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

Stable Aqueous Suspension and Self-Assembly of Graphite Nanoplatelets Coated with Various Polyelectrolytes

Exfoliated graphite nanoplatelets (xGnPs) with an average thickness of 1–10 nm present an inexpensive alternative to carbon nanotubes in many applications. In this paper, stable aqueous suspension of xGnP was achieved by noncovalent functionalization of xGnP with polyelectrolytes. The surfactants and polyelectrolytes were compared with respect to their ability to suspend graphite nanoplatelets. The surface charge of the nanoplatelets was characterized with zeta potential measurements, and the bonding strength of the polymer chains to the surface of xGnP was characterized with Raman spectroscopy. This robust method opens up the possibility of using this inexpensive nanomaterial in many applications, including electrochemical devices, and leads to simple processing techniques such as layer-by-layer deposition. Therefore, the formation of xGnP conductive coatings using layer-by-layer deposition was also demonstrated

Consensus of self-driven agents with avoidance of collisions

In recent years, many efforts have been addressed on collision avoidance of collectively moving agents. In this paper, we propose a modified version of the Vicsek model with adaptive speed, which can guarantee the absence of collisions. However, this strategy leads to an aggregated state with slowly moving agents. We therefore further introduce a certain repulsion, which results in both faster consensus and longer safe distance among agents, and thus provides a powerful mechanism for collective motions in biological and technological multi-agent systems.Comment: 8 figures, and 7 page

Use of the WARMF Model to Identify Sources of Oxygen Impairment and Potential Management Strategies for the San Joaquin River Watershed

Eutrophication of the San Joaquin River (SJR) has resulted in low dissolved oxygen (DO) conditions, which has led to a regulatory response and development of total maximum daily load (TMDL) allocations. Due to the dynamic nature of processes governing oxygen depletion in the SJR, a model was needed to help stakeholders understand the fate and transport of nutrients and oxygendemanding substances that cause the low DO conditions. Here, the Watershed Analysis Risk Management Framework (WARMF) model was used to simulate nutrient removal and control strategies, accounting for the secondary effects of growth and transformation between sources and discharge. Using the management alternatives in the WARMF Consensus Module, simulations were run to test the global removal of nutrient inputs on downstream phytoplankton growth, a major contributor of oxygen demand in the SJR. In the simulations, removal of ammonia had the greatest impact on downstream phytoplankton, causing a 32% reduction, while removal of phosphate and nitrate caused reductions of 25% and 13%, respectively. When ammonia and nitrate were both removed, phytoplankton reduction was 62%. These model results suggest that nitrogen control programs would be more effective than phosphorus programs. Using the Data Module in WARMF, input files were modified to determine the impacts of individual tributaries and agricultural drainages. In each simulation, the contributing loads for individual inputs were removed while maintaining flow. According to the model output, the largest impact on phytoplankton occurred with the removal of mass loads from Salt Slough (32% less than baseline). The effect of removing the mass loads from Mud Slough had a slightly lower impact (26% less than baseline). The WARMF model proved useful for exploration of planning and management alternatives, providing an expert decision-making tool that is available to stakeholders

Performance Evaluation of Whole Body Counting Facilities in the Marshall Islands (2002-2005)

The United States Department of Energy (U.S. DOE) has recently implemented a series of strategic initiatives to address long-term radiological surveillance needs at former U.S. nuclear test sites in the Marshall Islands (https://eed.llnl.gov/mi/). Local atoll governments have been actively engaged in developing shared responsibilities for protecting the health and safety of resettled and resettling population at risk from exposure to elevated levels of residual fallout contamination in the environment. Under the program, whole body counting facilities have been established at three locations in the Marshall Islands. These facilities are operated and maintained by Marshallese technicians with scientists from the Lawrence Livermore National Laboratory (LLNL) providing technical support services including data quality assurance and performance testing. We have also established a mirror whole body counting facility at the Lawrence Livermore National Laboratory as a technician training center. The LLNL facility also allows program managers to develop quality assurance and operational procedures, and test equipment and corrective actions prior to deployment at remote stations in the Marshall Islands. This document summarizes the results of external performance evaluation exercises conducted at each of the facilities (2002-2005) under the umbrella of the Oak Ridge National Laboratory Intercomparison Studies Program (ISP). The ISP was specifically designed to meet intercomparison requirements of the United States (U.S.) Department of Energy Laboratory Accreditation Program (DOELAP). In this way, the Marshall Islands Radiological Surveillance Program has attempted to establish quality assurance measures in whole body counting that are consistent with standard requirements used to monitor DOE workers in the United States. Based on ANSI N13.30, the acceptable performance criteria for relative measurement bias and precision for radiobioassay service laboratory quality control, performance evaluation, and accreditation is -25% to +50% and less than or equal to 40%, respectively

Childhood socioeconomic status and childhood maltreatment: Distinct associations with brain structure

The present study examined the relationship between childhood socioeconomic status (SES), childhood maltreatment, and the volumes of the hippocampus and amygdala between the ages of 25 and 36 years. Previous work has linked both low SES and maltreatment with reduced hippocampal volume in childhood, an effect attributed to childhood stress. In 46 adult subjects, only childhood maltreatment, and not childhood SES, predicted hippocampal volume in regression analyses, with greater maltreatment associated with lower volume. Neither factor was related to amygdala volume. When current SES and recent interpersonal stressful events were also considered, recent interpersonal stressful events predicted smaller hippocampal volumes over and above childhood maltreatment. Finally, exploratory analyses revealed a significant sex by childhood SES interaction, with women’s childhood SES showing a significantly more positive relation (less negative) with hippocampus volume than men’s. The overall effect of childhood maltreatment but not SES, and the sex-specific effect of childhood SES, indicate that different forms of stressful childhood adversity affect brain development differently

Genome wide screens in yeast to identify potential binding sites and target genes of DNA-binding proteins

Knowledge of all binding sites for transcriptional activators and repressors is essential for computationally aided identification of transcriptional networks. The techniques developed for defining the binding sites of transcription factors tend to be cumbersome and not adaptable to high throughput. We refined a versatile yeast strategy to rapidly and efficiently identify genomic targets of DNA-binding proteins. Yeast expressing a transcription factor is mated to yeast containing a library of genomic fragments cloned upstream of the reporter gene URA3. DNA fragments with target-binding sites are identified by growth of yeast clones in media lacking uracil. The experimental approach was validated with the tumor suppressor protein p53 and the forkhead protein FoxI1 using genomic libraries for zebrafish and mouse generated by shotgun cloning of short genomic fragments. Computational analysis of the genomic fragments recapitulated the published consensus-binding site for each protein. Identified fragments were mapped to identify the genomic context of each binding site. Our yeast screening strategy, combined with bioinformatics approaches, will allow both detailed and high-throughput characterization of transcription factors, scalable to the analysis of all putative DNA-binding proteins