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At least 235 records · Page 13
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Development of a Universal Second-Tier Newborn Screening LC–MS/MS Method for Amino Acids, Lysophosphatidylcholines, and Organic Acids

First-tier MS-based newborn screening by flow injection analysis can have high presumptive positive rates, often due to isomeric/isobaric compounds or poor biomarker specificity. These presumptive positive samples can be analyzed by second-tier screening assays employing separations such as liquid chromatography–mass spectrometry (LC–MS/MS), which increases test specificity and drastically reduces false positive referrals. The ability to screen for multiple disorders in a single multiplexed test simplifies workflows and maximizes public health laboratories’ resources. In this study, we developed and validated a highly multiplexed second-tier method for dried blood spots using a hydrophilic interaction liquid chromatography (HILIC) column coupled to an MS/MS system. The LC–MS/MS method was capable of simultaneously detecting second-tier biomarkers for maple syrup urine disease, homocystinuria, methylmalonic acidemia, propionic acidemia, glutaric acidemia type 1, glutaric acidemia type 2, guanidinoacetate methyltransferase deficiency, short-chain acyl-CoA dehydrogenase deficiency, adrenoleukodystrophy, and Pompe disease.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Evaluation of Differential Peptide Loading on Tandem Mass Tag-Based Proteomic and Phosphoproteomic Data Quality

Global and phosphoproteome profiling has demonstrated great utility for the analysis of clinical specimens. One major barrier to the broad clinical application of proteomic profiling is the large amount of biological material required, particularly for phosphoproteomics—currently on the order of 25 mg wet tissue weight, depending on tissue type. For hematopoietic cancers such as acute myeloid leukemia (AML), the sample requirement is in excess of 10 million (1E7) peripheral blood mononuclear cells (PBMCs). Throughout the course of a prospective study, this requirement will certainly exceed what is obtainable from many of the individual patients/timepoints. For this reason, we were interested in examining the impact of differential peptide loading across multiplex channels on proteomic data quality. Methods: To achieve this, we tested a range of channel loading amounts (20, 40, 100, 200, and 400 µg of tryptic peptides, or approximately the material obtainable from 5E5, 1E6, 2.5E6, 5E6, and 1E7 AML patient cells) to assess proteome coverage, quantification reproducibility and accuracy in experiments utilizing isobaric tandem mass tag (TMT) labeling. As expected, we found that fewer missing values are observed in TMT channels with higher peptide loading amounts compared to those with lower loading. Moreover, channels with lower loading amounts have greater quantitative variability than channels with higher loading amounts. Statistical analysis of the differences in means among the five loading groups showed that the 20 µg loading group was significantly different from the 400 µg loading group. However, no significant differences were detected among the 40, 100, 200 and 400 µg loading groups. Conclusions: These assessment data demonstrate the practical limits of loading differential quantities of peptides across channels in TMT multiplexes, and provide a basis for designing the optimal clinical proteomics study when specimen quantities are limited.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Engineering an efficient and bright split Corynactis californica green fluorescent protein

Split green fluorescent protein (GFP) has been used in a panoply of cellular biology applications to study protein translocation, monitor protein solubility and aggregation, detect protein–protein interactions, enhance protein crystallization, and even map neuron contacts. Recent work shows the utility of split fluorescent proteins for large scale labeling of proteins in cells using CRISPR, but sets of efficient split fluorescent proteins that do not cross-react are needed for multiplexing experiments. We present a new monomeric split green fluorescent protein (ccGFP) engineered from a tetrameric GFP found in Corynactis californica, a bright red colonial anthozoan similar to sea anemones and scleractinian stony corals. Split ccGFP from C. californica complements up to threefold faster compared to the original Aequorea victoria split GFP and enable multiplexed labeling with existing A. victoria split YFP and CFP.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Multivariate chemogenomic screening prioritizes new macrofilaricidal leads

Development of direct acting macrofilaricides for the treatment of human filariases is hampered by limitations in screening throughput imposed by the parasite life cycle. In vitro adult screens typically assess single phenotypes without prior enrichment for chemicals with antifilarial potential. We developed a multivariate screen that identified dozens of compounds with submicromolar macrofilaricidal activity, achieving a hit rate of >50% by leveraging abundantly accessible microfilariae. Adult assays were multiplexed to thoroughly characterize compound activity across relevant parasite fitness traits, including neuromuscular control, fecundity, metabolism, and viability. Seventeen compounds from a diverse chemogenomic library elicited strong effects on at least one adult trait, with differential potency against microfilariae and adults. Our screen identified five compounds with high potency against adults but low potency or slow-acting microfilaricidal effects, at least one of which acts through a novel mechanism. We show that the use of microfilariae in a primary screen outperforms model nematode developmental assays and virtual screening of protein structures inferred with deep learning. These data provide new leads for drug development, and the high-content and multiplex assays set a new foundation for antifilarial discovery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Fiber optic computing using distributed feedback

Abstract The widespread adoption of machine learning and other matrix intensive computing algorithms has renewed interest in analog optical computing, which has the potential to perform large-scale matrix multiplications with superior energy scaling and lower latency than digital electronics. However, most optical techniques rely on spatial multiplexing, requiring a large number of modulators and detectors, and are typically restricted to performing a single kernel convolution operation per layer. Here, we introduce a fiber-optic computing architecture based on temporal multiplexing and distributed feedback that performs multiple convolutions on the input data in a single layer. Using Rayleigh backscattering in standard single mode fiber, we show that this technique can efficiently apply a series of random nonlinear projections to the input data, facilitating a variety of computing tasks. The approach enables efficient energy scaling with orders of magnitude lower power consumption than GPUs, while maintaining low latency and high data-throughput.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2020

Second Generation Readout For Large Format Photon Counting Microwave Kinetic Inductance Detectors

We present the development of a second generation digital readout system for photon counting microwave kinetic inductance detector (MKID) arrays operating in the optical and near-infrared wavelength bands. Our system retains much of the core signal processing architecture from the first generation system but with a significantly higher bandwidth, enabling the readout of kilopixel MKID arrays. Each set of readout boards is capable of reading out 1024 MKID pixels multiplexed over 2 GHz of bandwidth; two such units can be placed in parallel to read out a full 2048 pixel microwave feedline over a 4 GHz–8 GHz band. As in the first generation readout, our system is capable of identifying, analyzing, and recording photon detection events in real time with a time resolution of order a few microseconds. Here, we describe the hardware and firmware, and present an analysis of the noise properties of the system. We also present a novel algorithm for efficiently suppressing IQ mixer sidebands to below −30 dBc.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2023

Broadband bandpass Purcell filter for circuit quantum electrodynamics

In circuit quantum electrodynamics, qubits are typically measured using dispersively coupled readout resonators. Coupling between each readout resonator and its electrical environment, however, reduces the qubit lifetime via the Purcell effect. Inserting a Purcell filter counters this effect while maintaining high readout fidelity but reduces measurement bandwidth and, thus, limits multiplexing readout capacity. In this Letter, we develop and implement a multi-stage bandpass Purcell filter that yields better qubit protection while simultaneously increasing measurement bandwidth and multiplexed capacity. Here, we report on the experimental performance of our transmission-line-based implementation of this approach, a flexible design that can easily be integrated with current scaled-up, long coherence time superconducting quantum processors.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2020

Bragg grating inscription in photosensitive materials by an optical replication process

This paper examines the feasibility of producing multiplexed Bragg gratings for use as diffractive optical elements (DOEs) by coherent optical replication of a surface-relief phase grating into thick-film photosensitive materials. The use of such DOEs in light guide plates (LGPs) for optical imaging systems ensures energy-efficient light input into and output from LGPs and allows one to minimise their weight and size. We present simulation results for the interference structure fabrication process and recording of a series of multiplexed diffraction gratings in photosensitive materials. (paper)

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2025

Cryosectioning-enhanced super-resolution microscopy for single-protein imaging across cells and tissues

DNA-points accumulation for imaging in nanoscale topography (DNA-PAINT) enables nanoscale imaging with virtually unlimited multiplexing and molecular counting. Here, we address challenges, such as variable imaging performance and target accessibility, that can limit its broader applicability. Specifically, we enhance its capacity for robust single-protein imaging and molecular counting by optimizing the integration of total internal reflection fluorescence microscopy with physical sectioning, in particular, Tokuyasu cryosectioning. Our method, tomographic and kinetically enhanced DNA-PAINT (tkPAINT), achieves 3 nm localization precision across diverse samples, enhanced imager binding, and improved cellular integrity. tkPAINT can facilitate molecular counting with DNA-PAINT inside the nucleus, as demonstrated through its quantification of the in situ abundance of RNA Polymerase II in both HeLa cells as well as mouse tissues. Anticipating that tkPAINT could become a versatile tool for the exploration of biomolecular organization and interactions across cells and tissues, we also demonstrate its capacity to support multiplexing, multimodal targeting of proteins and nucleic acids, and three-dimensional (3D) imaging.

Science & Technology - Other Topics↗
DOE OSTI2024

Photon number resolving detection with a single-photon detector and adaptive storage loop

Abstract Photon number resolving (PNR) measurements are beneficial or even necessary for many applications in quantum optics. Unfortunately, PNR detectors are usually large, slow, expensive, and difficult to operate. However, if the input signal is multiplexed, photon ‘click’ detectors, that lack an intrinsic PNR capability, can still be used to realize photon number resolution. Here, we investigate the operation of a single click detector, together with a storage line with tunable outcoupling. Using adaptive feedback to adjust the storage outcoupling rate, the dynamic range of the detector can in certain situations be extended by up to an order of magnitude relative to a purely passive setup. An adaptive approach can thus allow for photon number variance below the quantum shot noise limit under a wider range of conditions than using a passive multiplexing approach. This can enable applications in quantum enhanced metrology and quantum computing.

Sullivan, Nicholas M. (ORCID:0000000248447296)↗
DOE OSTI2021

MS2Planner: improved fragmentation spectra coverage in untargeted mass spectrometry by iterative optimized data acquisition

Motivation: Untargeted mass spectrometry experiments enable the profiling of metabolites in complex biological samples. The collected fragmentation spectra are the metabolite’s fingerprints that are used for molecule identification and discovery. Two main mass spectrometry strategies exist for the collection of fragmentation spectra: data-dependent acquisition (DDA) and data-independent acquisition (DIA). In the DIA strategy, all the metabolites ions in predefined mass-to-charge ratio ranges are co-isolated and co-fragmented, resulting in multiplexed fragmentation spectra that are challenging to annotate. In contrast, in the DDA strategy, fragmentation spectra are dynamically and specifically collected for the most abundant ions observed, causing redundancy and sub-optimal fragmentation spectra collection. Yet, DDA results in less multiplexed fragmentation spectra that can be readily annotated. Results: We introduce the MS2Planner workflow, an Iterative Optimized Data Acquisition strategy that optimizes the number of high-quality fragmentation spectra over multiple experimental acquisitions using topological sorting. Our results showed that MS2Planner increases the annotation rate by 38.6% and is 62.5% more sensitive and 9.4% more specific compared to DDA. Availability and implementation MS2Planner code is available at https://github.com/mohimanilab/MS2Planner. The generation of the inclusion list from MS2Planner was performed with python scripts available at https://github.com/lfnothias/IODA_MS.

47 OTHER INSTRUMENTATION↗
DOE OSTI2023

Strategies to identify and edit improvements in synthetic genome segments episomally

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments at low copy number. However, all stages of whole-genome engineering have the potential to impose mutations on the synthetic genome that can reduce or eliminate the fitness of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to recoding an Escherichia coli strain to replace seven codons with synonymous alternatives genome wide. Ten 44 402–47 179 bp de novo synthesized DNA segments contained in a BAC from the recoded strain were unable to complement deletion of the corresponding 33–61 wild-type genes using a single antibiotic resistance marker. Next-generation sequencing (NGS) was used to identify 1–7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the recoded segment contained in the BAC when both the recoded and wild-type copies of the mutated genes had to exist by necessity during the repair process. Finally, two web-based tools were used to predict the impact of a subset of non-recoding missense mutations on strain fitness using protein structure and function calls.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Field test of continuous-variable quantum key distribution with a true local oscillator

A continuous-variable quantum key distribution (CV QKD) using a true local (located at the receiver) oscillator (LO) has been proposed to remove any possibility of side-channel attacks associated with transmission of the LO as well as reduce the cross-pulse contamination. Here we report an implementation of true LO-CV QKD using “off-the-shelf” components and conduct QKD experiments using the fiber optical network at Oak Ridge National Laboratory. A phase reference and quantum signal are time multiplexed and then wavelength division multiplexed with the classical communications that “coexist” with each other on a single optical network fiber. Importantly, this is the first demonstration of CV QKD with a receiver-based true LO over a deployed fiber network, a crucial step for its application in real-world situations.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2024

A Cryogenic readout integrated circuit with analog pile-up and in-Pixel ADC for high frame rate Skipper CCD-in-CMOS Sensors

The Skipper CCD-in-CMOS Parallel Read-Out Circuit V2 (SPROCKET2) is designed to enable high frame rate readout of Skipper CCD-in-CMOS image sensors. The SPROCKET2 pixel is fabricated in a 65 nm CMOS process and occupies a 60$\mu$m $\times$ 60$\mu$m footprint. SPROCKET2 is intended to be heterogeneously integrated with a pixelated Skipper CCD-in-CMOS sensor, such that one readout pixel is connected to a multiplexed array of 16 active image sensor pixels, to match their spatial geometry. Our design benefits from the Skipper CCD-in-CMOS sensor's non-destructive readout capability to achieve exceptionally low noise through multi-sampling and averaging while optimizing for total power consumption. The pixel readout utilizes correlated double sampling to minimize 1/f noise and includes "pile-up" of ten successive samples in the analog domain before digitizing at a rate of 66.7 ksps. Measurement results of in-pixel serial SAR ADC show DNL and INL of ~0. 44 LSB and 0.58 LBS respectively. A large area array of 20,000 SPROCKET2 ADC pixels (multiplexed 1:16 to 320,000 sensor pixels) is currently under test. By reading out data over a 10 Gbps optical link, this pixel design enables a frame rate of $\sim$ 4 kfps for large sensing areas with minimal sensing deadtime. In the highest gain mode, the pixelated ADC has an input-referred resolution of 10$\mu$V with a simulated power consumption of 50$\mu$W. The pixel operates with constant current draw to minimize power-rail crosstalk.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2024

Fast Neutron Irradiation of a Multichannel JFET-Based Optical Encoder

Modern electrical components are susceptible to damage from high levels of radiation and extreme temperatures found near reactors in terrestrial nuclear power plants and in aerospace applications. Radiation-hardened electronics are being developed, largely for the aerospace industry, but they sometimes rely on application-specific, small-batch semiconductor fabrication processes. These processes tend to be prohibitively expensive to develop and maintain outside major industrial facilities or governmental agencies. Recently, commercially available, nonradiation-rated junction-gate field-effect transistors (JFETs) were shown to maintain their functionality at gamma doses exceeding 1 MGy, suggesting that nonrated, commercially available electrical components could be used to develop systems that are tolerant to ionizing radiation. However, gamma ray survival is not indicative of neutron dose survival, and few studies characterize JFETs under neutron irradiation. To address this knowledge gap, a JFET-based analog multiplexer and optical pulsewidth modulation (PWM) encoder was developed and irradiated using a 252 Cf source to 1.6×10 13 n/cm 2 . The multiplexed optical encoder (MOE) system maintained functionality throughout testing and showed little evidence of radiation effects. These results indicate that circuitry tolerant to fast neutron damage can be developed using low-cost, nonradiation-rated, commercially available JFETs, which could provide a lower production cost alternative to specialized semiconductor processes when designing and building electronics better able to survive neutron irradiation.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗
DOE OSTI2026

Constitutive and inducible oleoresin defenses share genetic architectures and mechanisms in Pinus taeda

The oleoresin defense system of loblolly pine (Pinus taeda) protects trees from insects and pathogens and is an important source of renewable biofuels and chemicals, but the genetic basis of oleoresin production is poorly understood. We characterized the genetic architecture of oleoresin flow, resin canal number, stem wood terpene content, and monoterpene composition in two clonal populations of P. taeda. We used quantitative genetic analyses, genome-wide association studies (GWASs), multiplex network learning, and gene expression profiling to elucidate shared gene networks underlying defense traits and to identify high-quality candidates for breeding and engineering loblolly pine. Genetic analyses revealed polygenic inheritance and trait-to-trait correlations provide strong evidence for shared genes regulating constitutive and induced oleoresin flow. We identified 236 single nucleotide polymorphisms associated with oleoresin flow, resin canal number, and terpene composition and highlight candidate genes likely involved in terpene biosynthesis, cambial meristem reprogramming, and pathogen perception and immune signaling. Fourteen GWAS candidates were methyl jasmonate-responsive in tissues where resin canals initiate and terpene production occurs. Integrating quantitative genetics, GWAS, gene expression, and multiplex network analyses enabled the prioritization of high-quality candidate genes. This work advances the development of more resilient loblolly pine optimized for ecological performance, renewable chemical, and biofuel production.

genome-wide association study↗
DOE OSTI2023

A Landing Pad System for Multicopy Gene Integration in Issatchenkia orientalis

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user’s choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

Fatma, Zia↗
DOE OSTI2022

Investigation of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions and performance of a rapid diagnostic test for identifying asymptomatic malaria infection in northern Ethiopia, 2015

Background: Rapid diagnostic tests (RDTs) are widely used for malaria diagnosis of both symptomatic and asymptomatic infections. Although RDTs are a reliable and practical diagnostic tool, the sensitivity of histidine-rich protein 2 (HRP2)-based RDTs can be reduced if pfhrp2 or pfhrp3 (pfhrp2/3) gene deletions exist in the Plasmodium falciparum parasite population. This study evaluated dried blood spot (DBS) samples collected from a national household survey to investigate the presence of pfhrp2/3 deletions and the performance of the RDT used in the cross-sectional survey in a low transmission setting. Methods: The 2015 Ethiopia Malaria Indicator Survey tested household members by RDT and collected DBS samples. DBS (n = 2648) from three regions in northern Ethiopia were tested by multiplex bead-based antigen detection assay after completion of the survey. The multiplex assay detected pan-Plasmodium lactate dehydrogenase (LDH), pAldolase, and HRP2 antigens in samples. Samples suspected for pfhrp2/3 gene deletions (pLDH and/or pAldolase positive but low or absent HRP2) were further investigated by molecular assays for gene deletions. Antigen results were also compared to each individual’s RDT results. Dose–response logistic regression models were fit to estimate RDT level of detection (LOD) antigen concentrations at which 50, 75, 90, and 95% of the RDTs returned a positive result during this survey. Results: Out of 2,648 samples assayed, 29 were positive for pLDH or pAldolase antigens but low or absent for HRP2 signal, and 15 of these samples (51.7%) were successfully genotyped for pfhrp2/3. Of these 15 P. falciparum infections, eight showed single deletions in pfhrp3, one showed a single pfhrp2 deletion, and six were pfhrp2/3 double-deletions. Six pfhrp2 deletions were observed in Tigray and one in Amhara. Twenty-five were positive for HRP2 by the survey RDT while the more sensitive bead assay detected 30 HRP2-positive samples. A lower concentration of HRP2 antigen generated a positive test result by RDT compared to pLDH (95% LOD: 16.9 ng/mL vs. 319.2 ng/mL, respectively). Conclusions: There is evidence of dual pfhrp2/3 gene deletions in the Tigray and Amhara regions of Ethiopia in 2015. As the prevalence of malaria was very low (< 2%), it is difficult to make strong conclusions on RDT performance, but these results challenge the utility of biomarkers in household surveys in very low transmission settings.

60 APPLIED LIFE SCIENCES↗