DNA stabilized silver nanoclusters for ratiometric and visual detection of Hg2+ and its immobilization in hydrogels

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.bios.2013.04.002." © 2013. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/DNA oligomers are particularly interesting templates for making silver nanoclusters (AgNCs) as different emission colors can be obtained by varying the DNA sequence. Many AgNCs have been used as Hg2+ sensors since Hg2+ induces fluorescence quenching. From an analytical chemistry standpoint, however, these ‘light off’ sensors are undesirable. In this work, taking advantage of the fact that some AgNCs are not as effectively quenched by Hg2+, we design a sensor with AgNCs containing two emission peaks. The red peak is strongly quenched by Hg2+ while the green peak shows a concomitant increase, producing an orange-to-green visual fluorescence transformation. Using this AgNC, we demonstrate ratiometric detection with a detection limit of 4 nM Hg2+. This sensor is further immobilized in a hydrogel matrix and this gel is also capable of detecting Hg2+ with a visual response.University of Waterloo || Canadian Foundation for Innovation || Natural Sciences and Engineering Research Council || Ontario Ministry of Research and Innovation |

Blue emitting gold nanoclusters templated by poly-cytosine DNA at low pH and poly-adenine DNA at neutral pH

Blue fluorescent gold nanoclusters were prepared in the presence of poly-cytosine DNAs at low pH and poly-adenine at neutral pH using citrate as the reducing agent; various buffer conditions affecting the synthesis have been explored.University of Waterloo || Canadian Foundation for Innovation || Natural Sciences and Engineering Research Council || Ontario Ministry of Research and Innovation |

Correlation of photobleaching, oxidation and metal induced fluorescence quenching of DNA-templated silver nanoclusters

Few-atom noble metal nanoclusters have attracted a lot of interest due to their potential applications in biosensor development, imaging and catalysis. DNA-templated silver nanoclusters (AgNCs) are of particular interest as different emission colors can be obtained by changing the DNA sequence. A popular analytical application is fluorescence quenching by Hg2+, where d10–d10 metallophilic interaction has often been proposed for associating Hg2+ with nanoclusters. However, it cannot explain the lack of response to other d10 ions such as Zn2+ and Cd2+. In our effort to elucidate the quenching mechanism, we studied a total of eight AgNCs prepared by different hairpin DNA sequences; they showed different sensitivity to Hg2+, and DNA with a larger cytosine loop size produced more sensitive AgNCs. In all the cases, samples strongly quenched by Hg2+ were also more easily photobleached. Light of shorter wavelengths bleached AgNCs more potently, and photobleached samples can be recovered by NaBH4. Strong fluorescence quenching was also observed with high redox potential metal ions such as Ag+, Au3+, Cu2+ and Hg2+, but not with low redox potential ions. Such metal induced quenching cannot be recovered by NaBH4. Electronic absorption and mass spectrometry studies offered further insights into the oxidation reaction. Our results correlate many important experimental observations and will fuel the further growth of this field.University of Waterloo || Canadian Foundation for Innovation || Ontario Ministry of Research & Innovation || Natural Sciences and Engineering Research Council |

Predictive Models of Assistance Dog Training Outcomes Using the Canine Behavioral Assessment and Research Questionnaire and a Standardized Temperament Evaluation

Assistance dogs can greatly improve the lives of people with disabilities. However, a large proportion of dogs bred and trained for this purpose are deemed unable to successfully fulfill the behavioral demands of this role. Often, this determination is not finalized until weeks or even months into training, when the dog is close to 2 years old. Thus, there is an urgent need to develop objective selection protocols that can identify dogs most and least likely to succeed, from early in the training process. We assessed the predictive validity of two candidate measures employed by Canine Companions for Independence (CCI), a national assistance dog organization headquartered in Santa Rosa, CA. For more than a decade, CCI has collected data on their population using the Canine Behavioral Assessment and Research Questionnaire (C-BARQ) and a standardized temperament assessment known internally as the In-For-Training (IFT) test, which is conducted at the beginning of professional training. Data from both measures were divided into independent training and test datasets, with the training data used for variable selection and cross-validation. We developed three predictive models in which we predicted success or release from the training program using C-BARQ scores (N = 3,569), IFT scores (N = 5,967), and a combination of scores from both instruments (N = 2,990). All three final models performed significantly better than the null expectation when applied to the test data, with overall accuracies ranging from 64 to 68%. Model predictions were most accurate for dogs predicted to have the lowest probability of success (ranging from 85 to 92% accurate for dogs in the lowest 10% of predicted probabilities), and moderately accurate for identifying the dogs most likely to succeed (ranging from 62 to 72% for dogs in the top 10% of predicted probabilities). Combining C-BARQ and IFT predictors into a single model did not improve overall accuracy, although it did improve accuracy for dogs in the lowest 20% of predicted probabilities. Our results suggest that both types of assessments have the potential to be used as powerful screening tools, thereby allowing more efficient allocation of resources in assistance dog selection and training.Ministry of Food, Agriculture and Fisheries of Denmark (DFFE) [3304-FVFP-09-F-011]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Expression of HIV-1 antigens in plants as potential subunit vaccines

Background Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. Results Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. Conclusion Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine

Predictive Models of Assistance Dog Training Outcomes Using the Canine Behavioral Assessment and Research Questionnaire and a Standardized Temperament Evaluation

Assistance dogs can greatly improve the lives of people with disabilities. However, a large proportion of dogs bred and trained for this purpose are deemed unable to successfully fulfill the behavioral demands of this role. Often, this determination is not finalized until weeks or even months into training, when the dog is close to 2 years old. Thus, there is an urgent need to develop objective selection protocols that can identify dogs most and least likely to succeed, from early in the training process. We assessed the predictive validity of two candidate measures employed by Canine Companions for Independence (CCI), a national assistance dog organization headquartered in Santa Rosa, CA. For more than a decade, CCI has collected data on their population using the Canine Behavioral Assessment and Research Questionnaire (C-BARQ) and a standardized temperament assessment known internally as the In-For-Training (IFT) test, which is conducted at the beginning of professional training. Data from both measures were divided into independent training and test datasets, with the training data used for variable selection and cross-validation. We developed three predictive models in which we predicted success or release from the training program using C-BARQ scores (N = 3,569), IFT scores (N = 5,967), and a combination of scores from both instruments (N = 2,990). All three final models performed significantly better than the null expectation when applied to the test data, with overall accuracies ranging from 64 to 68%. Model predictions were most accurate for dogs predicted to have the lowest probability of success (ranging from 85 to 92% accurate for dogs in the lowest 10% of predicted probabilities), and moderately accurate for identifying the dogs most likely to succeed (ranging from 62 to 72% for dogs in the top 10% of predicted probabilities). Combining C-BARQ and IFT predictors into a single model did not improve overall accuracy, although it did improve accuracy for dogs in the lowest 20% of predicted probabilities. Our results suggest that both types of assessments have the potential to be used as powerful screening tools, thereby allowing more efficient allocation of resources in assistance dog selection and training

Development of Complement Factor H-Based Immunotherapeutic Molecules in Tobacco Plants Against Multidrug-Resistant Neisseria gonorrhoeae

Novel therapeutics against the global threat of multidrug-resistant Neisseria gonorrhoeae are urgently needed. Gonococci possess several mechanisms to evade killing by human complement, including binding of factor H (FH), a key inhibitor of the alternative pathway. FH comprises 20 short consensus repeat (SCR) domains organized in a head-to-tail manner as a single chain. N. gonorrhoeae binds two regions in FH; domains 6 and 7 and domains 18 through 20. We designed a novel anti-infective immunotherapeutic molecule that fuses domains 18-20 of FH containing a D-to-G mutation in domain 19 at position 1119 (called FH*) with human IgG1 Fc. FH*/Fc retained binding to gonococci but did not lyse human erythrocytes. Expression of FH*/Fc in tobacco plants was undertaken as an alternative, economical production platform. FH*/Fc was expressed in high yields in tobacco plants (300-600 mg/kg biomass). The activities of plant- and CHO-cell produced FH*/Fc against gonococci were similar in vitro and in the mouse vaginal colonization model of gonorrhea. The addition of flexible linkers [e.g., (GGGGS)2 or (GGGGS)3] between FH* and Fc improved the bactericidal efficacy of FH*/Fc 2.7-fold. The linkers also improved PMN-mediated opsonophagocytosis about 11-fold. FH*/Fc with linker also effectively reduced the duration and burden of colonization of two gonococcal strains tested in mice. FH*/Fc lost efficacy: i) in C6(-/-) mice (no terminal complement) and ii) when Fc was mutated to abrogate complement activation, suggesting that an intact complement was necessary for FH*/Fc function in vivo. In summary, plant-produced FH*/Fc represent promising prophylactic or adjunctive immunotherapeutics against multidrug-resistant gonococci

Resistant starch and exercise independently attenuate weight regain on a high fat diet in a rat model of obesity

<p>Abstract</p> <p>Background</p> <p>Long-term weight reduction remains elusive for many obese individuals. Resistant starch (RS) and exercise may be useful for weight maintenance. The effects of RS, with or without exercise, on weight regain was examined during relapse to obesity on a high carbohydrate, high fat (HC/HF) diet.</p> <p>Methods</p> <p>Obesity-prone rats were fed <it>ad libitum </it>for 16 weeks then weight reduced on a low fat diet to induce a 17% body weight loss (weight reduced rats). Weight reduced rats were maintained on an energy-restricted low fat diet for 18 weeks, with or without a daily bout of treadmill exercise. Rats were then allowed free access to HC/HF diet containing low (0.3%) or high (5.9%) levels of RS. Weight regain, energy balance, body composition, adipocyte cellularity, and fuel utilization were monitored as rats relapsed to obesity and surpassed their original, obese weight.</p> <p>Results</p> <p>Both RS and exercise independently attenuated weight regain by reducing the energy gap between the drive to eat and suppressed energy requirements. Exercise attenuated the deposition of lean mass during relapse, whereas its combination with RS sustained lean mass accrual as body weight returned. Early in relapse, RS lowered insulin levels and reduced the deposition of fat in subcutaneous adipose tissue. Exercise cessation at five weeks of relapse led to increased weight gain, body fat, subcutaneous adipocytes, and decreased lean mass; all detrimental consequences to overall metabolic health.</p> <p>Conclusions</p> <p>These data are the first to show the complimentary effects of dietary RS and regular exercise in countering the metabolic drive to regain weight following weight loss and suggest that exercise cessation, in the context of relapse on a HC/HF diet, may have dire metabolic consequences.</p