Integrated risk management

The purpose of this report is to first present a basis or foundation for the building of an integrated risk management plan and them to present the plan. The integration referred to is across both the temporal and the hierarchical dimensions. Complexity, consequence, and credibility seem to be driving the need for the consideration of risk. Reduction of personal bias and reproducibility of the decision making process seem to be driving the consideration of a formal risk plan. While risk can be used as either a selection tool or a control tool, this paper concentrates on the selection usage. Risk relies on stated purpose. The tightness of the definition of purpose and success is directly reflected in the definition and control of risk. Much of a risk management plan could be designed by the answers to the questions of why, what, who, when, and where. However, any plan must provide the following information about a threat or risk: likelihood, consequence, predictability, reliability, and reproducibility. While the environment at NASA is seen as warm, but not hot, for the introduction of a risk program, some encouragement is seen if the following problems are addressed: no champion, no commitment of resource, confused definitions, lack of direction and focus, a hard sell, NASA culture, many choices of assessment methods, and cost. The plan is designed to follow the normal method of doing work and is structured to follow either the work break down structure or a functional structure very well. The parts of the plan include: defining purpose and success, initial threat assessment, initial risk assessment, reconciling threats and parameters, putting part of the information down and factoring the information back into the decision process as it comes back up, and developing inferences. Two major suggestions are presented. One is to build an office of risk management to be used as a resource by managers in doing the risk process. Another is to form a pilot program to try out the details in the plan and modify the method where needed

Identification and isolation of antigen-specific cytotoxic T lymphocytes with an automated microraft sorting system

The simultaneous measurement of T cell function with recovery of individual T cells would greatly facilitate characterizing antigen-specific responses both in vivo and in model systems

Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9

Microraft arrays have been used to screen and then isolate adherent and non-adherent cells with very high efficiency and excellent viability; however, manual screening and isolation limits the throughput and utility of the technology. In this work, novel hardware and software were developed to automate the microraft array platform. The developed analysis software identified microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity. The software enabled time-lapse imaging and the use of temporally varying characteristics as sort criteria. The automated hardware released microrafts with 98% efficiency and collected released microrafts with 100% efficiency. The automated system was used to examine the temporal variation in EGFP expression in cells transfected with CRISPR-Cas9 components for gene editing. Of 11,499 microrafts possessing a single cell, 220 microrafts were identified as possessing temporally varying EGFP-expression. Candidate cells (n=172) were released and collected from the microraft array and screened for the targeted gene mutation. Two cell colonies were successfully gene edited demonstrating the desired mutation

Single-cell sphingosine kinase activity measurements in primary leukemia

Sphingosine kinase (SK) is a promising therapeutic target in a number of cancers, including leukemia. Traditionally, SK has been measured in bulk cell lysates, but this technique obscures the cellular heterogeneity present in this pathway. For this reason, SK activity was measured in single cells loaded with a fluorescent sphingosine reporter. An automated capillary electrophoresis (CE) system enabled rapid separation and quantification of the phosphorylated and nonphosphorylated sphingosine reporter in single cells. SK activity was measured in tissue-cultured cells derived from chronic myelogenous leukemia (K562), primary peripheral blood mononuclear cells (PBMCs) from three patients with different forms of leukemia, and enriched leukemic blasts from a patient with acute myeloid leukemia (AML). Significant intercellular heterogeneity existed in terms of the degree of reporter phosphorylation (as much as an order of magnitude difference), the amount of reporter uptake, and the metabolites formed. In K562 cells, the average amount of reporter converted to the phosphorylated form was 39 ± 26% per cell. Of the primary PBMCs analyzed, the average amount of phosphorylated reporter was 16 ± 25%, 11 ± 26%, and 13 ± 23% in a chronic myelogenous leukemia (CML) patient, an acute myeloid leukemia (AML) patient, and a B-cell acute lymphocytic leukemia (B-ALL) patient, respectively. These experiments demonstrated the challenge of studying samples comprised of multiple cell types, with tumor blasts present at 5 to 87% of the cell population. When the leukemic blasts from a fourth patient with AML were enriched to 99% of the cell population, 19 ± 36% of the loaded sphingosine was phosphorylated. Thus the diversity in SK activity remained even in a nearly pure tumor sample. These enriched AML blasts loaded significantly less reporter (0.12 ± 0.2 amol) relative to that loaded into the PBMCs in the other samples (≥1 amol). The variability in SK signaling may have important implications for SK inhibitors as therapeutics for leukemia and demonstrates the value of single-cell analysis in characterizing the nature of oncogenic signaling in cancer

Ecto-5’-nucleotidase: Structure function relationships

Ecto-5’-nucleotidase (ecto-5’-NT) is attached via a GPI anchor to the extracellular membrane, where it hydrolyses AMP to adenosine and phosphate. Related 5’-nucleotidases exist in bacteria, where they are exported into the periplasmic space. X-ray structures of the 5’-nucleotidase from E. coli showed that the enzyme consists of two domains. The N-terminal domain coordinates two catalytic divalent metal ions, whereas the C-terminal domain provides the substrate specificity pocket for the nucleotides. Thus, the substrate binds at the interface of the two domains. Here, the currently available structural information on ecto-5’NT is reviewed in relation to the catalytic properties and enzyme function

Power spectrum analysis of ionospheric fluctuations with the Murchison Widefield Array

Low-frequency, wide field-of-view (FOV) radio telescopes such as the Murchison Widefield Array (MWA) enable the ionosphere to be sampled at high spatial completeness. We present the results of the first power spectrum analysis of ionospheric fluctuations in MWA data, where we examined the position offsets of radio sources appearing in two data sets. The refractive shifts in the positions of celestial sources are proportional to spatial gradients in the electron column density transverse to the line of sight. These can be used to probe plasma structures and waves in the ionosphere. The regional (10–100 km) scales probed by the MWA, determined by the size of its FOV and the spatial density of radio sources (typically thousands in a single FOV), complement the global (100–1000 km) scales of GPS studies and local (0.01–1 km) scales of radar scattering measurements. Our data exhibit a range of complex structures and waves. Some fluctuations have the characteristics of traveling ionospheric disturbances, while others take the form of narrow, slowly drifting bands aligned along the Earth's magnetic field

Purification and Structural Characterization of Siderophore (Corynebactin) from Corynebacterium diphtheriae

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin

Microsporidia::Why Make Nucleotides if You Can Steal Them?

Microsporidia are strict obligate intracellular parasites that infect a wide range of eukaryotes including humans and economically important fish and insects. Surviving and flourishing inside another eukaryotic cell is a very specialised lifestyle that requires evolutionary innovation. Genome sequence analyses show that microsporidia have lost most of the genes needed for making primary metabolites, such as amino acids and nucleotides, and also that they have only a limited capacity for making adenosine triphosphate (ATP). Since microsporidia cannot grow and replicate without the enormous amounts of energy and nucleotide building blocks needed for protein, DNA, and RNA biosynthesis, they must have evolved ways of stealing these substrates from the infected host cell. Providing they can do this, genome analyses suggest that microsporidia have the enzyme repertoire needed to use and regenerate the imported nucleotides efficiently. Recent functional studies suggest that a critical innovation for adapting to intracellular life was the acquisition by lateral gene transfer of nucleotide transport (NTT) proteins that are now present in multiple copies in all microsporidian genomes. These proteins are expressed on the parasite surface and allow microsporidia to steal ATP and other purine nucleotides for energy and biosynthesis from their host. However, it remains unclear how other essential metabolites, such as pyrimidine nucleotides, are acquired. Transcriptomic and experimental studies suggest that microsporidia might manipulate host cell metabolism and cell biological processes to promote nucleotide synthesis and to maximise the potential for ATP and nucleotide import. In this review, we summarise recent genomic and functional data relating to how microsporidia exploit their hosts for energy and building blocks needed for growth and nucleic acid metabolism and we identify some remaining outstanding questions