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

Genotypic Diversity and Host-Specificity of Frankia Bacteria Associated with Sympatric Populations of Alnus rubra and Alnus rhombifolia in Oregon

Biological nitrogen fixation is one of the most critical processes contributing to ecosystem productivity and stability on a global scale. In temperate climates of the northern hemisphere, plant-root associated bacteria of the genus Frankia are the major nitrogen fixers in forest environments. Trees belonging to the genus Alnus are the most widespread hosts of Frankia in the Pacific Northwest, and a myriad of biotic and abiotic factors can influence the robustness of this symbiosis. Host identity and bacterial strain are important features that can impact Alnus-Frankia association, but little is known about the interplay of intrageneric hosts that co-occur in natural settings. In this study we investigated the genetic diversity and host specificity of Frankia bacteria associated with sympatrically occurring populations of Alnus rubra (red alder) and Alnus rhombifolia (white alder) in Oregon. Based on sequence analysis of the nifH gene recovered from root nodules we found low overall bacterial diversity. One dominant Frankia genotype was associated with both host species, indicating a lack of strong host specificity in this system. Our results suggest that certain intrageneric plant hosts with overlapping distributions show cross-compatibility with symbiotic actinorhizal bacteria, and that low strain diversity of these bacteria can persist across mixed host populations

An Evaluation of the Performance and Acceptability of Three LED Fluorescent Microscopes in Zambia: Lessons Learnt for Scale-Up

The World Health Organization recommends the roll-out of light-emitting diode (LED) fluorescent microscopes (FM) as an alternative to light microscopes in resource-limited settings. We evaluated the acceptability and performance of three LED FMs after a short orientation among laboratory technicians from government health centers in Zambia. Sixteen technicians with varied light microscopy experience were oriented to FMs and divided into groups; each group read a different set of 40 slides on each LED FM (Primo Star iLED™, Lumin™, FluoLED™) and on a reference mercury-vapor FM (Olympus BX41TF). Slide reading times were recorded. An experienced FM technician examined each slide on the Olympus BX41TF. Sensitivity and specificity compared to TB culture were calculated. Misclassification compared to the experienced technician and inter-rater reliability between trainees was assessed. Trainees rated microscopes on technical aspects. Primo Star iLED™, FluoLED™ and Olympus BX41TF had comparable sensitivities (67%, 65% and 65% respectively), with the Lumin™ significantly worse (56%; p<0.05). Specificity was low for trainees on all microscopes (75.9%) compared to the experienced technician on Olympus BX41TF (100%). Primo Star iLED™ had significantly less misclassification (21.1% p<0.05) than FluoLED™ (26.5%) and Lumin™ (26.8%) and significantly higher inter-rater reliability (0.611; p<0.05), compared to FluoLED™ (0.523) and Lumin™ (0.492). Slide reading times for LED FMs were slower than the reference, but not significantly different from each other. Primo Star iLED™ rated highest in acceptability measures, followed by FluoLED™ then Lumin™. Primo Star iLED™ was consistently better than FluoLED™ and Lumin™, and performed comparably to the Olympus BX41TF in all analyses, except reading times. The Lumin™ compared least favorably and was thought unacceptable for use. Specificity and inter-rater reliability were low for all microscopes suggesting that a brief orientation was insufficient in this setting. These results provide important data for resource-limited settings to consider as they scale-up LED FMs

Classification Models of Idiopathic Pulmonary Fibrosis Patients

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal interstitial lung disease with no current cure. Progression of IPF is difficult to predict as the clinical course can be highly variable and range from a rapidly deteriorating state to a relatively stable state, or may be characterized by a slow progressive decline. Therefore, the need for an accurate diagnosis and improved tools for monitoring and managing IPF is of paramount importance, all for understanding the mitochondrial structure and the function played in the IPF. Mitochondrial DNA copy number (MtDCN) has been correlated with mortality in IPF patients and is a source of potentially clinically relevant information. We investigated the effects of various expiratory variables on MtDCN via multiple linear regression models. The models and their theoretical framework are presented under a descriptive and then analytic approach to investigate the complex and impact causes of IPF. Generalized linear model (GLM) based boosting is fitted before and after imputing the missing data. The Bayesian Hierarchical logistic models with categorical response variables that were created using carefully chosen cut-off points to classify the patients. This research provides an opportunity for novel patient surveillances

Influence of forest-to-silvopasture conversion and drought on components of evapotranspiration

The northeastern U.S. is projected to experience more frequent short-term (1-2 month) droughts interspersed among larger precipitation events. Agroforestry practices such as silvopasture may mitigate these impacts of climate change while maintaining economic benefits of both agricultural and forestry practices. This study evaluated the effects of forest-to-silvopasture (i.e., 50% thinning) conversion on the components of evapotranspiration (transpiration, rainfall interception, and soil evaporation) during the growing season of 2016. The study coincided with a late-summer drought throughout the northeastern U.S., which allowed us to also evaluate the effects of forest-to-silvopasture conversion on drought responses of multiple tree species, including Pinus strobus, Tsuga canadensis, and Quercus rubra. In the reference forest and silvopasture, we observed declining soil moisture and tree water use during the drought for all three tree species. However, the decline in P. strobus water use in response to declining soil moisture in the silvopasture was not as steep as compared with the reference forest, resulting in greater water use in the silvopasture for this species. In contrast, we did not detect different water-use responses between forest and silvopasture in T. canadensis or Q. rubra. This suggests that forest-to-silvopasture conversion via thinning can alleviate drought stress for P. strobus and that this species may be more sensitive to moisture stress when competition for water is high in denser stands. Evapotranspiration was 35% lower in the silvopasture compared with the reference forest, primarily a result of lower transpiration and rainfall interception. While soil evaporation was greater in the silvopasture, this was not enough to offset the considerably lower transpiration and interception. We observed greater radial tree growth 1-3 years following conversion in the silvopasture as compared with the reference forest for T. canadensis and Q. rubra, but not for P. strobus. Overall, our results suggest that forest conversion to silvopasture (in lieu of clearcutting for new pasture) may mitigate the impacts of agricultural land use intensification and climate change on ecosystem services, especially in terms of sustaining hydrologic regulation functions. Further study is required to determine the generality of these results and whether these benefits extend beyond the first few years post-conversion

Screening for tuberculosis and testing for human immunodeficiency virus in Zambian prisons

Objective: To improve the Zambia Prisons Service's implementation of tuberculosis screening and human immunodeficiency virus (HIV) testing. Methods: For both tuberculosis and HIV, we implemented mass screening of inmates and community-based screening of those residing in encampments adjacent to prisons. We also established routine systems – with inmates as peer educators – for the screening of newly entered or symptomatic inmates. We improved infection control measures, increased diagnostic capacity and promoted awareness of tuberculosis in Zambia's prisons. Findings: In a period of 9 months, we screened 7638 individuals and diagnosed 409 new patients with tuberculosis. We tested 4879 individuals for HIV and diagnosed 564 cases of infection. An additional 625 individuals had previously been found to be HIV-positive. Including those already on tuberculosis treatment at the time of screening, the prevalence of tuberculosis recorded in the prisons and adjacent encampments – 6.4% (6428/100 000) – is 18 times the national prevalence estimate of 0.35%. Overall, 22.9% of the inmates and 13.8% of the encampment residents were HIV-positive. Conclusion: Both tuberculosis and HIV infection are common within Zambian prisons. We enhanced tuberculosis screening and improved the detection of tuberculosis and HIV in this setting. Our observations should be useful in the development of prison-based programmes for tuberculosis and HIV elsewhere

Tree migration-rates : narrowing the gap between inferred post-glacial rates and projected rates

Faster-than-expected post-glacial migration rates of trees have puzzled ecologists for a long time. In Europe, post-glacial migration is assumed to have started from the three southern European peninsulas (southern refugia), where large areas remained free of permafrost and ice at the peak of the last glaciation. However, increasing palaeobotanical evidence for the presence of isolated tree populations in more northerly microrefugia has started to change this perception. Here we use the Northern Eurasian Plant Macrofossil Database and palaeoecological literature to show that post-glacial migration rates for trees may have been substantially lower (60–260 m yr–1) than those estimated by assuming migration from southern refugia only (115–550 m yr–1), and that early-successional trees migrated faster than mid- and late-successional trees. Post-glacial migration rates are in good agreement with those recently projected for the future with a population dynamical forest succession and dispersal model, mainly for early-successional trees and under optimal conditions. Although migration estimates presented here may be conservative because of our assumption of uniform dispersal, tree migration-rates clearly need reconsideration. We suggest that small outlier populations may be a key factor in understanding past migration rates and in predicting potential future range-shifts. The importance of outlier populations in the past may have an analogy in the future, as many tree species have been planted beyond their natural ranges, with a more beneficial microclimate than their regional surroundings. Therefore, climate-change-induced range-shifts in the future might well be influenced by such microrefugia

The ‘digital glimpse’ as imagining home

This paper proposes the concept of the ‘digital glimpse’, which develops the existing framing of imaginative travel. Here it articulates the experiences of mobile workers digitally connecting into family life and everyday rituals when physically absent with work. The recent embedding of digital communication technologies into personal relationships and family life is reconfiguring how absence is experienced and practiced by workers on the move, and through this, new digital paradigms for life on-the-move are emerging. This paper explores how such social relationships are maintained at-a-distance through digital technology – using evidence from qualitative interviews with mobile workers and their families. Digital technology now enables expressive forms of ‘virtual travel’, including video calling, picture sharing, and instant messaging. This has implications for the ways in which families can manage the social and relational pressures of being apart. Experiences of imaginative travel created through novel media can enrich the experience and give a greater sense of connection for both those who are at home and those who are away. While technology is limited in its ability to replicate a sense of co-presence, ‘digital glimpses’ are an emergent set of sociotechnical practices that can reduce the negative impact of absence on family relationships