Statistical tools for transgene copy number estimation based on real-time PCR

Background As compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination. Results Three experimental designs and four data quality control integrated statistical models are presented. For the first method, external calibration curves are established for the transgene based on serially-diluted templates. The Ct number from a control transgenic event and putative transgenic event are compared to derive the transgene copy number or zygosity estimation. Simple linear regression and two group T-test procedures were combined to model the data from this design. For the second experimental design, standard curves were generated for both an internal reference gene and the transgene, and the copy number of transgene was compared with that of internal reference gene. Multiple regression models and ANOVA models can be employed to analyze the data and perform quality control for this approach. In the third experimental design, transgene copy number is compared with reference gene without a standard curve, but rather, is based directly on fluorescence data. Two different multiple regression models were proposed to analyze the data based on two different approaches of amplification efficiency integration. Our results highlight the importance of proper statistical treatment and quality control integration in real-time PCR-based transgene copy number determination. Conclusion These statistical methods allow the real-time PCR-based transgene copy number estimation to be more reliable and precise with a proper statistical estimation. Proper confidence intervals are necessary for unambiguous prediction of trangene copy number. The four different statistical methods are compared for their advantages and disadvantages. Moreover, the statistical methods can also be applied for other real-time PCR-based quantification assays including transfection efficiency analysis and pathogen quantification

New Concepts in Median Nail Dystrophy, Onychomycosis, and Hand, Foot, and Mouth Disease Nail Pathology

Nails are underutilized as diagnostic tools, despite being involved in many dermatologic conditions. This paper explores new concepts in the treatment of median nail dystrophy (MND), onychomycosis, and the nail pathology of hand, foot, and mouth disease (HFMD). A Pubmed database literature search was conducted for MND treatment, onychomycosis treatment, and HFMD nail pathology. Only papers published after January 2008 were reviewed. The results showed that 0.1% tacrolimus ointment can be an effective treatment for MND. Early studies on laser therapy indicate that it is a safe and efficacious treatment option for onychomycosis, compared to conventional oral antifungal agents. Vicks VapoRub (The Proctor & Gamble Company, Cincinnati, OH) is effective against onychomycosis and is a reasonable option in patients who choose to forgo conventional treatments. Lastly, there is evidence to support a correlation between HFMD and onychomadesis

Research Knowledge of Advanced Standing and Traditional Students: Implications for BSW Education

The advanced standing model of social work education, which affords graduate credit to qualified BSW students who pursue their MSW, has not been without issue or controversy, including questions of potential differences in performance on various educational outcomes. Specifically related to research curriculum, the importance of which is often not wholly embraced by students, this article reports the results of a secondary data analysis comparing research knowledge among advanced standing and traditional MSW students as well as among the various undergraduate majors (i.e., BSW, psychology, and sociology). Results suggest that research knowledge is similar and low across student subgroups. Important differences in research knowledge were found among student groups based on undergraduate major, with BSW undergraduates without advanced standing, on average, scoring lower than any other group. Implications for BSW research curriculum are considere

Self-reported symptoms and managment by midwestern breast cancer survivors

Lymphedema (LE) is a life-long potential consequence of breast cancer treatment that may affect quality of life of breast cancer survivors in long-term survivorship. Previous studies reported that about 2 million women living with breast cancer are at a lifetime risk for LE development. Information from self-reported lymphedema symptoms and its management will provide potential early detection and intervention to manage LE. The purposes of this study were: To report the frequency of occurrence of commonly self- reported LE symptoms following breast cancer diagnosis and treatment. To find the most commonly reported self-management actions taken for the five LE symptoms.Research supported by NIH/NINR NR05342/NR010293, University of Missouri PRIME funds, and Ellis Fischel Cancer Center research funds

Acoustic Processing of Fluidic Samples for Planetary Exploration

The search for life on other planetary bodies is driven by our quest to determine if Earth is unique with respect to harboring life. In order to answer this question, instrumentation with suitable sensitivity is required to measure biosignatures. In addition to accurate measurements by in-situ instruments, specialized sample acquisition and sample handling hardware is required to maximize the scientific information obtained from an acquired sample. This paper discusses a class of compact sample processing instrumentation using solid-state mechanisms that use acoustic waves to process samples prior to delivery to the instrument. Some of the acoustic sample processes that can be used to aid in preparation of liquid and liquid/solid mixtures include: mixing, milling, cavitating, lysing, heating, streaming, stirring, lofting, concentrating, segregating, and filtering. We will review these acoustic processes and show how they are generated using electromechanical systems. In addition to processing, these transduction systems could also use acoustics to interrogate physical properties such as the state of the sample, the acoustic velocity, and its attenuation. In order to generate these processes and sensing capabilities at these frequencies, a transduction mechanism is required to produce stress waves from electrical signals and vice versa. One commonly used technique is to use piezoelectric transducers that generate a stress that is linearly proportional to the voltage across the transducer and a voltage that is proportional to the stress on a transducer’s face. A variety of transducer modes are available to excite the sample, including thickness, transverse, radial, and shear extensional, and these can be used to build composite resonance structures including ultrasonic horns, tuning forks, bimorph, and unimorph benders to increase stress generated in the sample. We discuss how to model the acoustic interactions with the sample and the sample chamber in order to produce the required stress waves and illustrate the use of network models of piezoelectric transducers to accomplish this modeling. We demonstrate how to build up these models using Mason’s equivalent circuit for a piezoelectric and network models for acoustic layers in a design. Finally, to illustrate this acoustic processing ability, we will discuss a few systems that we have developed for sample handling systems for other planetary bodies like Mars and ocean worlds Enceladus and Europa

Genomic analysis of expressed sequence tags in American black bear Ursus americanus

<p>Abstract</p> <p>Background</p> <p>Species of the bear family (<it>Ursidae</it>) are important organisms for research in molecular evolution, comparative physiology and conservation biology, but relatively little genetic sequence information is available for this group. Here we report the development and analyses of the first large scale Expressed Sequence Tag (EST) resource for the American black bear (<it>Ursus americanus</it>).</p> <p>Results</p> <p>Comprehensive analyses of molecular functions, alternative splicing, and tissue-specific expression of 38,757 black bear EST sequences were conducted using the dog genome as a reference. We identified 18 genes, involved in functions such as lipid catabolism, cell cycle, and vesicle-mediated transport, that are showing rapid evolution in the bear lineage Three genes, Phospholamban (<it>PLN</it>), cysteine glycine-rich protein 3 (<it>CSRP3</it>) and Troponin I type 3 (<it>TNNI3</it>), are related to heart contraction, and defects in these genes in humans lead to heart disease. Two genes, biphenyl hydrolase-like (<it>BPHL</it>) and <it>CSRP3</it>, contain positively selected sites in bear. Global analysis of evolution rates of hibernation-related genes in bear showed that they are largely conserved and slowly evolving genes, rather than novel and fast-evolving genes.</p> <p>Conclusion</p> <p>We provide a genomic resource for an important mammalian organism and our study sheds new light on the possible functions and evolution of bear genes.</p

Kinetics of antigen expression and epitope presentation during virus infection

Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity.NHMRC (National Health and Medical Research Council of Australia

Coral restoration in a changing world - a global synthesis of methods and techniques

Coral reef ecosystems have suffered an unprecedented loss of habitat-forming hard corals in recent decades, due to increased nutrient outputs from agriculture, elevated levels of suspended sediment caused by deforestation and development, destructive fishing practices, over-harvesting of reef species, outbreaks of corallivorous crown-of-thorns starfish (COTS, Acanthaster planci), coral disease and tropical storms. However, in recent years climate change has emerged as the primary threat to coral reefs. While reefs have a natural capacity for recovery, recurring events like mass coral bleaching and extreme weather events is increasing in frequency, intensity and severity, and are eroding the time for recovery between catastrophic events. Marine conservation has primarily focused on passive habitat protection over active restoration, in contrast to terrestrial ecosystems where active restoration is common practice. Further, active restoration is well accepted for wetlands and shellfish reefs however coral reef restoration has remained controversial both in academia and amongst marine managers. This is despite recent research suggesting that optimal conservation outcomes include both habitat protection and restoration. Critics often argue that coral restoration detracts focus from mitigating climate change and other threats to the marine environment, while proponents of coral restoration counter that interventions can serve to protect coral biodiversity and endangered species in the short-term, while mitigation of large-scale threats such as climate change and water quality take effect. Despite this disconnect between coral restoration practitioners, coral reef managers and scientists, active coral restoration is increasingly used as a tool to attempt to restore coral populations. The field has largely developed through independent work of isolated groups, and has fallen victim to ‘growing pains’ associated with ecological restoration in many other ecosystems. Partly this is due to a reluctance to share outcomes of projects, and in some cases a lack of monitoring or appropriate reporting of project outcomes. To mitigate this, we aimed to synthesise the available knowledge in a comprehensive global review of coral restoration methods, incorporating data from a traditional literature search of the scientific literature, complemented with information gathered from online sources and through a survey of coral restoration practitioners. We identified 329 case studies on coral restoration, of which 195 were from the scientific literature, 79 were sourced from the grey literature (i.e. reports and online descriptions), and 55 were responses to our survey of restoration practitioners. We identified ten coral restoration intervention types: coral gardening - transplantation phase (23% of records), direct transplantation (21%), artificial reefs (19%), coral gardening - nursery phase (17%), coral gardening (both phases, 7%), substrate enhancement with electricity (4%), substrate stabilisation (4%), algae removal (2%), larval enhancement (1%) and microfragmentation (<1%). The majority of interventions involve coral fragmentation or transplantation of coral fragments (70%). While 52 countries are represented in the dataset, the majority of projects were conducted in the USA, Philippines, Thailand and Indonesia (together representing 40% of projects). Coral restoration case studies are dominated by short-term projects, with 66% of all projects reporting less than 18 months of monitoring of the restored sites. Overall, the median length of projects was 12 months. Similarly, most projects are relatively small in spatial scale, with a median size of restored area of 500 m2. A diverse range of species are represented in the dataset, with 221 different species from 89 coral genera. Overall, coral restoration projects focused primarily (65% of studies) on fast-growing branching corals. Among all the published documents, the top five species (22% of studies) were Acropora cervicornis, Pocillopora damicornis, Stylophora pistillata, Porites cylindrica and Acropora palmata. Over a quarter of projects (26%) involved the coral genus Acropora, while 9% of studies included a single species - Acropora cervicornis. Much of the focus on Acropora cervicornis and Acropora palmata is likely to have resulted from these important reef-forming species being listed as threatened on the United States Endangered Species List and as Endangered on the International Union for Conservation of Nature Red List of Endangered Species (IUCN 2018). We have dedicated a section to each intervention type covered in this review, and describe the potential and limitations of each intervention type in detail there. However, collating this information has highlighted the following main points which apply to coral restoration in general. 1. On average, survival in restored corals is relatively high. All coral genera with sufficient replication from which to draw conclusions (>10 studies listing that genus) report an average survival between 60-70%. 2. Differences in survival and growth are largely species and/or location specific, so the selection of specific methods should be tailored to the local conditions, costs, availability of materials, and to the specific objectives of each project. 3. Projects are overall small and short, however substantial scaling up is required for restoration to be a useful tool in supporting the persistence of reefs in the future. While there is ample evidence detailing how to successfully grow corals at smaller scales, few interventions demonstrate a capacity to be scaled up much beyond one hectare. Notable exceptions include methods which propagate sexually derived coral larvae. 4. To date, coral restoration has been plagued by the same common problems as ecological restoration in other ecosystems. Mitigating these will be crucial to successfully scale up projects, and to retain public trust in restoration as a tool for resilience based management. a. Lack of clear objectives - There is a clear mismatch between the stated objectives of projects, and the design of projects and monitoring of outcomes. Poorly articulated or overinflated objectives risk alienating the general public and scientists, by over-promising and under-delivering. Social and economic objectives have inherent value and do not need to be disguised with ecological objectives. b. Lack of appropriate monitoring - A large proportion of projects do not monitor metrics relevant to their stated objectives, or do not continue monitoring for long enough to provide meaningful estimates of success. Further, there is a clear need for standardisation in the metrics that are used, to allow comparisons between projects. c. Lack of appropriate reporting - The outcomes of a large proportion of projects are not documented, which restricts knowledge-sharing and adaptive learning. While we attempted to access some of the unreported projects through our survey, it is clear we have only scratched the surface of existing knowledge. d. Poorly designed projects - An effect of inadequate monitoring and reporting is that projects are poorly suited to their specific area and conditions. Improved knowledge-sharing and development of best practice coral restoration guidelines aims to mitigate this problem

Refining tree recruitment models

We used a micrometeorological dispersal model to simulate seed and seedling distributions derived from subcanopy balsam fir (Abies balsamea (L.) Mill.) source trees in a trembling aspen (Populus tremuloides Michx.) dominated forest. Our first objective was to determine the effect of substituting basal area for cone production as a proxy for seed output. The results showed that the r2 from the regression of predicted versus observed densities increased by ∼5% for seeds and ∼15% for seedling simulations. Our second objective was to determine the effects of changing the median horizontal wind speed. The median speed in this forest environment varies according to the proportion of leaves abscised. For values of the median expected wind speed between the extremes of leafless and full-canopy forests, the r2 of predicted versus observed varied between 0.35 and 0.49 for seeds and between 0.33 and 0.62 for seedling simulations. We demonstrated that the simple one-dimensional model can have added precision if the dispersal parameters are chosen so as to allow more fine-scale variation