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Item Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains(Public Library of Science, 2015-06) Orrú, Christina D.; Groveman, Bradley R.; Raymond, Lynne D.; Hughson, Andrew G.; Nonno, Romolo; Zou, Wenquan; Ghetti, Bernardino; Gambetti, Pierluigi; Caughey, Byron; Department of Pathology and Laboratory Medicine, IU School of MedicinePrions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.Item Bioinformatics Analysis and Annotation of Microtubule Binding and Associated Proteins (MAPs) - Creating a Database of MAPs(2005-08) Shenoy, Narmada; Guenther, BrianMicrotubules have many roles in the cytoskeletal infrastructure. This infrastructure underlies vital processes of cellular life such as motility, division, morphology, and intracellular organization and transport. These different roles are carried out by the creation of different microtubule (MT) systems (such as basal bodies, centrioles, flagellum, kinetochores, and mitotic spindles). The changing roles require the cytoskeleton to be both dynamic and static in nature. Guiding these processes are a network of proteins that direct cellular behavior through their ability to bind microtubules (MTs) in a spatial- and temporal-specific manner. The identification and characterization of the suite of microtubule binding and associated proteins (MAPs) involved in MT systems is important for the understanding of the biological form and function of each MT system. This research involved the analysis and annotation of four MAPs – Ensconsin in Humans, Hook (homolog 3) in Humans, Protein Regulator of Cytokinesis 1 (PRC1) in Humans and Anaphase Spindle Elongation protein (ASE1) in yeast. A bioinformatics approach was used for the annotation and analysis. A protocol for analysis and annotation of MAPs was developed. During the process, some limitations in using bioinformatics tools and procedures were encountered. These limitations were overcome, the initial protocol was improved on and a modified protocol of analysis was developed. A database was designed and built to hold annotated information on the MAPs. We seek to disseminate this database and its functionalities as a web resource to the scientific community. It will provide an excellent forum for researchers to obtain relevant information on MT binding and associated proteins (MAPs). Infection by parasitic protozoa causes incalculable morbidity and mortality to humans and agricultural animals. In this research, we have also focused on MAPs in parasitic organisms of the Apicomplexan and Trypanosomatid genera. The protocol for analysis incorporates steps to analyze MAPs from these organisms as well. Malaria (a potentially life threatening disease) is caused by Plasmodium, an Apicomplexan parasite. This parasite is transmitted to people by the female Anopheles mosquito, which feeds on human blood. African Sleeping Sickness is an acute disease 8 caused by Trypanosoma brucei that typically leads to death within weeks or months if not treated. Microtubule-associated proteins (MAPs) and their alteration of the unique microtubule (MT) systems play major roles in these organisms throughout their life cycle and are required for their pathogenic mechanisms. Each parasite contains unique MT systems that will test our annotation process as well as prepare the DB for addition of other novel MT systems, such as those contained with plants. Additionally, these single cell organisms have a multistage life cycle that provide similar annotation challenges to those encountered when one considers multi-cellular organisms. Therefore, a researcher working on any MT system within the database will find useful information regardless of the organism that they are studying. This will leave us with a sub-set of MAPs from parasitic organisms in our database that are potential drug-targets.Item CGM-measured glucose values have a strong correlation with C-peptide, HbA1c and IDAAC, but do poorly in predicting C-peptide levels in the two years following onset of diabetes(Springer-Verlag, 2015-06) Buckingham, Bruce; Cheng, Peiyao; Beck, Roy W.; Kollman, Craig; Ruedy, Katrina J.; Weinzimer, Stuart A.; Slover, Robert; Bremer, Andrew A.; Fuqua, John; Tamborlane, William; Diabetes Research in Children Network (DirecNet) and Type 1 Diabetes TrialNet Study Groups; Department of Pediatrics, IU School of MedicineAIMS/HYPOTHESIS: The aim of this work was to assess the association between continuous glucose monitoring (CGM) data, HbA1c, insulin-dose-adjusted HbA1c (IDAA1c) and C-peptide responses during the first 2 years following diagnosis of type 1 diabetes. METHODS: A secondary analysis was conducted of data collected from a randomised trial assessing the effect of intensive management initiated within 1 week of diagnosis of type 1 diabetes, in which mixed-meal tolerance tests were performed at baseline and at eight additional time points through 24 months. CGM data were collected at each visit. RESULTS: Among 67 study participants (mean age [± SD] 13.3 ± 5.7 years), HbA1c was inversely correlated with C-peptide at each time point (p < 0.001), as were changes in each measure between time points (p < 0.001). However, C-peptide at one visit did not predict the change in HbA1c at the next visit and vice versa. Higher C-peptide levels correlated with increased proportion of CGM glucose values between 3.9 and 7.8 mmol/l and lower CV (p = 0.001 and p = 0.02, respectively) but not with CGM glucose levels <3.9 mmol/l. Virtually all participants with IDAA1c < 9 retained substantial insulin secretion but when evaluated together with CGM, time in the range of 3.9-7.8 mmol/l and CV did not provide additional value in predicting C-peptide levels. CONCLUSIONS/INTERPRETATION: In the first 2 years after diagnosis of type 1 diabetes, higher C-peptide levels are associated with increased sensor glucose levels in the target range and with lower glucose variability but not hypoglycaemia. CGM metrics do not provide added value over the IDAA1c in predicting C-peptide levels.Item Cumulative ultraviolet radiation flux in adulthood and risk of incident skin cancers in women(Nature Publishing Group, 2014-04-01) Wu, S; Han, J; Vleugels, R A; Puett, R; Laden, F; Hunter, D J; Qureshi, A A; Department of Epidemiology, Richard M. Fairbanks School of Public HealthBackground: Solar ultraviolet (UV) exposure estimated based on residential history has been used as a sun exposure indicator in previous case–control and descriptive studies. However, the associations of cumulative UV exposure based on residential history with different skin cancers, including melanoma, squamous cell carcinoma (SCC), and basal cell carcinoma (BCC), have not been evaluated simultaneously in prospective studies. Methods: We conducted a cohort study among 108 578 women in the Nurses' Health Study (1976–2006) to evaluate the relative risks of skin cancers with cumulative UV flux based on residential history in adulthood. Results: Risk of SCC and BCC was significantly lower for women in lower quintiles vs the highest quintile of cumulative UV flux (both P for trend <0.0001). The association between cumulative UV flux and risk of melanoma did not reach statistical significance. However, risk of melanoma appeared to be lower among women in lower quintiles vs the highest quintile of cumulative UV flux in lag analyses with 2–10 years between exposure and outcome. The multivariable-adjusted hazard ratios per 200 × 10−4 Robertson–Berger units increase in cumulative UV flux were 0.979 (95% confidence interval (CI): 0.933, 1.028) for melanoma, 1.072 (95% CI: 1.041, 1.103) for SCC, and 1.043 (95% CI: 1.034, 1.052) for BCC. Conclusions: Associations with cumulative UV exposure in adulthood among women differed for melanoma, SCC, and BCC, suggesting a potential variable role of UV radiation in adulthood in the carcinogenesis of the three major skin cancers.Item Direct detection and quantification of microRNAs(Elsevier, 2009-04-01) Hunt, Eric A.; Goulding, Ann M.; Deo, Sapna K.; Department of Chemistry & Chemical Biology, School of ScienceItem Effects of Chronic Alcohol and Repeated Deprivations on Dopamine D1 and D2 Receptor Levels in the Extended Amygdala of Inbred Alcohol-Preferring Rats(Wiley Blackwell (Blackwell Publishing), 2006-01) Sari, Youssef; Bell, Richard L.; Zhou, Feng C.; Department of Anatomy & Cell Biology, IU School of MedicineBackground Dopaminergic (DA) activity in the extended amygdala (EA) has been known to play a pivotal role in mediating drug and alcohol addiction. Alterations of DA activity within the EA after chronic exposure to alcohol or substances of abuse are considered a major mechanism for the development of alcoholism and addiction. To date, it is not clear how different patterns of chronic alcohol drinking affect DA receptor levels. Therefore, the current studies investigated the effects of chronic ethanol consumption, with or without deprivations, on D1 and D2 receptor densities within the EA. Methods Inbred alcohol-preferring (iP) rats were divided into 3 groups with the following treatments: (1) water for 14 weeks; (2) continuous alcohol (C-Alc) for 14 weeks [24-hour concurrent access to 15 and 30% (v/v) ethanol]; or (3) repeatedly deprived of alcohol (RD-Alc) (24-hour concurrent access to 15 and 30% ethanol for 6 weeks, followed by 2 cycles of 2 weeks of deprivation of and 2 weeks of reexposure to ethanol access). At the end of 14 weeks, the rats were killed for autoradiographic labeling of D1 and D2 receptors. Results Compared with the water control group, both the C-Alc and the RD-Alc groups displayed increases in D1 receptor binding density in the anterior region of the Acb core, whereas the RD-Alc group displayed additional increases in D1 receptor binding density in anterior regions of the lateral and intercalated nuclei of the amygdala. Additionally, both C-Alc and RD-Alc rats displayed increases in D2 receptor binding density in anterior regions of the Acb shell and core, whereas RDAlc rats displayed additional increases in D2 receptor binding density in the dorsal striatum. Conclusion The results of this study indicate that 14-week extended alcohol drinking with continuous chronic or repeated deprivations increase binding sites of D1 and D2 receptors in specific regions of the EA with greater sensitivity in the anterior regions. The repeated deprivation has greater effect on altering D1 and D2 receptor binding sites in the Acb, dorsal striatum, and subamygdala regions. The current result indicates that the two drinking paradigms may have common as well as differential mechanisms on alteration of dopamine receptor–binding sites in specific regions of the EA.Item Established and emerging biomarkers for the prediction of type 1 diabetes: a systematic review(Elsevier, 2014-08) WATKINS, RENECIA A.; EVANS-MOLINA, CARMELLA; BLUM, JANICE S.; DIMEGLIO, LINDA A.; Department of Pediatrics, IU School of MedicineType 1 diabetes (T1D) is an autoimmune disease with a prolonged and variable latent period that culminates in the destruction of pancreatic β-cells and the development of hyperglycemia. There is a need for diagnostic biomarkers to detect more accurately detect individuals with prediabetes to expedite targeting for prevention and intervention strategies. To assess the current ability to predict the insidious development of T1D, we conducted a comprehensive systematic review for established and prospective predictive markers of T1D using the Medline, OVID, and EMBASE databases. Resulting citations were screened for relevance to subject. Our research generated five major categories of markers that are either currently used or forthcoming: genetic, autoantibodies, risk score quantification, cellular immunity, and β-cell function. The current standard used to assess T1D onset or predisposition focuses on autoimmune pathology and disease-associated autoantibodies. Research studies in general go beyond autoantibody screening and assess genetic predisposition, and quantitate risk of developing disease based on additional factors. However, there are few currently used techniques that assess the root of T1D: β-cell destruction. Thus, novel techniques are discussed with the potential to gauge degrees of β-cell stress and failure via protein, RNA, and DNA analyses.Item Evaluating the Conversation After the Ryan White Exhibit: A Systematic Examination of Research and Evaluation Methodologies(Office of the Vice Chancellor for Research, 2016-04-08) Day, BrittneyMy project is an exploration of methodologies for assessing visitor response to a museum exhibition centered on socially charged issues. My hypothesis is twofold: (a) family conversations continue after visitors leave a socially charged museum exhibit; (b) by analyzing these conversations, meaningful trends can be identified. I seek to establish a rigorous methodology for testing my hypothesis that can then be applied to the evaluation of similar socially charged museum exhibits. For my project, I structured a methodology that outlines the procedures for evaluating conversations that parents have with their children after they leave the Ryan White exhibit at the Children’s Museum of Indianapolis. The Ryan White exhibit raises awareness for issues such as HIV/AIDS, a difficult subject for many parents to discuss with their children. My project will begin with an evaluation design, which includes my project hypotheses, a brief project description, objectives, methods, and a timeline. I will reach out to the community via web postings and phone calls to find 6 subjects who fit my specified criteria: (a) Parents born between 1970 and 1980 (i.e. the same sociological generation as Ryan White); (b) parents must have one or more children ages 6-14. The sampling of participants will be completed on a voluntary basis. Before beginning the interviews, I will then complete training for the Collaborative Institutional Training Initiative (CITI), for the purpose of protecting the participants of my study. I will conduct 6, thirty minute, semi-structured interviews with my participants and analyze the results. This project will establish a foundation upon which a further research can be conducted on this subject. My finished product will exemplify the methodologies of evaluation for a socially charged museum exhibit, and will help scholars of Visitors Studies understand the complete dialogue of exhibits.Item Exhaled nitric oxide during infancy as a risk factor for asthma and airway hyperreactivity(European Respiratory Society, 2015-01) Chang, Daniel; Yao, Weiguo; Tiller, Christina J.; Kisling, Jeffrey; Slaven, James E.; Yu, Zhangsheng; Kaplan, Mark H.; Tepper, Robert S.; Department of Pediatrics, IU School of MedicineChildhood asthma is often characterised by elevated exhaled nitric oxide (eNO), decreased lung function, increased airway reactivity and atopy; however, our understanding of when these phenotypic airway characteristics develop remains unclear. This study evaluated whether eNO, lung function, airway reactivity and immune characteristics during infancy are risk factors of asthma at age 5 years. Infants with eczema, enrolled prior to wheezy illness (n=116), had eNO, spirometry, airway reactivity and allergen sensitisation assessed at entry to the study and repeated at age 5 years (n=90). Increasing eNO at entry was associated with an increased risk of asthma (p=0.037) and increasing airway reactivity (p=0.015) at age 5 years. Children with asthma at 5 years of age had a greater increase in eNO between infancy and age 5 years compared with those without asthma (p=0.002). Egg sensitisation at entry was also associated with an increased risk of asthma (p=0.020), increasing eNO (p = 0.002) and lower forced expiratory flows (p=0.029) as a 5 year-old. Our findings suggest that, among infants at high risk for developing asthma, eNO early in life may provide important insights into the subsequent risk of asthma and its airway characteristics.Item Isolation and Analysis of a Mycobacteriophage Specific to Mycobacterium Smegmatis(2015-12-15) Collins, Angela J.; Ghazali, Danish M.; Li, Yi; Ha, Soo; Clase, Kari L.; Hatfull, GrahamBACKGROUND: Bacteriophages are viruses that kill bacteria. It is estimated that there are roughly 1031 types of bacteriophages in the world, and that every bacterium has an average of 10 bacteriophages that can infect it. A mycobacteriophage is a type of bacteriophage which has a mycobacterial species as its host. There are 30 known clusters, or types, or mycobacteriophages which all contain very distinct genetic structures, but there are still countless mycobacteriophage populations that need to be isolated. Now that microorganisms are becoming resistant to many antibiotics, the study of bacteriophages is increasingly important because they have an incredibly untapped potential to treat resistant bacteria, eradicate bacterial contaminants in food products, promote ulcer healing, control bacterial growth during fermentation, and much more. The goal of this project was to discover new bacteriophages that might contribute to novel medical solutions. MATERIALS AND METHODS: We plated 29 agar plates for 8 different samples. One sample resulted in plaque growth, from which we performed 9 streak tests, underwent a dilution series and filtration to eliminate the contamination, and harvested a High Titer Lysate. After isolating and purifying the phage genomic DNA, we analyzed the sample and quantified the DNA using a Nanodrop spectrophotometer, and we used agarose gel electrophoresis to separate and analyze the DNA fragments. Finally, we used electron mycrocopy to visualize the physical structure and confirm the successful isolation of a single mycobacteriophage population. DISCUSSION: The cloudiness of the plaque produced by the bacteriophage suggests it is a temperate phage, meaning it switchs between two behavior types regarding replication and survival. Analysis of the physical structure of the mycobacteriophage reveals more information about its genomic DNA; its large capsid diameter suggests lengthy indwelling genomic DNAand its relatively long tail suggests a large tape-measure gene. We were unble to analyze the agarose gel electrophoresis results due to time constraints. However, the results of the genomic DNA sequence will hopefully prove that our bacteriophage is a distinct population. CONCLUSION: We successfully captured, isolated, and purified a single bacteriophage species from the local environment of West Lafayette, Indiana. Upon isolating a single bacteriophage species, we then isolated, purified, restricted, and analyzed the genomic DNA of the phage. Finally, we documented our findings on the Bacteriophage Database as FelixElFago (http://phagesdb.org/phages/FelixElFago/) and sent our DNA to the Sequencing Center.