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DOE OSTI2021

Inhibition of Clostridium difficile TcdA and TcdB toxins with transition state analogues

Clostridium difficile causes life-threatening diarrhea and is the leading cause of healthcare-associated bacterial infections in the United States. TcdA and TcdB bacterial toxins are primary determinants of disease pathogenesis and are attractive therapeutic targets. TcdA and TcdB contain domains that use UDP-glucose to glucosylate and inactivate host Rho GTPases, resulting in cytoskeletal changes causing cell rounding and loss of intestinal integrity. Transition state analysis revealed glucocationic character for the TcdA and TcdB transition states. We identified transition state analogue inhibitors and characterized them by kinetic, thermodynamic and structural analysis. Iminosugars, isofagomine and noeuromycin mimic the transition state and inhibit both TcdA and TcdB by forming ternary complexes with Tcd and UDP, a product of the TcdA- and TcdB-catalyzed reactions. Both iminosugars prevent TcdA- and TcdB-induced cytotoxicity in cultured mammalian cells by preventing glucosylation of Rho GTPases. Iminosugar transition state analogues of the Tcd toxins show potential as therapeutics for C. difficile pathology.

99 GENERAL AND MISCELLANEOUS↗

DOE OSTI2026

Data for Expression of a Bacterial Trehalose 6-Phosphate Synthase Gene otsA in Camelina sativa Seeds Promotes the Channelling of Carbon Towards Oil Accumulation

Improving seed oil yield is essential for developing Camelina sativa as a sustainable biofuel crop. Fatty acid synthesis depends on the production of acetyl-CoA from photosynthetically derived sugars. Trehalose 6-phosphate (T6P), a proxy for sucrose availability, can link sugar status to plant growth and development. Synthesised by trehalose 6-phosphate synthase (TPS) from UDP-glucose and glucose-6-phosphate, T6P plays a regulatory role in metabolism. Our previous studies on Arabidopsis transgenic lines constitutively expressing the E. coli otsA (encoding TPS) showed increased T6P levels and seed triacylglycerol, along with stunted growth. In the present study we express otsA in camelina under the control of a seed-specific Phaseolin promoter. Seeds of the resulting transgenic lines accumulated high levels of T6P, and a 15%–20% increase in total fatty acids and triacylglycerol compared to wild-type. Molecular analysis showed the transgenic seeds had reduced SnRK1 activity, elevated WRI1 protein levels, and increased the levels of WRI1 and its target genes, along with enhanced rates of fatty acid synthesis that increased seed weights relative to wild type. Notably, the increase in oil did not affect seed protein levels but did reduce the soluble metabolite fraction. Crucially, seed-specific expression of otsA mitigated the growth defects associated with constitutive otsA expression, and the transgenic lines showed normal seed development and germination. These findings demonstrate that targeted T6P modulation via seed-specific otsA expression is an effective metabolic engineering strategy to boost oil production in camelina and potentially in other oilseed crops and bioenergy crops such as energycane, sorghum and miscanthus.

Lipids↗

NASA NTRS1994

A new communication protocol family for a distributed spacecraft control system

In this paper we describe the concepts behind and architecture of a communication protocol family, which was designed to fulfill the communication requirements of ESOC's new distributed spacecraft control system SCOS 2. A distributed spacecraft control system needs a data delivery subsystem to be used for telemetry (TLM) distribution, telecommand (TLC) dispatch and inter-application communication, characterized by the following properties: reliability, so that any operational workstation is guaranteed to receive the data it needs to accomplish its role; efficiency, so that the telemetry distribution, even for missions with high telemetry rates, does not cause a degradation of the overall control system performance; scalability, so that the network is not the bottleneck both in terms of bandwidth and reconfiguration; flexibility, so that it can be efficiently used in many different situations. The new protocol family which satisfies the above requirements is built on top of widely used communication protocols (UDP and TCP), provides reliable point-to-point and broadcast communication (UDP+) and is implemented in C++. Reliability is achieved using a retransmission mechanism based on a sequence numbering scheme. Such a scheme allows to have cost-effective performances compared to the traditional protocols, because retransmission is only triggered by applications which explicitly need reliability. This flexibility enables applications with different profiles to take advantage of the available protocols, so that the best rate between sped and reliability can be achieved case by case.

Baldi, Andrea↗

NASA NTRSDate not supplied

Space Grant Final Paper

The purpose of this internship was to begin the development of an APK that would subscribe to NavCam and video stream via UDP. This project involved having the Astrobee Robot Software installed, the Astrobee Android Repository, and the use of an android emulator. While the final APK currently cannot stream via UDP, it can still subscribe to the topic, which for me was already a challenge.

Astrobee↗

DOE OSTI2023

Development of LpxH Inhibitors Chelating the Active Site Dimanganese Metal Cluster of LpxH

Abstract Despite the widespread emergence of multidrug‐resistant nosocomial Gram‐negative bacterial infections and the major public health threat it brings, no new class of antibiotics for Gram‐negative pathogens has been approved over the past five decades. Therefore, there is an urgent medical need for developing effective novel antibiotics against multidrug‐resistant Gram‐negative pathogens by targeting previously unexploited pathways in these bacteria. To fulfill this crucial need, we have been investigating a series of sulfonyl piperazine compounds targeting LpxH, a dimanganese‐containing UDP‐2,3‐diacylglucosamine hydrolase in the lipid A biosynthetic pathway, as novel antibiotics against clinically important Gram‐negative pathogens. Inspired by a detailed structural analysis of our previous LpxH inhibitors in complex with K. pneumoniae LpxH ( Kp LpxH), here we report the development and structural validation of the first‐in‐class sulfonyl piperazine LpxH inhibitors, JH‐LPH‐45 ( 8 ) and JH‐LPH‐50 ( 13 ), that achieve chelation of the active site dimanganese cluster of Kp LpxH. The chelation of the dimanganese cluster significantly improves the potency of JH‐LPH‐45 ( 8 ) and JH‐LPH‐50 ( 13 ). We expect that further optimization of these proof‐of‐concept dimanganese‐chelating LpxH inhibitors will ultimately lead to the development of more potent LpxH inhibitors for targeting multidrug‐resistant Gram‐negative pathogens.

Pharmacology & Pharmacy↗

DOE OSTI2023

Specificity determinants revealed by the structure of glycosyltransferase Campylobacter concisus PglA

Abstract In selected Campylobacter species, the biosynthesis of N‐linked glycoconjugates via the pgl pathway is essential for pathogenicity and survival. However, most of the membrane‐associated GT‐B fold glycosyltransferases responsible for diversifying glycans in this pathway have not been structurally characterized which hinders the understanding of the structural factors that govern substrate specificity and prediction of resulting glycan composition. Herein, we report the 1.8 Å resolution structure of Campylobacter concisus PglA, the glycosyltransferase responsible for the transfer of N ‐acetylgalatosamine (GalNAc) from uridine 5′‐diphospho‐ N ‐acetylgalactosamine (UDP‐GalNAc) to undecaprenyl‐diphospho‐ N , N ′‐diacetylbacillosamine (UndPP‐diNAcBac) in complex with the sugar donor GalNAc. This study identifies distinguishing characteristics that set PglA apart within the GT4 enzyme family. Computational docking of the structure in the membrane in comparison to homologs points to differences in interactions with the membrane‐embedded acceptor and the structural analysis of the complex together with bioinformatics and site‐directed mutagenesis identifies donor sugar binding motifs. Notably, E113, conserved solely among PglA enzymes, forms a hydrogen bond with the GalNAc C6″‐OH. Mutagenesis of E113 reveals activity consistent with this role in substrate binding, rather than stabilization of the oxocarbenium ion transition state, a function sometimes ascribed to the corresponding residue in GT4 homologs. The bioinformatic analyses reveal a substrate‐specificity motif, showing that Pro281 in a substrate binding loop of PglA directs configurational preference for GalNAc over GlcNAc. This proline is replaced by a conformationally flexible glycine, even in distant homologs, which favor substrates with the same stereochemistry at C4, such as glucose. The signature loop is conserved across all Campylobacter PglA enzymes, emphasizing its importance in substrate specificity.

Vuksanovic, Nemanja↗

DOE OSTI2026

Preliminary proof-of-concept of real-time divertor heat flux control from infrared cameras with nitrogen injection in the DIII-D tokamak

In future tokamak reactors like ITER and the Fusion Pilot Plant (FPP), real-time feedback control of heat flux to the plasma-facing components (PFC) will be critical for steady-state operation. This work presents the first experimental demonstration of real-time divertor heat flux estimation with infrared thermography and feedback control with impurity seeding on the DIII-D tokamak. The flexible infrastructure of the Plasma Control System (PCS) on DIII-D makes this new capability possible. The PCS software runs on a gateway computer system, and five real-time compute nodes. An array of low latency streaming digitizers from D-TACQ Solutions connects to these real-time computers to collect and process data, and send commands to actuators during plasma discharges. This system handles the signal IO from the tokamak and allows the PCS to utilize the diagnostic data necessary to perform control in real-time. Feedback control on heat flux was accomplished by feeding infrared camera data from the “Infrared TV” (IRTV) camera to a custom-developed User Datagram Protocol (UDP) server. This server transmits infrared data to a newly developed PCS algorithm that estimates the heat flux to PFC. Here, a proportional integral derivative (PID) controller minimizes the error between a heat flux reference and the real-time estimate by injecting nitrogen gas into the divertor.

Algorithm development↗

DOE OSTI2020

Glucuronidation of Methylated Quercetin Derivatives: Chemical and Biochemical Approaches

Botanical supplements derived from grapes are functional in animal model systems for the amelioration of neurological conditions, including cognitive impairment. Rats fed with grape extracts accumulate 3'-O-methyl-quercetin-3-O-β-Dglucuronide (3) in their brains, suggesting 3 as a potential therapeutic agent. To develop methods for the synthesis of 3 and the related 4'-O-methyl-quercetin-7-O-β-D-glucuronide (4), 3-O-methyl-quercetin-3'-O-β-D-glucuronide (5), and 4'-O-methylquercetin-3'-O-β-D-glucuronide (6), which are not found in the brain, we have evaluated both enzymatic semisynthesis and full chemical synthetic approaches. Biocatalysis by mammalian UDP-glucuronosyltransferases generated multiple glucuronidated products from 4'-O-methylquercetin, and is not cost-effective. Chemical synthetic methods, on the other hand, provided good results; 3, 5, and 6 were obtained in six steps at 12, 18, and 30% overall yield, respectively, while 4 was synthesized in five steps at 34% overall yield. Furthermore, a mechanistic study on the unexpected regioselectivity observed in the quercetin glucuronide synthetic steps is also presented.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Generation of Pseudomonas putida KT2440 Strains with Efficient Utilization of Xylose and Galactose via Adaptive Laboratory Evolution

While Pseudomonas putida KT2440 has great potential for biomass-converting processes, its inability to utilize the biomass abundant sugars xylose and galactose has limited its applications. Here, in this study, we utilized Adaptive Laboratory Evolution (ALE) to optimize engineered KT2440 with heterologous expression of xylD encoding xylonate dehydratase from Caulobacter crescentus and galETKM encoding UDP-glucose 4-epimerase, galactose-1-phosphate uridylyltransferase, galactokinase, and galactose-1-epimerase from Escherichia coli K-12 MG1655. Poor starting strain growth (<0.1 h –1 or none) was evolutionarily optimized to rates of up to 0.25 h –1 on xylose and 0.52 h –1 on galactose. Whole-genome sequencing, transcriptomic analysis, and growth screens revealed significant roles of kguT encoding a 2-ketogluconate operon repressor and 2-ketogluconate transporter, and gtsABCD encoding an ATP-binding cassette (ABC) sugar transporting system in xylose and galactose growth conditions, respectively. Finally, we expressed the heterologous indigoidine production pathway in the evolved and unevolved engineered strains and successfully produced 3.2 g/L and 2.2 g/L from 10 g/L of either xylose or galactose in the evolved strains whereas the unevolved strains did not produce any detectable product. Thus, the generated KT2440 strains have the potential for broad application as optimized platform chassis to develop efficient microorganism-based biomass-utilizing bioprocesses.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

Advances in Engineering Nucleotide Sugar Metabolism for Natural Product Glycosylation in Saccharomyces cerevisiae

Glycosylation is a ubiquitous modification present across all of biology, affecting many things such as physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Nucleotide sugars are important precursors needed to study glycosylation and produce glycosylated products. Saccharomyces cerevisiae is a potentially powerful platform for producing glycosylated biomolecules, but it lacks nucleotide sugar diversity. Nucleotide sugar metabolism is complex, and understanding how to engineer it will be necessary to both access and study heterologous glycosylations found across biology. This review overviews the potential challenges with engineering nucleotide sugar metabolism in yeast from the salvage pathways that convert free sugars to their associated UDP-sugars to de novo synthesis where nucleotide sugars are interconverted through a complex metabolic network with governing feedback mechanisms. Finally, recent examples of engineering complex glycosylation of small molecules in S. cerevisiae are explored and assessed.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases

Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modeling and simulations in conjunction with biochemical characterization to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Involvement of the Streptococcus mutans PgfE and GalE 4-epimerases in protein glycosylation, carbon metabolism, and cell division

Streptococcus mutans is a key pathogen associated with dental caries and is often implicated in infective endocarditis. This organism forms robust biofilms on tooth surfaces and can use collagen-binding proteins (CBPs) to efficiently colonize collagenous substrates, including dentin and heart valves. One of the best characterized CBPs of S. mutans is Cnm, which contributes to adhesion and invasion of oral epithelial and heart endothelial cells. These virulence properties were subsequently linked to post-translational modification (PTM) of the Cnm threonine-rich repeat region by the Pgf glycosylation machinery, which consists of 4 enzymes: PgfS, PgfM1, PgfE, and PgfM2. Inactivation of the S. mutans pgf genes leads to decreased collagen binding, reduced invasion of human coronary artery endothelial cells, and attenuated virulence in the Galleria mellonella invertebrate model. The present study aimed to better understand Cnm glycosylation and characterize the predicted 4-epimerase, PgfE. Using a truncated Cnm variant containing only 2 threonine-rich repeats, mass spectrometric analysis revealed extensive glycosylation with HexNAc2. Compositional analysis, complemented with lectin blotting, identified the HexNAc2 moieties as GlcNAc and GalNAc. Comparison of PgfE with the other S. mutans 4-epimerase GalE through structural modeling, nuclear magnetic resonance, and capillary electrophoresis demonstrated that GalE is a UDP-Glc-4-epimerase, while PgfE is a GlcNAc-4-epimerase. While PgfE exclusively participates in protein O-glycosylation, we found that GalE affects galactose metabolism and cell division. Furthermore, this study further emphasizes the importance of O-linked protein glycosylation and carbohydrate metabolism in S. mutans and identifies the PTM modifications of the key CBP, Cnm.

4-epimerases↗

DOE OSTI2021

A Communication Testbed for Testing Power Electronic Agent Systems

Power electronic systems (PES) incorporate complex intra-system communication, which are of vital importance for the successful operation of these systems. This paper proposes and outlines a communication testbed that will help in the development and testing of the communications between the components of PES. It allows for the comparison and evaluation of different communication methods, such as MQTT, Modbus, or User Datagram Protocol (UDP), and for the characterization of how these communication protocols perform.

Dean, Ben↗

DOE OSTI2023

ESnet/JLab FPGA Accelerated Transport

To increase the science rate for high data rates/volumes, Thomas Jefferson National Accelerator Facility (JLab) has partnered with Energy Sciences Network (ESnet) to define an edge to data center traffic shaping / steering transport capability featuring data event aware network shaping and forwarding. The keystone of this ESnet+JLab FPGA Accelerated Transport (EJFAT) is the joint development of an AI/ML directed dynamic compute work Load Balancer (LB) of UDP streamed data. The LB is a suite consisting of a Field Programmable Gate Array (FPGA) executing the dynamically configurable, low fixed latency LB data plane featuring real-time packet redirection and high throughput, and a control plane running on the FPGA host computer that monitors network and compute farm telemetry in order to make dynamic AI/ML guided decisions for destination compute host redirection/load balancing and destination resource provisioning. The LB provides for three-tier horizontal scaling across LB suites, core compute hosts, and CPUs within a host. The LB effectively provides seamless integration of edge/core computing to support direct experimental data processing for immediate use by JLab science programs and others such as the EIC as well as data centers of the future requiring high throughput and low latency for both hot and cooled data for both running experiment data acquisition systems and data center use cases.

97 MATHEMATICS AND COMPUTING↗

DOE OSTI2024

Real-Time Ethernet Interface for NSTX-U’s Thomson Scattering Diagnostic (2023)

Here, the multipoint Thomson scattering (MPTS) diagnostic system at the National Spherical Torus Experiment Upgrade (NSTX-U) facility is undergoing an upgrade to operate in real-time and interface with the plasma control system (PCS) for NSTX-U. Previous prototyping efforts have shown that spectral analysis and rapid calculations of electron temperature and density are possible on a real-time Linux machine when using up to a 100-Hz laser pulse repetition rate. A remaining challenge was transferring the real-time data to NSTX-U’s PCS, which utilizes the front panel data port (FPDP) protocol. The original proposed method was to convert the real-time data into analog values, but a new solution was developed to keep the output format digital by using an Ethernet controller with a field-programmable gate array (FPGA). This article focuses on a new input module that has been developed to accept incoming user datagram protocol (UDP) packets sent over Ethernet, convert into FPDP format, and integrate into the existing data stream under NSTX-U’s real-time framework.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

DOE OSTI2026

Deploying MARTe2 and MDSplus for Scalable Real-Time Control Systems: A Proof-of-Concept for the SMART Tokamak

The SMART tokamak is a spherical tokamak with flexible shaping that will need a modular and high-performance real-time control system. This work presents the implementation and validation of the MARTe2 framework at the Princeton Plasma Physics Laboratory (PPPL) as a testbed for supporting SMART (University of Seville, Spain). The system integrates MARTe2, MDSplus for data archiving, and user datagram protocol (UDP)-based communication with both synthetic and physical devices. A deterministic control loop was developed using custom control algorithms, showing the system’s ability to operate under strict real-time constraints. The resulting framework is scalable, maintainable, and adaptable to experimental tokamaks like SMART.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗

DOE OSTI2025

Expression of a Bacterial Trehalose 6-Phosphate Synthase Gene otsA in Camelina sativa Seeds Promotes the Channelling of Carbon Towards Oil Accumulation

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Identification of xylan arabinosyl 2- O -xylosyltransferases catalyzing the addition of 2- O -xylosyl residue onto arabinosyl side chains of xylan in grass species

Grass xylan, the major hemicellulose in both primary and secondary cell walls, is heavily decorated with α-1,3-linked arabinofuranosyl (Araf) residues that may be further substituted at O-2 with xylosyl (Xyl) or Araf residues. Although xylan 3-O-arabinosyltransferases (XATs) catalyzing 3-O-Araf addition onto xylan have been characterized, glycosyltransferases responsible for the transfer of 2-O-Xyl or 2-O-Araf onto 3-O-Araf residues of xylan to produce the Xyl-Araf and Araf-Araf disaccharide side chains remain to be identified. In this report, we showed that a rice GT61 member, named OsXAXT1 (xylan arabinosyl 2-O-xylosyltransferase 1) herein, was able to mediate the addition of Xyl-Araf disaccharide side chains onto xylan when heterologously co-expressed with OsXAT2 in the Arabidopsis gux1/2/3 (glucuronic acid substitution of xylan 1/2/3) triple mutant that lacks any glycosyl substitutions. Recombinant OsXAXT1 protein expressed in human embryonic kidney 293 cells exhibited a xylosyltransferase activity catalyzing the addition of Xyl from UDP-Xyl onto arabinosylated xylooligomers. Further, consistent with its function as a xylan arabinosyl 2-O-xylosyltransferase, CRISPR-Cas9-mediated mutations of the OsXAXT1 gene in transgenic rice plants resulted in a reduction in the level of Xyl-Araf disaccharide side chains in xylan. Furthermore, we revealed that XAXT1 close homologs from several other grass species, including switchgrass, maize, and Brachypodium, possessed the same functions as OsXAXT1, indicating functional conservation of XAXTs in grass species. Together, our findings establish that grass XAXTs are xylosyltransferases catalyzing Xyl transfer onto O-2 of Araf residues of xylan to form the Xyl-Araf disaccharide side chains, which furthers our understanding of genes involved in xylan biosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗