Genetic variation among populations of, and evidence of deep divergence within, the Rio Grande Chirping Frog, Eleutherodactylus campi (Anura: Eleutherodactylidae).

Herein we report the first molecular assessment of intra-species genetic variation and interrelationships within the Rio Grande Chirping frog, Eleutherodactylus campi. We analyzed 548 base pairs of 16S rRNA gene for 71 ingroup individuals belonging to the genus Eleutherodactylus (including 42 E. campi sampled from 15 localities in the United States and Mexico) and four outgroup samples. By unveiling two highly divergent and geographically structured clades within E. campi this study provides a novel phylogenetic placement of E. campi populations north and south of the Rio Grande Valley as sister groups to each other. The observed level of genetic divergence between these two clades (5.8%) is, on average, comparable to or greater than the levels of divergence found between several currently valid amphibian species pairs. Estimates of Time to Most Common Ancestor (TMRCA) indicate that the phylogeographic split between the two E. campi clades may have occurred 7.6 MYA (i.e., late Miocene), consistent with the geologic history of southwestern North America. The study also confirms that south Texas served as the source population for populations of E. campi in its introduced range (i.e., Alabama, Louisiana, and Texas). Overall, this molecular study indicates that E. campi consists of two deeply divergent lineages corresponding to its populations north and south of Rio Grande Valley. These results suggest that the recovered lineages may represent independent species and thereby highlight the need for further research to clarify their status.

Design and Characterization of an Icosahedral Protein Cage Formed by a Double-Fusion Protein Containing Three Distinct Symmetry Elements.

Exploiting simple types of symmetry common to many natural protein oligomers as a starting point, several recent studies have succeeded in engineering complex self-assembling protein architectures reminiscent but distinct from those evolved in the natural world. Designing symmetric protein cages with a wide range of properties has been of particular interest for potential applications in the fields of medicine, energy, imaging, and more. In this study we genetically fused three naturally symmetric protein components together-a pentamer, trimer, and dimer-in a fashion designed to create a self-assembling icosahedral protein cage built from 60 copies of the protein subunit. The connection between the pentamer and dimer was based on a continuous shared α helix in order to control the relative orientation of those components. Following selection of suitable components by computational methods, a construct with favorable design properties was tested experimentally. Negative stain electron microscopy and solution-state methods indicated successful formation of a 60-subunit icosahedral cage, 2.5 MDa in mass and 30 nm in diameter. Diverse experimental studies also suggested substantial degrees of flexibility and asymmetric deformation of the assembled particle in solution. The results add further examples of successes and challenges in designing atomically precise protein materials.

Designed Protein Cages as Scaffolds for Building Multienzyme Materials.

The functions of enzymes can be strongly affected by their higher-order spatial arrangements. In this study we combine multiple new technologies-designer protein cages and sortase-based enzymatic attachments between proteins-as a novel platform for organizing multiple enzymes (of one or more types) in specified configurations. As a scaffold we employ a previously characterized 24-subunit designed protein cage whose termini are outwardly exposed for attachment. As a first-use case, we test the attachment of two cellulase enzymes known to act synergistically in cellulose degradation. We show that, after endowing the termini of the cage subunits with a short "sort-tag" sequence (LPXTG) and the opposing termini of the cellulase enzymes with a short polyglycine sequence tag, addition of sortase covalently attaches the enzymes to the cage with good reactivity and high copy number. The doubly modified cages show enhanced activity in a cellulose degradation assay compared to enzymes in solution, and compared to a combination of singly modified cages. These new engineering strategies could be broadly useful in the development of enzymatic material and synthetic biology applications.

An exploratory study of parental knowledge of early childhood oral health care in Southland, New Zealand.

OBJECTIVES: The primary objective was to clearly assess the oral health care knowledge of Southland parents. The secondary objective was to identify whether inequalities exist between parents with different ethnicity, education or income. DESIGN: An exploratory study based on a simple online/ paper questionnaire. SETTING: Participants were recruited through Southland early childcare centres. Researchers contacted 115 centres, 66 agreed to participate and 58 returned questionnaires. METHODS: Questionnaires were distributed to each centre to be completed by the parents. The questionnaire was able to be completed online or as a paper copy. Centres were supplied with dental brochures, which were distributed after the questionnaires were returned. Questionnaires were collated and the responses analysed. RESULTS: Six hundred and seventy questionnaires were returned, 213 online and 457 paper copies. The typical participant was a mother (93.9%), age 34 years (median), a non-smoker (86.3%), non-Maori (87.1%), with a university degree (33.9%) and an annual household income between $60,001 and $100,000 (36.5%). Twenty of the 47 questions were selected to reflect parental knowledge. Overall, 65.1% of the respondents answered all 20 questions correctly. Differences in knowledge were identified between mothers and other participants (65.4% vs. 59.4%), smokers and non-smokers (61.3% vs. 65.7%), Maori and non-Maori (61.6% vs. 65.6%) and education level (Primary 58.0% vs. Degree 68.7%) (P < 0.05). CONCLUSIONS: This study revealed differences in knowledge in early childhood oral health care. Participants who identified as non-mothers (fathers, step-fathers, legal care givers or other), smokers, Maori or low education displayed significantly less knowledge. Further education and oral health care promotion may be needed to improve this disparity.

Dental attendance, perceptions of cost and self-care of school year 12 and 13 students: A focus on Southland, New Zealand.

BACKGROUND AND OBJECTIVES: Adolescents and emerging adults can provide dentists with many challenges. Little information is available on their perceptions of dental costs once they turn 18 and dentistry is no longer State-funded. The aim of this study was to explore the use of dental care by Southland students in years 12 and 13, their perceptions of the cost of four common dental procedures, self-related oral health and dental self-care habits, time off school related to dental problems, and knowledge and views regarding fluoride. METHODS: After ethical approval, a 26-question survey was conducted of all Southland students in years 12 and 13. Data were statistically analysed in SPSS version 20 with the alpha value set at 0.05. RESULTS: The participation rate was 49.6%. Regular attendance for examinations was reported by 77.5% with non-attendance mainly related to attitudes around lack of importance or necessity. Reported dental attendance varied according to gender, ethnicity and decile rating of school attended. Although some were accurate in their estimations of dental costs, the standard deviation for all procedures was large. The majority thought that costs put people off going to the dentist. While 74.8% brushed their teeth at least twice daily, only 26.6% flossed regularly. Knowledge regarding fluoride was lacking. CONCLUSIONS: It may be advantageous to include education regarding costs of dental care with patients of this age. This may motivate them to improve their self-care and ensure that their oral health is of a high standard before their dental needs are no longer State-funded.