of EE at the point-of-choice at fast food restaurants, will lead to lower calorie food choices compared to presenting simple caloric information or no information at all. Methods: A randomized repeated measures experiment was conducted. Participants ordered a fast food meal from Burger King with menus that contained only the names of the food choices (Lunch 1). One week later (Lunch 2), study participants were given one of three menus that varied: no information, calorie information, or calorie information and EE. Study participants included 62 college aged students. Additionally, the study controlled for dietary restraint by blocking participants, before randomization, to the three groups. Results: A repeated measures analysis of variance was conducted. The study was not sufficiently powered, and while the study was designed to determine large effect sizes, a small effect size of .026, was determined. No significant differences were found in the foods ordered among the various menu conditions. Conclusion: Menu labeling alone might not be enough to reduce calories at the point-of-choice at restaurants. Additional research is necessary to determine if calorie information and EE at the point-of-choice would lead to fewer calories chosen at a meal. Studies should also look at long-term, repeated exposure to determine the effectiveness of calories and or EE at the point-of-choice at fast food restaurants.