The knowledge-based software assistant

Where the Knowledge Based Software Assistant (KBSA) is now, four years after the initial report, is discussed. Also described is what the Rome Air Development Center expects at the end of the first contract iteration. What the second and third contract iterations will look like are characterized

Specification Reformulation During Specification Validation

The goal of the ARIES Simulation Component (ASC) is to uncover behavioral errors by 'running' a specification at the earliest possible points during the specification development process. The problems to be overcome are the obvious ones the specification may be large, incomplete, underconstrained, and/or uncompilable. This paper describes how specification reformulation is used to mitigate these problems. ASC begins by decomposing validation into specific validation questions. Next, the specification is reformulated to abstract out all those features unrelated to the identified validation question thus creating a new specialized specification. ASC relies on a precise statement of the validation question and a careful application of transformations so as to preserve the essential specification semantics in the resulting specialized specification. This technique is a win if the resulting specialized specification is small enough so the user my easily handle any remaining obstacles to execution. This paper will: (1) describe what a validation question is; (2) outline analysis techniques for identifying what concepts are and are not relevant to a validation question; and (3) identify and apply transformations which remove these less relevant concepts while preserving those which are relevant

Spatial and Temporal Dynamics of Dissolved Oxygen Concentrations and Bioactivity in the Hyporheic Zone

Dissolved oxygen (DO) concentrations and consumption rates are primary indicators of heterotrophic respiration and redox conditions in the hyporheic zone (HZ). Due to the complexity of hyporheic flow and interactions between hyporheic hydraulics and the biogeochemical processes, a detailed, mechanistic, and predictive understanding of the biogeochemical activity in the HZ has not yet been developed. Previous studies of microbial activity in the HZ have treated the metabolic DO consumption rate constant (KDO) as a temporally fixed and spatially homogeneous property that is determined primarily by the concentration of bioavailable carbon. These studies have generally treated bioactivity as temporally steady state, failing to capture the temporal dynamics of a changeable system. We demonstrate that hyporheic hydraulics controls rate constants in a hyporheic system that is relatively abundant in bioavailable carbon, such that KDO is a linear function of the local downwelling flux. We further demonstrate that, for triangular dunes, the downwelling velocities are lognormally distributed, as are the KDO values. By comparing measured and modeled DO profiles, we demonstrate that treating KDO as a function of the downwelling flux yields a significant improvement in the accuracy of predicted DO profiles. Additionally, our results demonstrate the temporal effect of carbon consumption on microbial respiration rates

An Easy and Efficient Method for Native and Immunoreactive Echinococcus granulosus Antigen 5 Enrichment from Hydatid Cyst Fluid

Background: Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis. Methodology/Principal Findings: Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results. Conclusions/Significance: The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera.This work was supported by Regione Autonoma della Sardegna (http://www.regione.sardegna.it/)Pubblicat

Prescribing Prevalence of Medications With Potential Genotype-Guided Dosing in Pediatric Patients

Importance: Genotype-guided prescribing in pediatrics could prevent adverse drug reactions and improve therapeutic response. Clinical pharmacogenetic implementation guidelines are available for many medications commonly prescribed to children. Frequencies of medication prescription and actionable genotypes (genotypes where a prescribing change may be indicated) inform the potential value of pharmacogenetic implementation. Objective: To assess potential opportunities for genotype-guided prescribing in pediatric populations among multiple health systems by examining the prevalence of prescriptions for each drug with the highest level of evidence (Clinical Pharmacogenetics Implementation Consortium level A) and estimating the prevalence of potential actionable prescribing decisions. Design, setting, and participants: This serial cross-sectional study of prescribing prevalences in 16 health systems included electronic health records data from pediatric inpatient and outpatient encounters from January 1, 2011, to December 31, 2017. The health systems included academic medical centers with free-standing children's hospitals and community hospitals that were part of an adult health care system. Participants included approximately 2.9 million patients younger than 21 years observed per year. Data were analyzed from June 5, 2018, to April 14, 2020. Exposures: Prescription of 38 level A medications based on electronic health records. Main outcomes and measures: Annual prevalence of level A medication prescribing and estimated actionable exposures, calculated by combining estimated site-year prevalences across sites with each site weighted equally. Results: Data from approximately 2.9 million pediatric patients (median age, 8 [interquartile range, 2-16] years; 50.7% female, 62.3% White) were analyzed for a typical calendar year. The annual prescribing prevalence of at least 1 level A drug ranged from 7987 to 10 629 per 100 000 patients with increasing trends from 2011 to 2014. The most prescribed level A drug was the antiemetic ondansetron (annual prevalence of exposure, 8107 [95% CI, 8077-8137] per 100 000 children). Among commonly prescribed opioids, annual prevalence per 100 000 patients was 295 (95% CI, 273-317) for tramadol, 571 (95% CI, 557-586) for codeine, and 2116 (95% CI, 2097-2135) for oxycodone. The antidepressants citalopram, escitalopram, and amitriptyline were also commonly prescribed (annual prevalence, approximately 250 per 100 000 patients for each). Estimated prevalences of actionable exposures were highest for oxycodone and ondansetron (>300 per 100 000 patients annually). CYP2D6 and CYP2C19 substrates were more frequently prescribed than medications influenced by other genes. Conclusions and relevance: These findings suggest that opportunities for pharmacogenetic implementation among pediatric patients in the US are abundant. As expected, the greatest opportunity exists with implementing CYP2D6 and CYP2C19 pharmacogenetic guidance for commonly prescribed antiemetics, analgesics, and antidepressants

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Nitrous Oxide from Streams and Rivers: A Review of Primary Biogeochemical Pathways and Environmental Variables

Atmospheric concentrations of the powerful greenhouse gas nitrous oxide (N2O) have increased dramatically over the last 100 years, and part of these emissions come from streams and rivers. N2O production has been more carefully studied in soils, but runoff of reactive nitrogen, likely from fertilizer, influences lotic N2O emissions. N2O production and consumption are strongly microbially mediated and mostly involve oxidation and reduction of the reactive nitrogen species ammonia, nitrate, and nitrite. Of the four main pathways leading to N2O production in soils and sediments, incomplete denitrification is likely the globally dominant N2O generating pathway and is favored by elevated nitrate concentrations, suboxic conditions, and sufficient organic carbon to promote reduction. The two pathways that oxidize ammonia, nitrifier denitrification and nitrification, are favored with higher concentrations of dissolved oxygen and ammonia. It is often difficult to distinguish these two pathways in field settings, but most evidence suggests that nitrifier-denitrification is likely the globally more significant of the two. The fourth reaction pathway is dissimilatory nitrate reduction to ammonia (DNRA), in which N2O may be produced from intermediate nitrite. This pathway is more recently discovered, and its global relevance remains uncertain. The key variables influencing N2O cycling, concentrations of the primary reactants (nitrate and ammonia), organic carbon, and dissolved oxygen (DO), may vary temporally with season and time of day. Increasing nitrate and ammonia generally result in higher N2O production. Elevated carbon availability generally promotes denitrification. However, N2O yield is generally higher when carbon is less available or less reactive. Efforts to quantify N2O in lotic settings include mostly studies of N2O dissolved in or emitted from surface water, with fewer studies of N2O produced or emitted from sediments. With some exceptions and limits, N2O emissions are generally positively correlated with nitrate concentration (and in some cases, ammonia concentration). Most studies observe more N2O emissions with low DO. Lotic N2O emissions were generally higher in the warmer months and at night. Most studies assume a denitrification source for N2O, except in the case of high DO and NH4+, in which nitrification is assumed. Lotic N2O production and consumption may take place in the hyporheic zone along groundwater flow paths and in the water column of streams and rivers. Because microbial nitrogen processing requires substrate, influx of reactants, appropriate redox conditions, and intermediate residence times, the hyporheic zone is likely the site of most N2O production. However, high rates of N2O production may also occur associated with suspended sediments in turbid streams and rivers. Models that combine hydromorphogical and chemical variables are most likely to provide the best predictions of N2O emissions. Such models and some observations suggest that N2O emissions decrease downstream as sedimentary processes (likely denitrification) decrease relative to processes in the surface water (likely nitrification). Downstream sites could have large N2O emissions, however, due to inputs of nitrate or ammonia. Better quantification of lotic N2O processing will inform the emission factors incorporated into greenhouse gas budgets. Both quantification and mitigation of N2O emissions will benefit from future research that more closely examines the biogeochemical pathways and physical settings for N2O production and consumption

Controls on Nitrous Oxide Emissions from the Hyporheic Zones of Streams

The magnitude and mechanisms of nitrous oxide (N2O) release from rivers and streams are actively debated. The complex interactions of hydrodynamic and biogeochemical controls on emissions of this important greenhouse gas preclude prediction of when and where N2O emissions will be significant. We present observations from column and large-scale flume experiments supporting an integrative model of N2O emissions from stream sediments. Our results show a distinct, replicable, pattern of nitrous oxide generation and consumption dictated by subsurface (hyporheic) residence times and biological nitrogen reduction rates. Within this model, N2O emission from stream sediments requires subsurface residence times (and microbially mediated reduction rates) be sufficiently long (and fast reacting) to produce N2O by nitrate reduction but also sufficiently short (or slow reacting) to limit N2O conversion to dinitrogen gas. Most subsurface exchange will not result in N2O emissions; only specific, intermediate, residence times (reaction rates) will both produce and release N2O to the stream. We also confirm previous observations that elevated nitrate and declining organic carbon reactivity increase N2O production, highlighting the importance of associated reaction rates in controlling N2O accumulation. Combined, these observations help constrain when N2O release will occur, providing a predictive link between stream geomorphology, hydrodynamics, and N2O emissions

Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance

Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide “letters”, the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson–Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so.Results: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson–Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2’-deoxyisocytidine and 2’-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted.Conclusion: AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology

Ultra-rapid detection of SARS-CoV-2 in public workspace environments.

Managing the pandemic caused by SARS-CoV-2 requires new capabilities in testing, including the possibility of identifying, in minutes, infected individuals as they enter spaces where they must congregate in a functioning society, including workspaces, schools, points of entry, and commercial business establishments. Here, the only useful tests (a) require no sample transport, (b) require minimal sample manipulation, (c) can be performed by unlicensed individuals, (d) return results on the spot in much less than one hour, and (e) cost no more than a few dollars. The sensitivity need not be as high as normally required by the FDA for screening asymptomatic carriers (as few as 10 virions per sample), as these viral loads are almost certainly not high enough for an individual to present a risk for forward infection. This allows tests specifically useful for this pandemic to trade-off unneeded sensitivity for necessary speed, simplicity, and frugality. In some studies, it was shown that viral load that creates forward-infection risk may exceed 105 virions per milliliter, easily within the sensitivity of an RNA amplification architecture, but unattainable by antibody-based architectures that simply target viral antigens. Here, we describe such a test based on a displaceable probe loop amplification architecture