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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.

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MISIP: a data standard for the reuse and reproducibility of any stable isotope probing-derived nucleic acid sequence and experiment

DNA/RNA-stable isotope probing (SIP) is a powerful tool to link in situ microbial activity to sequencing data. Every SIP dataset captures distinct information about microbial community metabolism, process rates, and population dynamics, offering valuable insights for a wide range of research questions. Data reuse maximizes the information derived from the labor and resource-intensive SIP approaches. Yet, a review of publicly available SIP sequencing metadata showed that critical information necessary for reproducibility and reuse was often missing. Here, we outline the Minimum Information for any Stable Isotope Probing Sequence (MISIP) according to the Minimum Information for any (x) Sequence (MIxS) framework and include examples of MISIP reporting for common SIP experiments. Our objectives are to expand the capacity of MIxS to accommodate SIP-specific metadata and guide SIP users in metadata collection when planning and reporting an experiment. The MISIP standard requires 5 metadata fields—isotope, isotopolog, isotopolog label, labeling approach, and gradient position—and recommends several fields that represent best practices in acquiring and reporting SIP sequencing data (e.g., gradient density and nucleic acid amount). The standard is intended to be used in concert with other MIxS checklists to comprehensively describe the origin of sequence data, such as for marker genes (MISIP-MIMARKS) or metagenomes (MISIP-MIMS), in combination with metadata required by an environmental extension (e.g., soil). The adoption of the proposed data standard will improve the reuse of any sequence derived from a SIP experiment and, by extension, deepen understanding of in situ biogeochemical processes and microbial ecology.

Simpson, Abigayle

Application of FISH based G2-PCC assay for the cytogenetic assessment of high radiation dose exposures: Potential implications for rapid triage biodosimetry

The main goal of this study is to test the utility of calyculin A induced G2-PCC assay as a biodosimetry triage tool for assessing a wide range of low and acute high radiation dose exposures of photons. Towards this initiative, chromosome aberrations induced by low and high doses of x-rays were evaluated and characterized in G2-prematurely condensed chromosomes (G2-PCCs) by fluorescence in situ hybridization (FISH) using human centromere and telomere specific PNA (peptide nucleic acid) probes. A dose dependent increase in the frequency of dicentric chromosomes was observed in the G2-PCCs up to 20 Gy of x-rays. The combined yields of dicentrics and rings in the G2-PCCs showed a clear dose dependency up to 20 Gy from 0.02/cell for 0.1 Gy to 14.98/cell for 20 Gy. Centric rings were observed more frequently than acentric ring chromosomes in the G2-PCCs at all the radiation doses from 1 Gy to 20 Gy. A head-to-head comparison was also performed by FISH on the yields of chromosome aberrations induced by different doses of x-rays (0 Gy -7.5 Gy) in colcemid arrested metaphase chromosomes and calyculin A induced G2-PCCs. In general, the frequencies of dicentrics, rings and acentric fragments were slightly higher in G2-PCCs than in colcemid arrested metaphase chromosomes at all the radiation doses, but the differences were not statistically significant. To reduce the turnaround time for absorbed radiation dose estimation, attempt was made to obtain G2-PCCs by reducing the culture time to 36 hrs. The absorbed doses estimated in x-rays irradiated (0,1,2 and 4 Gy) G2-PCCs after 36 hrs of culture were grossly like that of G2-PCCs and colcemid arrested metaphase chromosomes prepared after 48 hrs of culture. Our study indicates that the shortened version of calyculin A induced G2-PCC assay coupled with the FISH staining technique can serve as an effective triage biodosimetry tool for large-scale radiological/nuclear incidents.

Science & Technology - Other Topics

Fluorescent, phosphorescent, magnetic resonance contrast and radioactive tracer labelling of extracellular vesicles

This review focusses on the significance of fluorescent, phosphorescent labelling and tracking of extracellular vesicles (EVs) for unravelling their biology, pathophysiology, and potential diagnostic and therapeutic uses. Various labeling strategies, such as lipid membrane, surface protein, luminal, nucleic acid, radionuclide, quantum dot labels, and metal complex-based stains, are evaluated for visualizing and characterizing EVs. Direct labelling with fluorescent lipophilic dyes is simple but generally lacks specificity, while surface protein labelling offers selectivity but may affect EV-cell interactions. Luminal and nucleic acid labelling strategies have their own advantages and challenges. Here, each labelling approach has strengths and weaknesses, which require a suitable probe and technique based on research goals, but new tetranuclear polypyridylruthenium(II) complexes as phosphorescent probes have strong phosphorescence, selective staining, and stability. Future research should prioritize the design of novel fluorescent probes and labelling platforms that can significantly enhance the efficiency, accuracy, and specificity of EV labeling, while preserving their composition and functionality. It is crucial to reduce false positive signals and explore the potential of multimodal imaging techniques to gain comprehensive insights into EVs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH

Tre-DST: A Drug Susceptibility Test for Mycobacterium tuberculosis Using Solvatochromic Trehalose Probes

In 2024, an estimated 10 million people developed Tuberculosis (TB), nearly half a million of whom were infected with drug-resistant tuberculosis (DR-TB). Early detection of infection and drug resistance enables rapid engagement in effective care. Bacterial culture and nucleic acid testing remain the primary diagnostic methods, with smear microscopy being phased out. However, these methods present significant limitations for diagnosing drug resistance, such as lengthy time-to-result for phenotypic tests, as well as the need for prior knowledge of resistance mutations and prohibitive cost for molecular tests. To address this, we developed a rapid phenotypic TB drug susceptibility test, termed Tre-DST, based on novel metabolically incorporated trehalose probes, which specifically detect live mycobacteria. We used the nonpathogenic Mycobacterium smegmatis and the virulence-attenuated Mycobacterium tuberculosis (Mtb) H37Ra or auxotrophic Mtb to demonstrate a strong correlation between cost-effective plate reader results and flow cytometry data, suggesting that the plate reader is a suitable fluorescence detector for Tre-DST. We determined that adding a 1-week incubation step allowed Mtb samples originally seeded at 10 4 CFU/mL to become detectable, over 2 weeks earlier than colony-forming unit analysis. We found that Tre-DST reports on drug susceptibility in a drug-agnostic manner, demonstrating loss of fluorescence with frontline TB drugs as well as the newer drug bedaquiline. Tre-DST distinguished RIF- and INH-resistant auxotrophs from susceptible controls and accurately reported the resistance activity. Ultimately, because Tre-DST is agnostic to mechanisms of drug resistance, this assay is likely compatible with all WHO-recommended and future DR-TB drugs as a diagnostic in reference laboratories.

diagnostics

Neutrons in Structural Biology: Challenges and Opportunities (Workshop Report)

Gaining a thorough understanding of biological systems requires building our knowledge about biological processes from the level of atoms and electrons, and up to whole organisms. Such comprehensive knowledge will allow for a predictive understanding of complex biological systems behavior. It will guide us in the design and development of novel therapeutics and vaccines to tackle existing health threats and to prepare for future pandemics, and it will provide information necessary to create new biomaterials and bio-inspired technologies through manipulation of biological macromolecules, their assemblies, single cells and even microorganisms. Reaching these goals will require a synergistic combination of multiple experimental techniques with molecular calculations and predictive simulations, and the design and development of new techniques and capabilities that bridge current knowledge and technology gaps. Neutron scattering provides unique information about the biomacromolecular structure and function and can play a major role in achieving these goals. A workshop was held to engage the scientific community in identifying pressing challenges in biochemistry, structural biology, enzymology and structure-guided drug design not solved with the current neutron scattering technologies or utilizing other structural biology techniques such as X-ray crystallography, NMR, and cryo-EM. The workshop brought together structural biology, biochemistry and computational experts, as well as early career researchers and students, creating a forum for discussing scientific advancement and collaboration. The workshop included a one-day satellite training workshop where graduate students and postdoctoral researchers were educated in the application of neutron crystallography and small-angle scattering in structural biology. Furthermore, the Instrument Scientific Advisory Board (ISAB) for the development of a macromolecular neutron diffractometer at ORNL’s Second Target Station was introduced at the workshop. The major outcome was that neutrons can provide atomic-level understanding of biomacromolecular structure, function and dynamics which is of paramount importance for addressing the identified challenges. Neutron crystallography, in particular, can resolve long-standing biochemical issues regarding enzyme function by delineating the underlying chemistry and can have a major impact on the design of small-molecule therapeutics, especially in combination with molecular computation (quantum chemistry and molecular dynamics simulations) and the emerging artificial intelligence (AI)-assisted drug design technologies. The unique properties of neutrons, including their high sensitivity to hydrogen and their non-destructive nature, make them ideal probes of biological matter. There is a palpable need in the scientific community to expand and enhance the impact of neutron sciences on biology. Neutron crystallography is the only structural biology method capable of determining positions of all hydrogen atoms in proteins, nucleic acids and their complexes at near-physiological temperatures and of unstable species at cryogenic temperatures. Moreover, neutron analysis is non-ionizing, non-destructive and does not perturb the structure or redox chemistry of active site metal centers and clusters in proteins, which can be invaluable for studying radiation-sensitive metalloprotein complexes. Further, neutron energies used in scattering applications are similar to atomic motions, permitting neutron spectroscopies to characterize the dynamics of biomacromolecules on the picosecond to microsecond timescales. The different sensitivities of neutrons to protium (H) and deuterium (D) isotopes of hydrogen allow enhanced visibility of specific parts of biological complexes through isotopic labeling. The impact of neutrons will be most powerful when neutron scattering is combined with complementary experimental techniques that use photons and electrons, and with high-performance computing. The interconnection and mutuality of the experimental and theoretical capabilities will drive discoveries in biological and health sciences to generate more complete picture of complex biological systems. The major limitation in the field of biological neutron crystallography has been signal-to-noise, demanding large samples that are difficult to produce for the majority of biomacromolecules and limiting the applicability of this technique in biological sciences. A neutron crystallography instrument at the Second Target Station will revolutionize biological science with neutrons by engaging a large scientific community of structural biologists, enabling successful neutron diffraction experiments from radically smaller biomacromolecular crystals, resolving unanswered biochemical questions, and meaningfully contributing to rational drug design. The meeting highlighted 10 grand challenges that will be addressed with this advanced capability over the next decade and beyond, and the recommendations required to help address them are given below.

59 BASIC BIOLOGICAL SCIENCES

Precise Linker Length and Dynamic Bond Exchange Control Penetrant Diffusion in Dense Vitrimers

Polymer networks with dynamic covalent bonds have been investigated for their self-healing ability, recyclability, and potential as more sustainable materials. Recent results have indicated that in some cases, bond exchange can enhance the transport of penetrants in dense networks, pointing to their potential for separations of membranes. Here, imine dynamic bonds in ethylene oxide (EO) networks with precise linker lengths were synthesized to investigate the transport of N,N′-bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenedicarboximide (BTBP), a large, anisotropic dye molecule. Networks with mesh sizes smaller than, comparable to, and greater than the size of the penetrant axes were investigated to probe the effects of bond exchange and network confinement on transport. Mesh sizes, which ranged from 0.5 to 1.62 nm, were determined from shear rheology, glass transitions by calorimetry, and probe diffusion coefficients by fluorescence recovery after photobleaching. Permanent networks with identical EO chain lengths were prepared as control samples, and up to a 3 orders of magnitude increase in diffusion coefficient is observed in the dynamic systems for short linkers containing 13 backbone atoms. The longest linkers with 71 backbone atoms show no difference between the permanent and dynamic networks. Linkers shorter than 11 backbone atoms, corresponding to a mesh size smaller than the penetrant small axis, diffusion is no longer observable on the experimental time scale, indicating a sharp cutoff attributed to the precise linkers and narrow mesh size distribution. The dynamic imine exchange time scales were compared to the diffusive hopping times of penetrants and indicate that exchange can occur during a diffusive displacement. Furthermore, these findings provide insights into the factors affecting penetrant transport in dense polymers and inspire the development of next-generation selective polymer membranes.

Diffusion