Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay

Impartial comparative analysis of measurement of leukocyte telomere length/DNA content by Southern blots and qPCR.

Telomere length/DNA content has been measured in epidemiological/clinical settings with the goal of testing a host of hypotheses related to the biology of human aging, but often the conclusions of these studies have been inconsistent. These inconsistencies may stem from various reasons, including the use of different telomere length measurement techniques. Here, we report the first impartial evaluation of measurements of leukocyte telomere length by Southern blot of the terminal restriction fragments and quantitative PCR (qPCR) of telomere DNA content, expressed as the ratio of telomeric product (T)/single copy gene (S) product. Blind measurements on the same samples from 50 donors were performed in two independent laboratories on two different occasions. Both the qPCR and Southern blots displayed highly reproducible results as shown by r values > 0.9 for the correlations between results obtained by either method on two occasions. The inter-assay CV measurement for the qPCR was 6.45%, while that of the Southern blots was 1.74%. The relation between the results generated by Southern blots versus those generated by qPCR deviated from linearity. We discuss the ramifications of these findings with regard to measurements of telomere length/DNA content in epidemiological/clinical circumstances

Home Away from Home: Diaspora Tourism and Transnational Attachment of Second-Generation Chinese-Americans

Globalization has allowed contemporary immigrants to live in two worlds and maintain virtual and physical contact with their homeland through leisure and diaspora tourism. This study examined the lived experience of second-generation Chinese-Americans as they traveled to their parents’ country of origin and explored the relationship between their transnational homeland attachment and diaspora tourism experience. Using a phenomenological approach, twenty-six second-generation Chinese-Americans who had the experience of traveling in China were interviewed. Four themes were identified from their travel experience: destination image, authenticity, family history, and homeland attachment. Findings revealed how being a secondgeneration immigrant influenced the way they saw and experienced China as both destination and homeland

The Specific Characteristics in Children with Obstructive Sleep Apnea and Cor Pulmonale

Background. The prevalence of obstructive sleep apnea (OSA) in the pediatric population is currently estimated at 1-2% of all children. The purpose of this study was to investigate the clinical and hemodynamic characteristics in pediatric patients with cor pulmonale and OSA. Methods. Thirty children with the diagnosis of OSA were included. These patients consisted of 26 male and 4 female children with a mean age of 7 ± 4 years old. Five of those children were found to be associated with cor pulmonale, and 25 had OSA but without cor pulmonale. Results. The arousal index was much higher in children with OSA and cor pulmonale. The children with OSA and cor pulmonale had much lower mean and minimal oxygen saturation and a higher incidence of bradycardia events. All 5 patients with OSA and cor pulmonale underwent an adenotonsillectomy, and the pulmonary arterial pressure dropped significantly after the surgery. Conclusion. This study demonstrated that the OSA pediatric patients with cor pulmonale had the different clinical manifestations and hemodynamic characteristics from those without cor pulmonale. The adenotonsillectomy had excellent results in both the OSA pediatric patients with and without cor pulmonale

Real-Time, Digital LAMP with Commercial Microfluidic Chips Reveals the Interplay of Efficiency, Speed, and Background Amplification as a Function of Reaction Temperature and Time

Real-time, isothermal, digital nucleic acid amplification is emerging as an attractive approach for a multitude of applications including diagnostics, mechanistic studies, and assay optimization. Unfortunately, there is no commercially available and affordable real-time, digital instrument validated for isothermal amplification; thus, most researchers have not been able to apply digital, real-time approaches to isothermal amplification. Here, we generate an approach to real-time digital loop-mediated isothermal amplification (LAMP) using commercially available microfluidic chips and reagents and open-source components. We demonstrate this approach by testing variables that influence LAMP reaction speed and the probability of detection. By analyzing the interplay of amplification efficiency, background, and speed of amplification, this real-time digital method enabled us to test enzymatic performance over a range of temperatures, generating high-precision kinetic and end-point measurements. We were able to identify the unique optimal temperature for two polymerase enzymes while accounting for amplification efficiency, nonspecific background, and time to threshold. We validated this digital LAMP assay and pipeline by performing a phenotypic antibiotic susceptibility test on 17 archived clinical urine samples from patients diagnosed with urinary tract infections. We provide all the necessary workflows to perform digital LAMP using standard laboratory equipment and commercially available materials. This real-time digital approach will be useful to others in the future to understand the fundamentals of isothermal chemistries, including which components determine amplification fate, reaction speed, and enzymatic performance. Researchers can also adapt this pipeline, which uses only standard equipment and commercial components, to quickly study and optimize assays using precise, real-time digital quantification, accelerating development of critically needed diagnostics

Dynamic Metasurface Antennas for Energy Efficient Massive MIMO Uplink Communications

Future wireless communications are largely inclined to deploy a massive number of antennas at the base stations (BS) by exploiting energy-efficient and environmentally friendly technologies. An emerging technology called dynamic metasurface antennas (DMAs) is promising to realize such massive antenna arrays with reduced physical size, hardware cost, and power consumption. This paper aims to optimize the energy efficiency (EE) performance of DMAs-assisted massive MIMO uplink communications. We propose an algorithmic framework for designing the transmit precoding of each multi-antenna user and the DMAs tuning strategy at the BS to maximize the EE performance, considering the availability of the instantaneous and statistical channel state information (CSI), respectively. Specifically, the proposed framework includes Dinkelbach's transform, alternating optimization, and deterministic equivalent methods. In addition, we obtain a closed-form solution to the optimal transmit signal directions for the statistical CSI case, which simplifies the corresponding transmission design. The numerical results show good convergence performance of our proposed algorithms as well as considerable EE performance gains of the DMAs-assisted massive MIMO uplink communications over the baseline schemes

“To Plan or ot to Plan:” Serendipitous vs. Organized Travel

The recent invention and popularity of mobile navigation devices have increased the possibility of unplanned, serendipitous travel. To better understand the independent travel market, this study developed a scale for the construct “Style of Independent Travel” (SIT) in order to explore the serendipitous and organized dimensions of travel and their relationship to relevant travel behavior. The scale was included in a survey of visitors to the Rocky Knob area of southwest Virginia. Findings revealed that tourists had a higher preference for travelling spontaneously than following a fixed and organized itinerary and that serendipity and organization in travel was related to travel planning and visitor activities. However, there was no significant relationship between SIT, past visitation, and role of the destination as primary destination or stopover

Inter-domain traffic routing in vehicular delay tolerant networks

“Copyright © [2010] IEEE. Reprinted from IEEE International Conference on Communications (IEEE ICC 2010). ISSN:1550-3607. This material is posted here with permission of the IEEE. Internal or personal use of this material is permitted. However, permission to reprint/republish this material for advertising or promotional purposes or for creating new collective works for resale or redistribution must be obtained from the IEEE by writing to pubs [email protected]. By choosing to view this document, you agree to all provisions of the copyright laws protecting it.”In this paper, we consider the problem of dynamic inter-domain traffic routing between a VDTN and a non-DTN (e.g., Internet). The inter-domain traffic can be classified as inbound and outbound traffic. Our main contribution in this work is the intro- duction of several fault-tolerant routing algorithms for inbound and outbound traffic. Using simulations, we compare the performance of the proposed algorithms in terms of required resources, packet delivery time, and blocking probability.This work was supported in part by the Instituto de Telecomunicações, Next Generation Networks and Applications Group (NetGNA), Covilhã Delegation, Portugal in the framework of the VDTN@Lab Project

Metastases From Nested Variant Urothelial Carcinoma of the Urinary Bladder in Pancreatic Allograft Mimicking Graft Rejection

While not an uncommon tumor, urothelial carcinoma of the urinary bladder is rare in bladders draining pancreatic allografts. A case of urothelial carcinoma directly involving a pancreatic allograft with metastasis that occurred in a 49-year-old pancreas and kidney transplant recipient is described. Her initial clinical presentation and findings of CT scan of the abdomen suggested pancreatitis with features worrisome for rejection. A biopsy of her pancreatic allograft contained poorly differentiated carcinoma and cystoscopic biopsy disclosed an invasive high grade urothelial carcinoma arising in the background of extensive urothelial carcinoma in situ. Exploratory laparotomy revealed that the tumor invaded the right ovary and fallopian tube, cecum, and allograft with extensive retroperitoneal involvement. She underwent en bloc resection of distal ileum and cecum, resection of transplant pancreas, partial cystectomy, ileocolostomy anastomosis, and right salpingo-oophorectomy. Postoperatively, the patient was treated with four cycles of carboplatin and gemcitabine. She ultimately succumbed to her disease approximately 1 year after diagnosis. This case should alert physicians and radiologists to be aware of atypical presentation of urothelial carcinoma in bladder-drained pancreas grafts, the aggressiveness of such lesions, and the need for early biopsy to avoid diagnostic confusion with rejection