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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

Genomic Analysis of Metastatic Solid Tumors in Veterans: Findings From the VHA National Precision Oncology Program

The Veterans Health Administration (VHA) is the largest cancer care provider in the United States, with the added challenge of serving more than twice the percentage of patients with cancer in rural areas than the national average. The VHA established the National Precision Oncology Program in 2016 to implement and standardize the practice of precision oncology across the diverse VHA system. Tumor or peripheral blood specimens from veterans with advanced solid tumors who were eligible for treatment were submitted for next-generation sequencing (NGS) at two commercial laboratories. Annotated results were generated by the laboratories and independently using IBM Watson for Genomics. Levels-of-evidence treatment recommendations were based on OncoKB criteria. From July 2016 to June 2018, 3,698 samples from 72 VHA facilities were submitted for NGS testing, of which 3,182 samples (86%) were successfully sequenced. Most samples came from men with lung, prostate, and colorectal cancers. Thirty-four percent of samples were from patients who lived in a rural area. TP53, ATM, and KRAS were among the most commonly mutated genes. Approximately 70% of samples had at least one actionable mutation, with clinical trials identified as the recommended option in more than 50%. Mutations in genes associated with a neuroendocrine prostate cancer phenotype were expressed at increased frequency among veterans than in the general population. The most frequent therapies prescribed in response to NGS testing were immune checkpoint inhibitors, EGFR kinase inhibitors, and PARP inhibitors. Clinical implementation of precision oncology is feasible across the VHA health care system, including rural sites. As a result, veterans have unique occupational exposures that might inform the nature of the mutational signatures identified here. Importantly, these results underscore the importance of increasing clinical trial availability to veterans.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Effectiveness of a genetic test panel designed for gynecological cancer: an exploratory study

To increase diagnostic efficiency and cost-effectiveness, we performed an exploratory genetic test using a newly designed panel containing 28 actionable and druggable genes, alterations in which are frequently reported in gynecological cancers (TANRE-G, Targeted variants ANalysis RElated to Gynecological cancers). Samples consisted of the formalin-fixed, paraffin-embedded tissue of endometrial (4 cases), cervical (3 cases), and ovarian (4 cases) carcinomas. The sequencing procedure was performed using Ion PGM in our institute with related sequencing kits, and data were analyzed using ClinVar. The present system achieved more than 2500 reads in all tumor samples, and enabled a copy number variation analysis. Results showed that actionable and druggable mutations were detected in 82% (9/11) and 64% (7/11) of cases, respectively, which was similar to other commercially available genetic tests. The amplification of MYC and KRAS was also detected. The analysis cost for each sample was JPY 94,000 (USD 850). These results demonstrate the potential of the TANRE-G panel as an effective tool for examining genetic alterations in gynecological cancers.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2024

The extent of multiallelic, co‐editing of LIGULELESS1 in highly polyploid sugarcane tunes leaf inclination angle and enables selection of the ideotype for biomass yield

Summary Sugarcane ( Saccharum spp. hybrid) is a prime feedstock for commercial production of biofuel and table sugar. Optimizing canopy architecture for improved light capture has great potential for elevating biomass yield. LIGULELESS1 ( LG1 ) is involved in leaf ligule and auricle development in grasses. Here, we report CRISPR/Cas9‐mediated co‐mutagenesis of up to 40 copies/alleles of the putative LG1 in highly polyploid sugarcane (2 n = 100–120, x = 10–12). Next generation sequencing revealed co‐editing frequencies of 7.4%–100% of the LG1 reads in 16 of the 78 transgenic lines. LG1 mutations resulted in a tuneable leaf angle phenotype that became more upright as co‐editing frequency increased. Three lines with loss of function frequencies of ~12%, ~53% and ~95% of lg1 were selected following a randomized greenhouse trial and grown in replicated, multi‐row field plots. The co‐edited LG1 mutations were stably maintained in vegetative progenies and the extent of co‐editing remained constant in field tested lines L26 and L35. Next generation sequencing confirmed the absence of potential off targets. The leaf inclination angle corresponded to light transmission into the canopy and tiller number. Line L35 displaying loss of function in ~12% of the lg1 NGS reads exhibited an 18% increase in dry biomass yield supported by a 56% decrease in leaf inclination angle, a 31% increase in tiller number, and a 25% increase in internode number. The scalable co‐editing of LG1 in highly polyploid sugarcane allows fine‐tuning of leaf inclination angle, enabling the selection of the ideotype for biomass yield.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2002

Genetic basis of clinical catecholamine disorders

Norepinephrine and epinephrine are critical determinants of minute-to-minute regulation of blood pressure. Here we review the characterization of two syndromes associated with a genetic abnormality in the noradrenergic pathway. In 1986, we reported a congenital syndrome of undetectable tissue and circulating levels of norepinephrine and epinephrine, elevated levels of dopamine, and absence of dopamine-beta-hydroxylase (DBH). These patients appeared with ptosis and severe orthostatic hypotension and lacked sympathetic noradrenergic function. In two persons with DBH deficiency, we identified seven novel polymorphisms. Both patients are compound heterozygotes for a variant that affects expression of DBH protein via impairment of splicing. Patient 1 also has a missense mutation in DBH exon 2, and patient 2 carries missense mutations in exons 1 and 6. Orthostatic intolerance is a common syndrome affecting young women, presenting with orthostatic tachycardia and symptoms of cerebral hypoperfusion on standing. We tested the hypothesis that abnormal norepinephrine transporter (NET) function might contribute to its etiology. In our proband, we found an elevated plasma norepinephrine with standing that was disproportionate to the increase in levels of dihydroxphenylglycol, as well as impaired norepinephrine clearance and tyramine resistance. Studies of NET gene structure revealed a coding mutation converting a conserved alanine residue in transmembrane domain 9 to proline. Analysis of the protein produced by the mutant cDNA demonstrated greater than 98% reduction in activity relative to normal. The finding of genetic mutations responsible for DBH deficiency and orthostatic intolerance leads us to believe that genetic causes of other autonomic disorders will be found, enabling us to design more effective therapeutic interventions.

Non-NASA Center↗
DOE OSTI2014

Iterative derivation of effective potentials to sample the conformational space of proteins at atomistic scale

The current capacity of computers makes it possible to perform simulations of small systems with portable, explicit-solvent potentials achieving high degree of accuracy. However, simplified models must be employed to exploit the behavior of large systems or to perform systematic scans of smaller systems. While powerful algorithms are available to facilitate the sampling of the conformational space, successful applications of such models are hindered by the availability of simple enough potentials able to satisfactorily reproduce known properties of the system. We develop an interatomic potential to account for a number of properties of proteins in a computationally economic way. The potential is defined within an all-atom, implicit solvent model by contact functions between the different atom types. The associated numerical values can be optimized by an iterative Monte Carlo scheme on any available experimental data, provided that they are expressible as thermal averages of some conformational properties. We test this model on three different proteins, for which we also perform a scan of all possible point mutations with explicit conformational sampling. The resulting models, optimized solely on a subset of native distances, not only reproduce the native conformations within a few Angstroms from the experimental ones, but show the cooperative transition between native and denatured state and correctly predict the measured free-energy changes associated with point mutations. Moreover, differently from other structure-based models, our method leaves a residual degree of frustration, which is known to be present in protein molecules.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2002

Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-x(L)

Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in DSB repair in human cells. However, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We demonstrated previously that overexpression of BCL-2 or BCL-x(L) enhanced the frequency of X-ray-induced TK1 mutations, including loss of heterozygosity events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells and to determine whether ectopic expression of BCL-x(L) affects HDR. Using TK6-neo cells, we find that a single DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold, demonstrating efficient DSB repair by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3-4-fold more frequent in TK6 cells that stably overexpress the antiapoptotic protein BCL-X(L). Thus, HDR plays an important role in maintaining genomic integrity in human cells, and ectopic expression of BCL-x(L) enhances HDR of DSBs. This is the first study to highlight a function for BCL-x(L) in modulating DSB repair in human cells.

NASA Discipline Radiation Health↗
DOE OSTI2024

Comparative Uptake Patterns of Radioactive Iodine and [18F]-Fluorodeoxyglucose (FDG) in Metastatic Differentiated Thyroid Cancers

Background: Metastatic differentiated thyroid cancer (DTC) represents a molecularly heterogeneous group of cancers with varying radioactive iodine (RAI) and [ 18 F]-fluorodeoxyglucose (FDG) uptake patterns potentially correlated with the degree of de-differentiation through the so-called “flip-flop” phenomenon. However, it is unknown if RAI and FDG uptake patterns correlate with molecular status or metastatic site. Materials and Methods: A retrospective analysis of metastatic DTC patients (n = 46) with radioactive 131-iodine whole body scan (WBS) and FDG-PET imaging between 2008 and 2022 was performed. The inclusion criteria included accessible FDG-PET and WBS studies within 1 year of each other. Studies were interpreted by two blinded radiologists for iodine or FDG uptake in extrathyroidal sites including lungs, lymph nodes, and bone. Cases were stratified by BRAF V600E mutation status, histology, and a combination of tumor genotype and histology. The data were analyzed by McNemar’s Chi-square test. Results: Lung metastasis FDG uptake was significantly more common than iodine uptake (WBS: 52%, FDG: 84%, p = 0.04), but no significant differences were found for lymph or bone metastases. Lung metastasis FDG uptake was significantly more prevalent in the papillary pattern sub-cohort (WBS: 37%, FDG: 89%, p = 0.02) than the follicular pattern sub-cohort (WBS: 75%, FDG: 75%, p = 1.00). Similarly, BRAF V600E+ tumors with lung metastases also demonstrated a preponderance of FDG uptake (WBS: 29%, FDG: 93%, p = 0.02) than BRAF V600E- tumors (WBS: 83%, FDG: 83%, p = 1.00) with lung metastases. Papillary histology featured higher FDG uptake in lung metastasis (WBS: 39%, FDG: 89%, p = 0.03) compared with follicular histology (WBS: 69%, FDG: 77%, p = 1.00). Patients with papillary pattern disease, BRAF V600E+ mutation, or papillary histology had reduced agreement between both modalities in uptake at all metastatic sites compared with those with follicular pattern disease, BRAF V600E- mutation, or follicular histology. Low agreement in lymph node uptake was observed in all patients irrespective of molecular status or histology. Conclusions: The pattern of FDG-PET and radioiodine uptake is dependent on molecular status and metastatic site, with those with papillary histology or BRAF V600E+ mutation featuring increased FDG uptake in distant metastasis. Further study with an expanded cohort may identify which patients may benefit from specific imaging modalities to recognize and surveil metastases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

An innovative platform for quick and flexible joining of assorted DNA fragments

Successful synthetic biology efforts rely on conceptual and experimental designs in combination with testing of multi-gene constructs. Despite recent progresses, several limitations still hinder the ability to flexibly assemble and collectively share different types of DNA segments. We describe an advanced system for joining DNA fragments from a universal library that automatically maintains open reading frames (ORFs) and does not require linkers, adaptors, sequence homology, amplification or mutation (domestication) of fragments in order to work properly. Moreover, we find that this system, which is enhanced by a unique buffer formulation, provides unforeseen capabilities for testing, and sharing, complex multi-gene circuitry assembled from different DNA fragments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Evidence for Highly Variable, Region-Specific Patterns of T-Cell Epitope Mutations Accumulating in Mycobacterium tuberculosis Strains

Vaccines that confer protection through induction of adaptive T-cell immunity rely on understanding T-cell epitope (TCE) evolution induced by immune escape. This is poorly understood in tuberculosis (TB), an ancient, chronic disease, where CD4 T-cell immunity is of recognized importance. We probed 905 functionally validated, curated human CD4 T cell epitopes in 79 Mycobacterium tuberculosis (Mtb) whole genomes from India. This screen resulted in identifying 64 mutated epitopes in these strains initially using a computational pipeline and subsequently verified by single nucleotide polymorphism (SNP) analysis. SNP based phylogeny revealed the 79 Mtb strains to cluster to East African Indian (EAI), Central Asian Strain (CAS), and Beijing (BEI) lineages. Eighty-nine percent of the mutated T-cell epitopes (mTCEs) identified in the 79 Mtb strains from India has not previously been reported. These mTCEs were encoded by genes with high nucleotide diversity scores including seven mTCEs encoded by six antigens in the top 10% of rapidly divergent Mtb genes encoded by these strains. Using a T cell functional assay readout, we demonstrate 62% of mTCEs tested to significantly alter CD4 T-cell IFNγ and/or IL2 secretion with associated changes in predicted HLA-DR binding affinity: the gain of function mutations displayed higher predicted HLA-DR binding affinity and conversely mutations resulting in loss of function displayed lower predicted HLA-DR binding affinity. Most mutated antigens belonged to the cell wall/cell processes, and, intermediary metabolism and respiration families though all known Mtb proteins encoded mutations. Analysis of the mTCEs in an SNP database of 5,310 global Mtb strains identified 82% mTCEs to be significantly more prevalent in Mtb strains isolated from India, including 36 mTCEs identified exclusively in strains from India. These epitopes had a significantly higher predicted binding affinity to HLA-DR alleles that were highly prevalent in India compared to HLA-DR alleles rare in India, highlighting HLA-DR maybe an important driver of these mutations. This first evidence of region-specific TCE mutations potentially employed by Mtb to escape host immunity has important implications for TB vaccine design.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Novel mutation leading to splice donor loss in a conserved site of DMD gene causes Duchenne muscular dystrophy with cryptorchidism

Background As one of the most common congenital abnormalities in male births, cryptorchidism has been found to have a polygenic aetiology according to previous studies of common variants. However, little is known about genetic predisposition of rare variants for cryptorchidism, since rare variants have larger effective size on diseases than common variants. Methods In this study, a cohort of 115 Chinese probands with cryptorchidism was analysed using whole-genome sequencing, alongside 19 parental controls and 2136 unaffected men. Additionally, CRISPR-Cas9 editing of a conserved variant was performed in a mouse model, with MRI screening used to observe the phenotype. Results In 30 of 115 patients (26.1%), we identified four novel genes ( ARSH , DMD , MAGEA4 and SHROOM2 ) affecting at least five unrelated patients and four known genes ( USP9Y , UBA1 , BCORL1 and KDM6A ) with the candidate rare pathogenic variants affecting at least two cases. Burden tests of rare variants revealed the genome-wide significances for newly identified genes (p<2.5×10 −6 ) under the Bonferroni correction. Surprisingly, novel and known genes were mainly found on X chromosome (seven on X and one on Y) and all rare X-chromosomal segregating variants exhibited a maternal inheritance rather than de novo origin. CRISPR-Cas9 mouse modelling of a splice donor loss variant in DMD (NC_000023.11:g.32454661C>G), which resides in a conserved site across vertebrates, replicated bilateral cryptorchidism phenotypes, confirmed by MRI at 4 and 10 weeks. The movement tests further revealed symptoms of Duchenne muscular dystrophy (DMD) in transgenic mice. Conclusion Our results revealed the role of the DMD gene mutation in causing cryptorchidism. The results also suggest that maternal-X inheritance of pathogenic defects could have a predominant role in the development of cryptorchidism.

Chen, Jianhai (ORCID:0000000300932003)↗
DOE OSTI2021

Computation-guided optimization of split protein systems

Splitting bioactive proteins into conditionally reconstituting fragments is a powerful strategy for building tools to study and control biological systems. However, split proteins often exhibit a high propensity to reconstitute, even without the conditional trigger, limiting their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific or often ineffective. Here, we report a computational design strategy grounded in fundamental protein biophysics to guide experimental evaluation of a sparse set of mutants to identify an optimal functional window. We hypothesized that testing a limited set of mutants would direct subsequent mutagenesis efforts by predicting desirable mutant combinations from a vast mutational landscape. This strategy varies the degree of interfacial destabilization while preserving stability and catalytic activity. We validate our method by solving two distinct split protein design challenges, generating both design and mechanistic insights. Furthermore, this new technology will streamline the generation and use of split protein systems for diverse applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1999

Contractile function is unaltered in diaphragm from mice lacking calcium release channel isoform 3

Skeletal muscle expresses at least two isoforms of the calcium release channel in the sarcoplasmic reticulum (RyR1 and RyR3). Whereas the function of RyR1 is well defined, the physiological significance of RyR3 is unclear. Some authors have suggested that RyR3 participates in excitation-contraction coupling and that RyR3 may specifically confer resistance to fatigue. To test this hypothesis, we measured contractile function of diaphragm strips from adult RyR3-deficient mice (exon 2-targeted mutation) and their heterozygous and wild-type littermates. In unfatigued diaphragm, there were no differences in isometric contractile properties (twitch characteristics, force-frequency relationships, maximal force) among the three groups. Our fatigue protocol (30 Hz, 0.25 duty cycle, 37 degrees C) depressed force to 25% of the initial force; however, lack of RyR3 did not accelerate the decline in force production. The force-frequency relationship was shifted to higher frequencies and was depressed in fatigued diaphragm; lack of RyR3 did not exaggerate these changes. We therefore provide evidence that RyR3 deficiency does not alter contractile function of adult muscle before, during, or after fatigue.

Non-NASA Center↗
DOE OSTI2014

Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Cylindricity Error Evaluation Based on an Improved Harmony Search Algorithm

The cylindricity error is one of the basic form errors in mechanical parts, which greatly influences the assembly accuracy and service life of relevant parts. For the minimum zone method (MZM) in international standards, there is no specific formula to calculate the cylindricity error. Therefore, the evaluation methods of the cylindricity error under the MZM have been widely concerned by international scholars. To improve the evaluation accuracy and accelerate the iteration speed of the cylindricity, an improved harmony search (IHS) algorithm is proposed and applied to compute the cylindricity. On the basis of the standard harmony search algorithm, the logistic chaotic initialization is introduced into the generation of initial solution to improve the quality of solutions. During the iterative process, the global and local search capabilities are balanced by adopting the par and b w operators adaptively. After each iteration, the Cauchy mutation strategy is adopted to the best solution to further improve the calculation precision of the IHS algorithm. Finally, four test functions and three groups of cylindricity error examples were applied to validity verification of the IHS algorithm, the simulation test results show that the IHS algorithm has advantages of the computing accuracy and iteration speed compared with other traditional algorithms, and it is very effective for the application in the evaluation of the cylindricity error.

Yang, Yang↗
NASA NTRS1999

[Radiation Anticarcinogenesis by Thiazolidine Pro-drug]

The original goal of this work was to determine the capacity of selected aminothiols to modulate radiation induced cytotoxicity, mutagenesis and carcinogenesis in a human mammary epithelial cell line. The conclusions from this work are that WR-1065 is the "gold standard" for protection against radiation induced cytotoxicity, mutagenesis and carcinogenesis. While a potent radiation protector, WR-1065 is cytotoxic in vitro and in vivo. Our rationale for a study of the thiazolidine pro-drugs was that these compounds are neither toxic in vitro or in vivo. The results obtained during this funding period indicate that the thiazolidine pro-drugs are as potent as WR-1065 as protectors against radiation induced mutation induction, and thus presumably against radiation induced carcinogenesis. Our results indicate that the thiazolidine prodrugs are excellent candidates to test as non-toxic anticarcinogens for protecting astronauts from cancer induction during space travel.

Warters, Raymond L.↗
NASA NTRS2021

Generation-based Evolutionary Tool for the Optimization of Constellations (GenETOC)

With the rapid growth in the capabilities of smaller satellites, satellite architectures that replace a single, extremely capable spacecraft with multiple, cheaper ones are gaining in popularity. Unfortunately, the orbit design process for constellations can be significantly more involved, especiallywhen the relative placement of the individual spacecraft within the constellation is not constrained by mission and/or science objectives. Optimizing a satellite constellation in the presence of multiple, competing objectives is a highly complex problem to which many traditional mathematical optimization methods cannot be applied and few tools exist to help mission designers search for promising candidate mission designs. The Generation-based Evolutionary Tool for the Optimization of Constellations (GenETOC) has been created to search for near-optimal constellation design options. GenETOC combines a modified version of the Non-dominated Sorting Genetic Algorithm II (NSGA II) with STK Components libraries (a 3rdparty .NET package created by Analytical Graphics Inc.) to create a framework that enables a mission designer to generate a simulation that models the design problem and obtain a family of potential, near-optimal solutions that can be investigated more in detail. GenETOC was developed in C# using the .NET framework with Windows Presentation Foundation (WPF) serving as the framework from which to create the graphical user interface (GUI). GenETOC user inputs can be categorized into three major data components: definition of the problem (areas of interest, satellite decision parameters, and sensor configurations), definition of performance objectives, and specification of the genetic algorithm (GA) parameters. In the problem definition component, the user is prompted to define the areas of interest against which the performance metrics will be computed, define the sensor parameters and attach them to specific spacecraft, select which satellite orbital parameters will be added to the decision space of the GA, and specify the range of desired values for each optimization parameter. For performance objectives, the user is presented with a list of available coverage and revisit performance based calculation options from which two metrics are chosen to serve as the objective functions that the GA will use to evaluate solutions during the optimization process. Finally, the definition of the GA parameters provides user control over the number of generations (number of optimization iterations), the population size (number of candidate constellations created in each generation), and the adaptive mutation and crossover threshold values (control parameters for how frequently each process occurs during the optimization). GenETOC has been extensively tested to verify the individual components of the optimization process. The GA has been tested against a suite of GA test problems to confirm convergence to the known two and three-dimensional Pareto fronts. The coverage and revisit performance metrics obtained in GenETOC are compared with STK desktop scenarios, confirming the constellations are being appropriately modeled within GenETOC simulations. A walkthrough of a simple, example problem is provided to illustrate the workings of GenETOC and to demonstrate the output available to the mission designer.

mission design↗
NASA NTRS2003

Quantitative analysis of bristle number in Drosophila mutants identifies genes involved in neural development

BACKGROUND: The identification of the function of all genes that contribute to specific biological processes and complex traits is one of the major challenges in the postgenomic era. One approach is to employ forward genetic screens in genetically tractable model organisms. In Drosophila melanogaster, P element-mediated insertional mutagenesis is a versatile tool for the dissection of molecular pathways, and there is an ongoing effort to tag every gene with a P element insertion. However, the vast majority of P element insertion lines are viable and fertile as homozygotes and do not exhibit obvious phenotypic defects, perhaps because of the tendency for P elements to insert 5' of transcription units. Quantitative genetic analysis of subtle effects of P element mutations that have been induced in an isogenic background may be a highly efficient method for functional genome annotation. RESULTS: Here, we have tested the efficacy of this strategy by assessing the extent to which screening for quantitative effects of P elements on sensory bristle number can identify genes affecting neural development. We find that such quantitative screens uncover an unusually large number of genes that are known to function in neural development, as well as genes with yet uncharacterized effects on neural development, and novel loci. CONCLUSIONS: Our findings establish the use of quantitative trait analysis for functional genome annotation through forward genetics. Similar analyses of quantitative effects of P element insertions will facilitate our understanding of the genes affecting many other complex traits in Drosophila.

Non-NASA Center↗
DOE OSTI2020

A Positive Charge in the Outer Coordination Sphere of an Artificial Enzyme Increases CO 2 Hydrogenation

The protein scaffold around the active site of enzymes is known to influence catalytic activity, but specific scaffold features responsible for favorable influences are often not known. This study focuses on using an artificial metalloenzyme to probe one specific feature of the scaffold, the position of a positive charge in the outer coordination sphere around the active site. Previous work showed that a small molecular complex, [Rh(P Et 2 N glycine P Et 2 )2 ] – , immobilized covalently within a protein scaffold was activated for CO 2 hydrogenation. In this work, using an iterative design where the effect of arginine, histidine, or lysine residues placed in the outer coordination sphere of the catalytic active site were evaluated, we tested the hypothesis that positively charged groups facilitate CO 2 hydrogenation with seven unique constructs. Single-, double-, and triple-point mutations were introduced to directly compare catalytic activity, as monitored by turnover frequencies (TOFs) measured in real time with 1 H NMR spectroscopy, and evaluate related structural and electronic properties. Two of the seven constructs showed a 2- and 3-fold increase relative to the wild type, with overall rates ranging from 0.2 to 0.7 h –1 , and a crystal structure of the fastest of these shows the positive charge positioned next to the active site. A crystal structure of the arginine-containing complex shows that the arginines are positioned near the metal. Molecular dynamics (MD) studies also suggest that the positive charge is oriented next to the active site in the two constructs with faster rates but not in the others and that the positive charge near the active site holds the CO 2 near the metal, all consistent with a positive charge appropriately positioned in the scaffold benefiting catalysis. The MD studies also suggest that changes in the water distribution around the active site may contribute to catalytic activity, while modest structural changes and movement of the complex within the scaffold do not.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗