Dual transcriptional-translational cascade permits cellular level tuneable expression control.

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems

DOWNSTREAM IMPACTS OF SLUDGE MASS REDUCTION VIA ALUMINUM DISSOLUTION ON DWPF PROCESSING OF SAVANNAH RIVER SITE HIGH LEVEL WASTE - 9382

The SRS sludge that was to become a major fraction of Sludge Batch 5 (SB5) for the Defense Waste Processing Facility (DWPF) contained a large fraction of H-Modified PUREX (HM) sludge, containing a large fraction of aluminum compounds that could adversely impact the processing and increase the vitrified waste volume. It is beneficial to reduce the non-radioactive fraction of the sludge to minimize the number of glass waste canisters that must be sent to a Federal Repository. Removal of aluminum compounds, such as boehmite and gibbsite, from sludge can be performed with the addition of NaOH solution and heating the sludge for several days. Preparation of SB5 involved adding sodium hydroxide directly to the waste tank and heating the contents to a moderate temperature through slurry pump operation to remove a fraction of this aluminum. The Savannah River National Laboratory (SRNL) was tasked with demonstrating this process on actual tank waste sludge in our Shielded Cells Facility. This paper evaluates some of the impacts of aluminum dissolution on sludge washing and DWPF processing by comparing sludge processing with and without aluminum dissolution. It was necessary to demonstrate these steps to ensure that the aluminum removal process would not adversely impact the chemical and physical properties of the sludge which could result in slower processing or process upsets in the DWPF

EpiCollect: Linking Smartphones to Web Applications for Epidemiology, Ecology and Community Data Collection

Epidemiologists and ecologists often collect data in the field and, on returning to their laboratory, enter their data into a database for further analysis. The recent introduction of mobile phones that utilise the open source Android operating system, and which include (among other features) both GPS and Google Maps, provide new opportunities for developing mobile phone applications, which in conjunction with web applications, allow two-way communication between field workers and their project databases.Here we describe a generic framework, consisting of mobile phone software, EpiCollect, and a web application located within www.spatialepidemiology.net. Data collected by multiple field workers can be submitted by phone, together with GPS data, to a common web database and can be displayed and analysed, along with previously collected data, using Google Maps (or Google Earth). Similarly, data from the web database can be requested and displayed on the mobile phone, again using Google Maps. Data filtering options allow the display of data submitted by the individual field workers or, for example, those data within certain values of a measured variable or a time period.Data collection frameworks utilising mobile phones with data submission to and from central databases are widely applicable and can give a field worker similar display and analysis tools on their mobile phone that they would have if viewing the data in their laboratory via the web. We demonstrate their utility for epidemiological data collection and display, and briefly discuss their application in ecological and community data collection. Furthermore, such frameworks offer great potential for recruiting 'citizen scientists' to contribute data easily to central databases through their mobile phone

Etiology of Severe Non-malaria Febrile Illness in Northern Tanzania: A Prospective Cohort Study.

The syndrome of fever is a commonly presenting complaint among persons seeking healthcare in low-resource areas, yet the public health community has not approached fever in a comprehensive manner. In many areas, malaria is over-diagnosed, and patients without malaria have poor outcomes. We prospectively studied a cohort of 870 pediatric and adult febrile admissions to two hospitals in northern Tanzania over the period of one year using conventional standard diagnostic tests to establish fever etiology. Malaria was the clinical diagnosis for 528 (60.7%), but was the actual cause of fever in only 14 (1.6%). By contrast, bacterial, mycobacterial, and fungal bloodstream infections accounted for 85 (9.8%), 14 (1.6%), and 25 (2.9%) febrile admissions, respectively. Acute bacterial zoonoses were identified among 118 (26.2%) of febrile admissions; 16 (13.6%) had brucellosis, 40 (33.9%) leptospirosis, 24 (20.3%) had Q fever, 36 (30.5%) had spotted fever group rickettsioses, and 2 (1.8%) had typhus group rickettsioses. In addition, 55 (7.9%) participants had a confirmed acute arbovirus infection, all due to chikungunya. No patient had a bacterial zoonosis or an arbovirus infection included in the admission differential diagnosis. Malaria was uncommon and over-diagnosed, whereas invasive infections were underappreciated. Bacterial zoonoses and arbovirus infections were highly prevalent yet overlooked. An integrated approach to the syndrome of fever in resource-limited areas is needed to improve patient outcomes and to rationally target disease control efforts

CHARACTERIZATION AND ALUMINUM DISSOLUTION DEMONSTRATION WITH A 3 LITER TANK 51H SAMPLE

A 3-liter sludge slurry sample was sent to SRNL for demonstration of a low temperature aluminum dissolution process. The sludge was characterized before and after the aluminum dissolution. Post aluminum dissolution sludge settling and the stability of the decanted supernate were also observed. The characterization of the as-received 3-liter sample of Tank 51H sludge slurry shows a typical high aluminum HM sludge. The XRD analysis of the dried solids indicates Boehmite is the predominant crystalline form of aluminum in the sludge solids. However, amorphous phases of aluminum present in the sludge would not be identified using this analytical technique. The low temperature (55 C) aluminum dissolution process was effective at dissolving aluminum from the sludge. Over the three week test, {approx}42% of the aluminum was dissolved out of the sludge solids. The process appears to be selective for aluminum with no other metals dissolving to any appreciable extent. At the termination of the three week test, the aluminum concentration in the supernate had not leveled off indicating more aluminum could be dissolved from the sludge with longer contact times or higher temperatures. The slow aluminum dissolution rate in the test may indicate the dissolution of the Boehmite form of aluminum however; insufficient kinetic data exists to confirm this hypothesis. The aluminum dissolution process appears to have minimal impact on the settling rate of the post aluminum dissolution sludge. However, limited settling data were generated during the test to quantify the effects. The sludge settling was complete after approximately twelve days. The supernate decanted from the settled sludge after aluminum dissolution appears stable and did not precipitate aluminum over the course of several months. A mixture of the decanted supernate with Tank 11 simulated supernate was also stable with respect to precipitation

Converting simulated total dry matter to fresh marketable yield for field vegetables at a range of nitrogen supply levels

Simultaneous analysis of economic and environmental performance of horticultural crop production requires qualified assumptions on the effect of management options, and particularly of nitrogen (N) fertilisation, on the net returns of the farm. Dynamic soil-plant-environment simulation models for agro-ecosystems are frequently applied to predict crop yield, generally as dry matter per area, and the environmental impact of production. Economic analysis requires conversion of yields to fresh marketable weight, which is not easy to calculate for vegetables, since different species have different properties and special market requirements. Furthermore, the marketable part of many vegetables is dependent on N availability during growth, which may lead to complete crop failure under sub-optimal N supply in tightly calculated N fertiliser regimes or low-input systems. In this paper we present two methods for converting simulated total dry matter to marketable fresh matter yield for various vegetables and European growth conditions, taking into consideration the effect of N supply: (i) a regression based function for vegetables sold as bulk or bunching ware and (ii) a population approach for piecewise sold row crops. For both methods, to be used in the context of a dynamic simulation model, parameter values were compiled from a literature survey. Implemented in such a model, both algorithms were tested against experimental field data, yielding an Index of Agreement of 0.80 for the regression strategy and 0.90 for the population strategy. Furthermore, the population strategy was capable of reflecting rather well the effect of crop spacing on yield and the effect of N supply on product grading

An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology

G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein–protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor conformational states, explaining the pharmacological preferences of calcitonin receptor-RAMP complexes. This provides novel insight into our understanding of G protein-coupled receptor-protein interaction that is likely broadly applicable for this receptor class

Intraseasonal Dynamics and Dominant Sequences in H3N2 Influenza

Long-term influenza evolution has been well studied, but the patterns of sequence diversity within seasons are less clear. H3N2 influenza genomes sampled from New York State over ten years indicated intraseasonal changes in evolutionary dynamics. Using the mean Hamming distance of a set of amino acid or nucleotide sequences as an indicator of its diversity, we found that influenza sequence diversity was significantly higher during the early epidemic period than later in the influenza season. Diversity was lowest during the peak of the epidemic, most likely due to the high prevalence of a single dominant amino acid sequence or very few dominant sequences during the peak epidemic period, corresponding with rapid expansion of the viral population. The frequency and duration of dominant sequences varied by influenza protein, but all proteins had an abundance of one distinct sequence during the peak epidemic period. In New York State from 1995 to 2005, high sequence diversity during the early epidemic suggested that seasonal antigenic drift could have occurred primarily in this period, followed by a clonal expansion of typically one clade during the peak of the epidemic, possibly indicating a shift to neutral drift or purifying selection

A quantitative analysis of transmission efficiency versus intensity for malaria

The relationship between malaria transmission intensity and efficiency is important for malaria epidemiology, for the design of randomized control trials that measure transmission or incidence as end points, and for measuring and modelling malaria transmission and control. Five kinds of studies published over the past century were assembled and reanalysed to quantify malaria transmission efficiency and describe its relation to transmission intensity, to understand the causes of inefficient transmission and to identify functions suitable for modelling mosquito-borne disease transmission. In this study, we show that these studies trace a strongly nonlinear relationship between malaria transmission intensity and efficiency that is parsimoniously described by a model of heterogeneous biting. When many infectious bites are concentrated on a few people, infections and parasite population structure will be highly aggregated affecting the immunoepidemiology of malaria, the evolutionary ecology of parasite life history traits and the measurement and stratification of transmission for control using entomological and epidemiological data

Triatoma dimidiata Infestation in Chagas Disease Endemic Regions of Guatemala: Comparison of Random and Targeted Cross-Sectional Surveys

Chagas disease is a vector-borne parasitic zoonosis endemic throughout South and Central America and Mexico. Guatemala is engaged in the Central America Initiative to interrupt Chagas disease transmission. A major strategy is the reduction of Triatoma dimidiata domiciliary infestations through indoor application of residual insecticides. Successful control of T. dimidiata will depend on accurate identification of areas at greatest risk for infestation. Initial efforts focused primarily on targeted surveys of presumed risk factors and suspected infestation to define intervention areas. This policy has not been evaluated and might not maximize the effectiveness of limited resources if high prevalence villages are missed or low prevalence villages are visited unnecessarily. We compare findings from the targeted surveys to concurrent random surveys in two primary foci of Chagas disease transmission in Guatemala to evaluate the performance of the targeted surveys. Our results indicate that random surveys performed better than targeted surveys and should be considered over targeted surveys when reliability of risk factors has not been evaluated, identify useful environmental factors to predict infestation, and indicate that infestation risk varies locally. These findings are useful for decision-makers at national Chagas Disease control programs in Central America, institutions supporting development efforts, and funding agencies