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Riboswitches function as important translational regulators in bacteria. Comprehensive mutational analysis of transcriptional riboswitches has been used to probe the energetic intricacies of interplay between the aptamer and expression platform, but translational riboswitches have been inaccessible to massively parallel techniques. The Guanidine-II (Gdm-II) riboswitch is an exclusively translational class. We have integrated RelE cleavage with next generation sequencing to quantify ligand-dependent changes in translation initiation for all single and double mutations of the Pseudomonas aeruginosa Gdm-II riboswitch, a total of more than 23,000 variants. This extensive mutational analysis is consistent with the prominent features of the bioinformatic consensus. These data indicate, unexpectedly, that direct sequestration of the Shine-Dalgarno sequence is dispensable for riboswitch function. Additionally, this comprehensive dataset reveals important positions not identified in previous computational and crystallographic studies. Mutations in the variable linker region stabilize alternate conformations. The double mutant data reveal the functional importance of the previously modeled P0b helix formed by the 5’ and 3’ tails that serves as the basis for translational control. Additional mutations to GU wobble base pairs in both P1 and P2 reveal how the apparent cooperativity of the system involves an intricate network of communication between the two binding sites. This comprehensive examination of a translational riboswitch’s expression platform illuminates how the riboswitch is precisely tuned and tunable with regard to ligand sensitivity, amplitude of expression between ON and OFF states, and cooperativity of ligand binding.

Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD(+) reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the “aromatic box” that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function.

The sequences of the 5′ untranslated regions (5′-UTRs) of mRNA alter gene expression across domains of life. Transcriptional modulators can be easily assayed through transcription termination, but translational regulators often require indirect, laborious methods. We have leveraged RelE’s ribosome-dependent endonuclease activity to develop a quantitative assay to monitor translation initiation of cis-regulatory mRNAs. RelE cleavage accurately reports ligand-dependent changes in ribosome association for two translational riboswitches and provides quantitative information about each switch's sensitivity and range of response. RelE accurately reads out sequence-driven changes in riboswitch specificity and function and is quantitatively dependent upon ligand concentration. RelE cleavage similarly captures differences in translation initiation between yeast 5′-UTR isoforms. RelE cleavage can thus reveal a plethora of information about translation initiation in different domains of life.

The modularity of protein domains is well-known, but the existence of independent domains that confer function in RNA is less established. Recently, a family of RNA aptamers termed ykkC was discovered; they bind at least four ligands of very different chemical composition, including guanidine, phosphoribosyl pyrophosphate (PRPP), and guanosine tetraphosphate (ppGpp) (graphical abstract). Structures of these aptamers revealed an architecture characterized by two coaxial helical stacks. The first helix appears to be a generic scaffold, while the second helix forms the most contacts to the ligands. To determine if these two regions within the aptamer are modular units for ligand recognition, we swapped the ligand-binding coaxial stacks of a guanidine aptamer and a PRPP aptamer. This operation, in combination with a single mutation in the scaffold domain, achieved full switching of ligand specificity. This finding suggests that the ligand-binding helix largely dictates the ligand specificity of ykkC RNAs and that the scaffold coaxial stack is generally compatible with various ykkC ligand-binding modules. This work presents an example of RNA domain modularity comparable to that of a ligand-binding protein, showcasing the versatility of RNA as an entity capable of molecular evolution through adaptation of existing motifs.

Many under-developed organisms possess important traits that can boost the effectiveness and sustainability of microbial biotechnology. Photoautotrophic cyanobacteria can utilize the energy captured from light to fix carbon dioxide for their metabolic needs while living in environments not suited for growing crops. Various value-added compounds have been produced by cyanobacteria in the laboratory; yet, the products' titers and yields are often not industrially relevant and lag behind what have been accomplished in heterotrophic microbes. Genetic tools for biological process control are needed to take advantage of cyanobacteria's beneficial qualities, as tool development also lags behind what has been created in common heterotrophic hosts. To address this problem, we developed a suite of sensors that regulate transcription in the model cyanobacterium Synechocystis sp. PCC 6803 in response to metabolically relevant signals, including light and the cell's nitrogen status, and a family of sensors that respond to the inexpensive chemical, l-arabinose. Increasing the number of available tools enables more complex and precise control of gene expression. Expanding the synthetic biology toolbox for this cyanobacterium also improves our ability to utilize this important under-developed organism in biotechnology. Biotechnol. Bioeng. 2017;114: 1561-1569. © 2017 Wiley Periodicals, Inc.

Four samples of two commercially available iron brands used as substrate for iron permeable reactive barriers (PRBs) were tested for suitability for remediation of perchloroethylene (PCE), trichloroethylene (TCE), cis-dichloroethylene (cDCE) and vinyl chloride (VC). Kinetic studies indicate that rates of reaction are enhanced for cDCE and VC on Connelly iron (2.8 x 10(-4) to 6.9 x 10(-4) L/m2/hr and 2.0 x 10(-4) to 9.0 x 10(-4) L/m2/hr, for cDCE and VC, respectively) vs. Peerless iron (3.1 x 10(-5) to 4.6 x 10(-5) L/m2/hr and 2.4 x 10(-5) to 4.1 x 10(-5) L/m2/hr, for cDCE and VC, respectively). Carbon isotopic analyses of the residual chlorinated ethylene (CE) during degradation indicate significant fractionation occurs during reductive dechlorination, with, for example, up to 70% enrichment in carbon isotopic values observed when VC is more than 99% degraded. Comparison of fractionation factors (epsilon) indicates significant differences in carbon isotopic fractionation for different iron types and for different CEs. For the lower CEs (cDCE and VC) in particular, both slower reaction rates and larger fractionation are observed for degradation on Peerless vs. Connelly iron. This is the first study to establish a correlation between the rate of abiotic degradation on Fe(0) and the extent of isotopic fractionation, and the first to confirm consistent differences in these two parameters as a function of iron type. The possibility that these differences in kinetics and carbon isotopic fractionation for cDCE and VC are related to differences in branching ratios between competing hydrogenolysis and beta-elimination reactions during reductive dechlorination on the iron surfaces is discussed.

The current regulations, as set forth by the United States Nuclear Regulatory Commission (NRC), to insure that light-water nuclear reactor pressure vessels (RPVs) maintain their structural integrity, throughout their operating life, when subjected to planned normal reactor startup (heat-up) and shutdown (cool-down) transients are specified in Appendix G to 10 CFR Part 50, which incorporates by reference Appendix G to Section XI of the American Society of Mechanical Engineers (ASME) Code. The technical basis for these regulations are generally considered to be conservative and some plants are finding it operationally difficult to heat-up and cool-down within the accepted limits. Consequently, the nuclear industry has developed, and submitted to the ASME Code for approval, an alternative risk-informed methodology that reduces the conservatism and is consistent with methods previously used to develop a risk-informed revision to the regulations for accidental transients such as pressurized thermal shock (PTS). The objective of the alternative methodology is to increase operational flexibility while continuing to provide reasonable assurance of adequate protection to public health and safety. The NRC and its contractor at Oak Ridge National Laboratory (ORNL) are reviewing the industry proposed risk-informed methodology. Previous results of this review, have been reported at PVP, and a NRC report summarizing all results is currently in preparation. The objective of this paper is to discuss and illustrate mechanistic insights into trends shown previously associated with normal cool-down.Copyright © 2011 by ASME

Title 10 of the Code of Federal Regulations, Part 50 Appendix G (10CFR50 App. G) prescribes the pressure-temperature (P-T) limits for light water power reactors by incorporating American Society of Mechanical Engineers (ASME) Boiler & Pressure Vessel Code, Section XI, Appendix G (ASME App. G) by reference. The P-T limits in ASME App. G are based on the fracture toughness of the vessel limiting material accounting for irradiation effects. The fracture toughness of the limiting material is determined based on the plane strain fracture toughness, KIc, indexed to the material reference temperature, RT NDT. Prior to the 2000 Addenda of ASME App. G, fracture toughness was based on the arrest fracture toughness, KIa, but the approval of Code Case N-640 [1] in 1998 changed the fracture toughness basis to KIc. In addition to the P-T limits determined by ASME App. G, 10CFR50 App. G sets additional minimum temperature requirements that limit the operating pressure based on other factors such as the minimum unirradiated reference temperature, RTNDT(u), of the material in the closure head flange region. This paper presents an analysis of the closure head flange region minimum temperature requirements based on both KIa and KIc. Recommendations for potential alternative minimum temperature requirements are made for normal operation and hydrostatic test conditions.

The U.S. Nuclear Regulatory Commission (NRC) and the Electric Power Research Institute (EPRI) entered into a cooperative agreement to develop a modular computer code for determining the probability of rupture of reactor coolant pressure boundary piping. The goal of xLPR project (Ex tremely L ow P robability of R upture), as it is known, is to develop a flexible modular code that satisfies the assessment methodology in the NRC Standard Review Plan (SRP) 3.6.3 leak-before-break analysis. The NRC SRP 3.6.3 provides guidance on satisfying Title 10 of the Code of Federal Regulations Part 50 (10 CFR Part 50) Appendix A, General Design Criteria 4 that requires primary system pressure piping to exhibit an extremely low probability of rupture. The initial phase of the xLPR project is the Pilot Study where the base structure of the code will be developed and tested on a known case. The Pilot Study version of the xLPR code consists of an alpha version where proxy modules and inputs are used and a beta version where the state-of-the art modules and inputs are used. This paper will discuss the input parameters used for the alpha xLPR code.Copyright © 2010 by ASME

The current regulations, as set forth by the United States Nuclear Regulatory Commission (NRC), to insure that light-water nuclear reactor pressure vessels (RPVs) maintain their structural integrity when subjected to planned normal reactor startup (heat-up) and shut-down (cool-down) transients are specified in Appendix G to 10 CFR Part 50, which incorporates by reference Appendix G to Section XI of the American Society of Mechanical Engineers (ASME) Code. The technical basis for these regulations are now recognized by the technical community as being conservative and some plants are finding it increasingly difficult to comply with the current regulations. Consequently, the nuclear industry has developed, and submitted to the ASME Code for approval, an alternative risk-informed methodology that reduces the conservatism and is consistent with the methods previously used to develop a risk-informed revision to the regulations for accidental transients such as pressurized thermal shock (PTS). The objective of the alternative methodology is to provide a relaxation to the current regulations which will provide more operational flexibility, particularly for reactor pressure vessels with relatively high irradiation levels and radiation sensitive materials, while continuing to provide reasonable assurance of adequate protection to public health and safety. The NRC and its contractor at Oak Ridge National Laboratory (ORNL) have recently performed an independent review of the industry proposed methodology. The NRC / ORNL review consisted of performing probabilistic fracture mechanics (PFM) analyses for a matrix of cool-down and heat-up rates, permutated over various reactor geometries and characteristics, each at multiple levels of embrittlement, including 60 effective full power years (EFPY) and beyond, for various postulated flaw characterizations. The objective of this review is to quantify the risk of a reactor vessel experiencing non-ductile fracture, and possible subsequent failure, over a wide range of normal transient conditions, when the maximum allowable thermal-hydraulic boundary conditions, derived from both the current ASME code and the industry proposed methodology, are imposed on the inner surface of the reactor vessel. This paper discusses the results of the NRC/ORNL review of the industry proposal including the matrices of PFM analyses, results, insights, and conclusions derived from these analyses.Copyright © 2010 by ASME

We have demonstrated a high-power widely tunable SG-DBR semiconductor laser monolithically integrated with a SOA. Chip power in excess of 55 mW and SMSR > 40 dB across a 40 nm tuning range have been achieved. Over 13 dBm fiber-coupled power was demonstrated across 85 50-GHz ITU L-band channels.

This report documents the mechanical properties of steel plates for Navy use. These plates conform to American Society for Testing and Materials (ASTM) Standard A945, High-strength, Low-alloy (HSLA), Class 65. Plates of various thickness were purchased from two different producers to determine if the steel could be obtained on a production scale, as opposed to a custom order. Tensile and Charpy V-notch impact tests were performed on the steels, and the results are reported within. All of the plates met the minimum mechanical property requirements. The plates also met the minimum Charpy impact energy requirements. Future testing will include dynamic tear testing, static and dynamic fracture toughness testing. A plate supplied by a third producer will also be evaluated.

The austenite recrystallization behavior during the hot deformation of HSLA-100 was characterized in order to determine parameters for thermomechanical controlled processing. The steel was reheated to 2000 deg F (1093 deg C) and subjected to multiple isothermal deformations at 1800 deg F (982 deg C) and 1650 deg F (900 deg C) under various levels of deformation and hold times. At 10% deformation per hit, the steel took longer than 20 seconds to recrystallize at 1800 deg F (982 deg C) after three hits while at 1650 deg F (900 deg C) the steel did not recrystallize during the hold periods. At 20% deformation per hit, the steel began to recrystallize after the first hit after 20 seconds and recrystallized repeatedly after each subsequent hit during all of the hold periods at 1800 deg F (982 deg F) while at 1650 deg F (900 deg C), recrystallization occurred after three hits and the 20 second hold time.

Efficiency and maximum power obtainable from vertical cavity lasers (VCL) are low at present due to high series resistance. It is possible to circumvent this and maintain high cavity quality by using a highly conductive, extremely thin metallic layer located in a node (null) of the standing waves just above the active layer. The fabrication was as follows: Bottom mirror and active region were essentially the same as in previous VCLs, and n-doped Al x Ga1- x As/AlAs mirror with an active region about a quarterwave thick. After ~1000 Å of p-doped AlGaAs on top of the active region the epitaxial growth stopped. An ~50-Å Au layer was evaporated on top of this and a conventional dielectric mirror (TiO2/SiO2) completed the structure. Current is injected from thicker Au contacts just to the side of the cavity into the thin layer, then directly into the p-doped layer just above (~1000 Å) the active region. Optically pumped lasing occurred at 240-pj input to such a device. The l–V response of a separate similar device, 10 μm in diameter, showed a nearly constant ~100 ohm resistance. More precise fabrication should yield better performance, both optically and electrically and electrically, in a single device.