SEARCH · Engineering Papers

Results for “Intact”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13

All sources NASA NTRS DOE OSTI

DOE OSTI2024

Response of Graphite to Dynamic Loading and Hypervelocity Jet Impacts

The compressive strengths of three varieties of high purity graphite, PCEA, NBG-18, and NBG-25, as well as the depth of penetration of small-scale charges into these materials was experimentally determined. These grades are similar in density, ranging from 1.80 – 1.85 g/cc, and nominal apparent porosity, ranging from 18% to 20%, but provide a wide range in maximum grain or particle size from 10s to 1000s of µm. Two very different manufacturing methods are also represented; PCEA is extruded while NBG-18 and NBG-25 are iso-molded. The quasistatic and dynamic strengths of each grade were determined on a load frame and split-Hopkinson pressure bar, respectively. The depth of penetration (DOP) of two small-scale shaped charges, the Teledyne RP-1 and RP-4, was determined against graphite. The global response of the RP-4 impacts was markedly different as the PCEA samples remained intact while all the NBG-25 samples split into 2 or 3 pieces after the jet penetration had completed. However, for all tests, the trusted DOPs fell within 2 cm. Preliminary hydrocode modeling of the penetration events used existing models that were not designed for graphite. The results can be tuned to reasonably reproduce the DOP, but the wound channel geometry is not reproduced well. A model designed for graphite would need to represent graphite’s non-linear and energy dissipation characteristics.

36 MATERIALS SCIENCE↗

DOE OSTI2024

Pretest Modeling A Spent Nuclear Fuel Seismic Shake Test

The U.S. Department of Energy Spent Fuel and Waste Science and Technology (SFWST) program is planning to conduct a series of full-scale shake table tests to simulate hypothetical earthquake conditions and record the response of surrogate spent nuclear fuel (SNF) assemblies in a dry canister storage system mockup. The shake table motions will represent a range of hypothetical earthquake conditions at hypothetical locations in the continental U.S. to generally define the range of mechanical loads that SNF can be expected to experience during extended dry storage periods. This paper describes the pretest predictions made with LS-DYNA models of the mockup storage systems. The test will use two dry storage system configurations, a mockup vertical concrete cask (VCC) and a mockup horizontal storage module (HSM). The test will use a production-quality canister and basket. Within the canister will be four instrumented fuel assemblies with fuel rods containing surrogate mass and 28 instrumented dummy assemblies that are intended to match the mass and outer dimension of a fuel assembly. The finite element models include models of the VCC and HSM on the shake table to calculate the system level dynamic responses and separate single fuel assembly models to calculate stress and strain in fuel assembly components. Both types of models include nonlinear behavior like rod-to-rod contact and the ability for VCC’s to rock and slide. This paper presents the expected response of the VCC, HSM, and fuel assemblies to the shake table testing that is planned to start in April of 2024. The earthquake conditions represent seismic hazards in the 2,000-to-20,000-year return period range. The test data is expected to confirm the expectation that fuel rod cladding will remain intact, fuel assembly structural components like guide tubes will remain intact, and no significant VCC sliding or tipping will occur in the range of conditions to be tested with the shake table.

Klymyshyn, Nicholas A.↗

DOE OSTI2022

In situ diversity of metabolism and carbon use efficiency among soil bacteria

The central carbon (C) metabolic network harvests energy to power the cell and feed biosynthesis for growth. In pure cultures, bacteria use some but not all of the network’s major pathways, such as glycolysis and pentose phosphate and Entner-Doudoroff pathways. However, how these pathways are used in microorganisms in intact soil communities is unknown. Here, we analyzed the incorporation of 13 C from glucose isotopomers into phospholipid fatty acids. We showed that groups of Gram-positive and Gram-negative bacteria in an intact agricultural soil used different pathways to metabolize glucose. They also differed in C use efficiency (CUE), the efficiency with which a substrate is used for biosynthesis. Our results provide experimental evidence for diversity among microbes in the organization of their central carbon metabolic network and CUE under in situ conditions. These results have important implications for our understanding of how community composition affects soil C cycling and organic matter formation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Molecular insights into the evolution of woody plant decay in the gut of termites

Plant cell walls represent the most abundant pool of organic carbon in terrestrial ecosystems but are highly recalcitrant to utilization by microbes and herbivores owing to the physical and chemical barrier provided by lignin biopolymers. Termites are a paradigmatic example of an organism’s having evolved the ability to substantially degrade lignified woody plants, yet atomic-scale characterization of lignin depolymerization by termites remains elusive. We report that the phylogenetically derived termite Nasutitermes sp. efficiently degrades lignin via substantial depletion of major interunit linkages and methoxyls by combining isotope-labeled feeding experiments and solution-state and solid-state nuclear magnetic resonance spectroscopy. Exploring the evolutionary origin of lignin depolymerization in termites, we reveal that the early-diverging woodroach Cryptocercus darwini has limited capability in degrading lignocellulose, leaving most polysaccharides intact. Conversely, the phylogenetically basal lineages of “lower” termites are able to disrupt the lignin-polysaccharide inter- and intramolecular bonding while leaving lignin largely intact. These findings advance knowledge on the elusive but efficient delignification in natural systems with implications for next-generation ligninolytic agents.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Mine Flooding History of a Regional Below-Drainage Coalfield Dominated by Barrier Leakage (1970–2014)

A 44-year record of water level fluctuations in a series of adjacent closed underground mines documents the history of closure and mine flooding in the Fairmont Coalfield, one of the oldest coal mining districts in the Pittsburgh coal basin, West Virginia, USA. As closures proceeded and mines began to flood, US environmental regulations were first enacted mandating mine water control and treatment, rendering uncontrolled surface discharges unacceptable. The purpose of this study is to present this flooding history and to identify critical events that determined how mine pools evolved in this case. Also examined is the strategy developed to control and treat water from these mines. Flooding is visualized using both water level hydrographs and mine flooding maps with the latter constructed assuming mine water hydraulic continuity between one or more mines. The earliest flooding formed small pools within near-surface mines closed prior to 1962 yet still pumped following closure to minimize leaking into adjacent still-active workings. These subpools gradually enlarged and merged as more closures occurred and the need for protective pumping was removed, forming what is today referred to as the unconfined Fairmont Pool. Later, deeper mines, separated by intact updip barriers from the Fairmont Pool, were closed and flooded more gradually, supplied in large part by leakage from the Fairmont Pool. By 1985, all mines except 2 had closed and by 1994 all had fully flooded, with the Fairmont Pool interconnected to deeper single mine pools via barrier leakage. As protective pumping ceased, the Fairmont Pool rose to a water level 3 m higher than surface drainage elevation and in 1997 discharged from an undermined section of Buffalo Creek near the Monongahela River. The principal mine operator in the basin then designed a pumping system to transfer water from the Fairmont Pool to their existing treatment facilities to the north, thus terminating the discharge. It may be concluded that the progress of mine flooding was influenced by mining history and design, by the timing of closures, by barrier leakage conditions, and by geologic structure. A key element in how flooding proceeded was the presence of a series of intact barriers separating deep from shallow mines. The shallow mines closed and flooded early, but then lost sufficient water by barrier leakage into the deeper mines to delay the completion of flooding until after the deep mines had all closed and flooded as well. Intensive mine water control has continued from the 1997 breakout to the present. The final water control scheme was likely unanticipated and serendipitous; future district-wide mining efforts should be advised to consider in advance closeout strategies to control mine water postmining.

Donovan, Joseph J.↗

DOE OSTI2020

Nuclear Canister Corrosion Detection

The software titled "nccd" is used for running residual neural networks (ResNets) on images of nuclear canisters. The software provides only the code for the implementation (based on the fastai library), but it does not share the image data. The compute code allows the use of residual nets (and more generally of other deep learning models) for classifying images from nuclear canisters as corroded. or intact. An image is considered to be corroded if it contains pitting or stress corrosion cracks. The code trains ResNets on image tiles extracted from original images from nuclear canisters, and then implements a classification rule, which can be applied to a validation set to decide if each original image is corroded or intact. The software automates the process of using images taken from nuclear canisters to detect corrosion. It provides also scope for future research directions on the basis of the existing research and code.

Papamarkou, Theodore↗

DOE OSTI2019

Selective Equatorial Sclera Crosslinking in the Orbit Using a Metal-Coated Polymer Waveguide

Purpose: Photochemical crosslinking of the sclera is an emerging technique that may prevent excessive eye elongation in pathologic myopia by stiffening the scleral tissue. To overcome the challenge of uniform light delivery in an anatomically restricted space, we previously introduced the use of flexible polymer waveguides. We presently demonstrate advanced waveguides that are optimized to deliver light selectively to equatorial sclera in the intact orbit. Methods: Our waveguides consist of a polydimethylsiloxane cladding and a polyurethane core, coupled to an optical fiber. A reflective silver coating deposited on the top and side surfaces of the waveguide prevents light leakage to nontarget, periorbital tissue. Postmortem rabbits were used to test the feasibility of in situ equatorial sclera crosslinking. Tensometry measurements were performed on ex vivo rabbit eyes to confirm a biomechanical stiffening effect. Results: Metal-coated waveguides enabled efficient light delivery to the entire circumference of the equatorial sclera with minimal light leakage to the periorbital tissues. Blue light was delivered to the intact orbit with a coefficient of variation in intensity of 22%, resulting in a 45 ± 11% bleaching of riboflavin fluorescence. A 2-fold increase in the Young's modulus at 5% strain (increase of 92% P < 0.05, at 25 J/cm 2 ) was achieved for ex vivo crosslinked eyes. Conclusions: Flexible polymer waveguides with reflective, biocompatible surfaces are useful for sclera crosslinking to achieve targeted light delivery. We anticipate that our demonstrated procedure will be applicable to sclera crosslinking in live animal models and, potentially, humans in vivo.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II

Abstract Background In plants, a large diversity of polysaccharides comprise the cell wall. Each major type of plant cell wall polysaccharide, including cellulose, hemicellulose, and pectin, has distinct structures and functions that contribute to wall mechanics and influence plant morphogenesis. In recent years, pectin valorization has attracted much attention due to its expanding roles in biomass deconstruction, food and material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of its structure. Herein, we present a workflow of principles relevant for the characterization of polysaccharide primary structure using nature’s most complex polysaccharide, rhamnogalacturonan-II (RG-II), as a model. Results We outline how to isolate RG-II from celery and duckweed cell walls and from red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we applied mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG-II and derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Ara p ) residue of side chain B is substituted at O- 2 with rhamnose. We used electrospray-MS techniques to identify non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters on RG-II-derived oligosaccharides. We then showed the utility of proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) to investigate the structure of intact RG-II and to complement the RG-II dimerization studies performed using size-exclusion chromatography. Conclusions The complexity of pectic polysaccharide structures has hampered efforts aimed at their valorization. In this work, we used RG-II as a model to demonstrate the steps necessary to isolate and characterize polysaccharides using chromatographic, MS, and NMR techniques. The principles can be applied to the characterization of other saccharide structures and will help inform researchers on how saccharide structure relates to functional properties in the future.

09 BIOMASS FUELS↗

DOE OSTI2019

Digital Breast Tomosynthesis for Intraoperative Margin Assessment during Breast-Conserving Surgery

Background: Intraoperative margin assessment for breast cancer patients undergoing segmental mastectomy (SM) enables identification of positive margins, with immediate excision of additional tissue to obtain negative margins. Objective: The aim of this study was to determine the ability of digital breast tomosynthesis (DBT) to detect positive margins compared with an institution’s standard extensive processing (SEP). Methods: SM specimens underwent intraoperative SEP with two-dimensional (2D) imaging of the intact and sliced specimen, with review by a breast radiologist and gross assessment by a breast pathologist. Findings guided the surgeon to excise additional tissue. DBT images of intact specimens were prospectively obtained and retrospectively reviewed by a breast radiologist. A positive margin was defined as tumor at ink. Results: Ninety-eight patients underwent 99 SMs. With SEP, 14 (14%) SM specimens had 19 positive margins. SEP did not detect 3 of the 19 positive margins, for a sensitivity of 84%, specificity of 78%, positive predictive value (PPV) of 11%, and negative predictive value (NPV) of 99%. Moreover, DBT did not detect 5 of the 19 positive margins, for a sensitivity of 74% (p > 0.05), specificity of 91% (p < 0.05), PPV of 21.5%, and NPV of 99%. With SEP guidance to excise additional tissue, six cases had final positive margins, with SEP not identifying three of these cases and DBT not identifying two. Pathology from the second surgery of these patients showed either no additional malignancy or only focal ductal carcinoma in situ. Conclusions: DBT is an accurate method for detecting positive margins in breast cancer patients undergoing SM, performing similar to institutional labor-intensive, intraoperative standard processing.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

DOE OSTI2019

Robotic Extended Right Hemicolectomy with Complete Mesocolic Excision and D3 Lymph Node Dissection

Background: Recent studies have shown the benefits of complete mesocolic excision and extended lymphadenectomy (D3 lymph node dissection) in patients with colon cancer.1{sup –}3 Methods: We present the case of a 62-year-old male with hepatic flexure adenocarcinoma. No metastatic disease was identified by computed tomography. A robot-assisted extended right hemicolectomy with complete mesocolic excision, D3 lymph node dissection, and resection of the mesentery with intact visceral peritoneum was performed. Results: The trocars are placed in the right lower (8 mm), lower midline (8 mm), and left upper (12 mm) quadrants. The camera port is placed superior to the umbilicus, and the assistant port is placed in the left lower quadrant. The robotic right lower port is used to place the cecum on tension in order to outline the ileocolic pedicle. The assistant retracts the transverse colon cephalad to outline the superior mesenteric artery and vein. Using two robotic arms, the surgeon begins dissection over the superior mesenteric vein inferior to the ileocolic pedicle. Cephalad dissection along the superior mesenteric vein proceeds with reflection of the mesentery and D3 lymph nodes laterally to allow en bloc resection. The ileocolic and middle colic vessels are identified, ligated and divided at their origins. The plane is then developed between the right colon mesentery and the retroperitoneum, including Gerota’s fascia, duodenum, and head of the pancreas, in a medial-to-lateral fashion, with care taken to ensure an intact visceral peritoneum is maintained. The proximal transverse colon, hepatic flexure, and ascending colon are mobilized by taking down lateral attachments. The intervening mesentery is transected, and perfusion is assessed with indocyanine green fluorescence imaging. An intracorporeal, isoperistaltic, side-to-side anastomosis is performed using the 45-mm robotic stapler. The common enterotomy is sewn closed in two layers. Pathology showed T3N0 adenocarcinoma with all negative margins. Conclusion: Extended right hemicolectomy with complete mesocolic excision and D3 lymph node dissection is facilitated by a robotic approach, which improves visualization and instrument dexterity.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

DOE OSTI2023

Reference data, predictors, and probability grids for forest degradation classes in three sites in the Brazilian Amazon

Forest degradation by fires and selective logging is widespread in the Amazon region. We implemented a gradient boosted classification modeling framework to classify intact, logged, and burned forests at three Amazonian sites: Feliz Natal Municipality and Xingu Indigenous Territory in Mato Grosso State, and Saracá-Taquera National Forest in Pará State. We used forest degradation history from Landsat time-series as reference data and textural metrics derived from PlanetScope images as predictors. Textural metrics were computed using the Gray-Level Co-Occurrence Matrix (GLCM) textural technique. Included in the attached zip file are ten files: - a shapefile containing the reference data (fire and selective logging polygons and year of event) for each site; - a multiband tif file containing the 8 GLCM metrics used as predictors (Mean, Variance, Homogeneity, Contrast, Dissimilarity, Entropy, Angular Second Moment, Correlation) at the original PlanetScope resolution (3.125m) for each site; - a multiband tif file containing the 72 aggregated GLCM metrics used as predictors (Mean, Variance, Homogeneity, Contrast, Dissimilarity, Entropy, Angular Second Moment, and Correlation aggregated using the mean, first quartile, third quartile, maximum, median, minimum, root mean square, standard deviation, and skewness statistics) at 562m resolution for each site; - a multiband tif file containing the 3 probability grids for either intact, logged, or burned forests at the aggregation resolution (562m) for each site.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2019

Cryogenic specimens for nanoscale characterization of solid–liquid interfaces

We report new cryogenic characterization techniques for exploring the nanoscale structure and chemistry of intact solid–liquid interfaces have recently been developed. These techniques provide high-resolution information about buried interfaces from large samples or devices that cannot be obtained by other means. These advancements were enabled by the development of instrumentation for cryogenic focused ion beam liftout, which allows intact solid–liquid interfaces to be extracted from large samples and thinned to electron-transparent thicknesses for characterization by cryogenic scanning transmission electron microscopy or atom probe tomography. Future implementation of these techniques will complement current strides in imaging of materials in fluid environments by in situ liquid-phase electron microscopy, providing a more complete understanding of the morphology, surface chemistry, and dynamic processes that occur at solid–liquid interfaces.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

DOE OSTI2021

Proposed Risk-Informed Regulatory Framework for Approval of Microreactor Transportation Packages

Microreactors are very small nuclear reactors with a power output of about 20 megawatts electric or less that are designed to be factory-built, modular in nature, and highly portable. These compact reactors will be small enough to be transported by truck or even air and could help solve energy challenges in a number of areas, ranging from remote commercial or residential locations to military bases. Pacific Northwest National Laboratory is tasked to develop and evaluate transportation licensing options for microreactors. The work is funded by the National Reactor Innovation Center a National Department of Energy program led by Idaho National Laboratory for the Office of Nuclear Energy Research and Development which support demonstration of microreactor technology. Key transportation steps include the (1) initial movement of high-assay low enriched uranium fresh fuel, (2) transportation of an intact, but never-operated microreactor, and (3) transportation of an intact, previously-operated microreactor. The deliverables on the project consists of a documentation of applicable regulations and regulatory authority for transportation. The objective of this report is to propose a risk-informed regulatory framework for the licensing of the transportation of microreactors, including the transportation of irradiated nuclear fuel that is assumed to be an integral component of the microreactor transportation package. The framework lays out a viable regulatory pathway, including decision points for regulatory options and the supporting technical evaluations for those options in phases from near to long term. This report includes discussion of the (1) general microreactor design concepts including representative microreactor source terms, (2) options for regulatory approval of microreactor transportation based on current regulation and historical precedence, (3) regulatory basis for including risk information in microreactor transportation licensing activities, and (4) description of a risk-informed regulatory framework.

21 SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLANTS↗

DOE OSTI2021

Proposed Risk-Informed Regulatory Framework for Approval of Microreactor Transportation Packages

21 SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLANTS↗

DOE OSTI2024

Dissolution Flowsheet for Non-Aluminum Spent Nuclear Fuel Campaign 1

As part of the Accelerated Basin De-inventory (ABD) program, H Canyon plans to dissolve non-aluminum spent nuclear fuel (NASNF) in the 6.3D electrolytic dissolver. NASNF Campaign 1 plans to electrolytically dissolve 68 bundles of fuel assemblies from the Carolinas-Virginia Tube Reactor (CVTR), Heavy Water Components Test Reactor (HWCTR), and Experimental Boiling Water Reactor (EBWR). The fuel assemblies are intact Zircaloy or stainless steel (SS) clad UO 2 rods, tubes, and plates. The H Canyon electrolytic dissolver previously dissolved a variety of UO 2 core fuel types in SS, Zircaloy, Nichrome, or Incoloy cladding from 1969 to 1980. The objective of this study was to identify flowsheet conditions through literature review and laboratory experimentation to safely dissolve NASNF Campaign 1 bundles in the H Canyon electrolytic dissolver. Bench-scale electrolytic dissolution tests were performed to demonstrate a flowsheet for NASNF Campaign 1 bundles. The outer bundles are composed of SS or Al alloy, Al 6061-T6, and contain intact Zircaloy or SS clad UO 2 fuel assemblies. The key objectives of these tests were to determine bounding dissolver chemistries and the sparge requirement to ensure H 2 concentration remain less than 60 vol % of the lower flammability limit (LFL) during dissolution. The impact of HNO 3 concentration and the addition of fluoride on the dissolution efficiency of Zircaloy, 304L SS, Al 6061-T6, and Inconel 625 were examined. While SS, Al, and Inconel 625 readily dissolve utilizing electrolytic dissolution, Zircaloy disintegrated anodically; the surface of Zircaloy oxidized and the oxide layer spalled off and settled at the bottom of the dissolver as an insoluble material. The black flakes were identified as ZrO 2 and 85% of the Zr processed was converted to black ZrO 2 flakes when Zr was anodically disintegrated in 9.5 M HNO 3 .

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2023

Developing Methods to Assess Changes in Mechanical Properties of Shale Modified by Engineered Mineral Precipitation

Fractures in subsurface shale formations serve multiple purposes, for example, in the recovery of resources in hydraulic fracturing or as potential harmful leakage passages through caprocks that may contribute undesired fluids to the atmosphere or functional groundwater aquifers. A proposed method to seal or influence fracture properties is Ureolysis-Induced Calcium Carbonate Precipitation (UICP), a bio-mineralization technology driven by the enzymatic hydrolysis of urea, resulting in the formation of calcium carbonate. Sporosarcina pasteurii is a common microbe used as the source of the urease enzyme that catalyzes the chemical reaction. The resulting calcium carbonate can bridge the gaps in fractured shale and reduce fluid flow through fractures. However, there is little information on how this process affects the mechanical properties of the resulting biomineralized shale. This study represents the first step toward determining the influence of UICP treatment on shale material and its subsequent mechanical strength properties. This methods development study aims to determine the effect that temperature has on the tensile strength of intact, unfractured shale cores (2.54 cm (1 in) diameter, 5.08 cm (2 in) long). Tensile strength was determined indirectly using a modified Brazilian test where the splitting tensile strength is attained by applying a compressive load onto the core. Shale cores from Eagle Ford and Wolfcamp formations were tested at both room temperature and 60°C to determine if increased temperature influences the tensile strength of the rock. This data will help to assess the necessity of testing biomineralized cores at temperature. Though 60°C may not mimic subsurface temperatures of the shales used in this study, it was chosen due to limitations of the UICP process while still approaching temperatures of shale formations. This project aims to evaluate what effect temperature has on the mechanical properties of intact shale cores so that engineered or natural rock fractures that are sealed by biomineralization can be better understood.

Bedey, Kayla↗

DOE OSTI2019

Recognition of Peptidoglycan Fragments by the Transpeptidase PBP4 From Staphylococcus aureus

Peptidoglycan (PG) is an essential component of the cell envelope, maintaining bacterial cell shape and protecting it from bursting due to turgor pressure. The monoderm bacterium Staphylococcus aureus has a highly cross-linked PG, with ~90% of peptide stems participating in DD-cross-links and up to 15 peptide stems connected with each other. These cross-links are formed in transpeptidation reactions catalyzed by penicillin-binding proteins (PBPs) of classes A and B. Most S. aureus strains have three housekeeping PBPs with this function (PBP1, PBP2, and PBP3) but MRSA strains have acquired a third class B PBP, PBP2a, which is encoded by the mecA gene and required for the expression of high-level resistance to β-lactams. Another housekeeping PBP of S. aureus is PBP4, which belongs to the class C PBPs, and hence would be expected to have PG hydrolase (DD-carboxypeptidase or DD-endopeptidase) activity. However, previous works showed that, unexpectedly, PBP4 has transpeptidase activity that significantly contributes to both the high level of cross-linking in the PG of S. aureus and to the low level of β-lactam resistance in the absence of PBP2a. To gain insights into this unusual activity of PBP4, we studied by NMR spectroscopy its interaction in vitro with different substrates, including intact peptidoglycan, synthetic peptide stems, muropeptides, and long glycan chains with uncross-linked peptide stems. PBP4 showed no affinity for the complex, intact peptidoglycan or the smallest isolated peptide stems. Transpeptidase activity of PBP4 was verified with the disaccharide peptide subunits (muropeptides) in vitro, producing cyclic dimer and multimer products; these assays also showed a designed PBP4(S75C) nucleophile mutant to be inactive. Using this inactive but structurally highly similar variant, liquid-state NMR identified two interaction surfaces in close proximity to the central nucleophile position that can accommodate the potential donor and acceptor stems for the transpeptidation reaction. A PBP4:muropeptide model structure was built from these experimental restraints, which provides new mechanistic insights into mecA independent resistance to β-lactams in S. aureus.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Mapping Influenza-Induced Posttranslational Modifications on Histones from CD8+ T Cells

T cell function is determined by transcriptional networks that are regulated by epigenetic programming via posttranslational modifications (PTMs) to histone proteins and DNA. Bottom-up mass spectrometry (MS) can identify histone PTMs, whereas intact protein analysis by MS can detect species missed by bottom-up approaches. We used a novel approach of online two-dimensional liquid chromatography-tandem MS with high-resolution reversed-phase liquid chromatography (RPLC), alternating electron transfer dissociation (ETD) and collision-induced dissociation (CID) on precursor ions to maximize fragmentation of uniquely modified species. The first online RPLC separation sorted histone families, then RPLC or weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) separated species heavily clad in PTMs. Tentative identifications were assigned by matching proteoform masses to predicted theoretical masses that were verified with tandem MS. We used this innovative approach for histone-intact protein PTM mapping (HiPTMap) to identify and quantify proteoforms purified from CD8 T cells after in vivo influenza infection. Activation significantly altered PTMs following influenza infection, histone maps changed as T cells migrated to the site of infection, and T cells responding to secondary infections had significantly more transcription enhancing modifications. Thus, HiPTMap identified and quantified proteoforms and determined changes in CD8 T cell histone PTMs over the course of infection.

mass spectrometry↗