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At least 307 records · Page 17
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Structural Insights into the Dynamics of Water in SOD1 Catalysis and Drug Interactions

Superoxide dismutase 1 (SOD1) is a crucial enzyme that protects cells from oxidative damage by converting superoxide radicals into H 2 O 2 and O 2 . This detoxification process, essential for cellular homeostasis, relies on a precisely orchestrated catalytic mechanism involving the copper cation, while the zinc cation contributes to the structural integrity of the enzyme. This study presents the 2.3 Å crystal structure of human SOD1 (PDB ID: 9IYK), revealing an assembly of six homodimers and twelve distinct active sites. The water molecules form a complex hydrogen-bonding network that drives proton transfer and sustains active site dynamics. Our structure also uncovers subtle conformational changes that highlight the intrinsic flexibility of SOD1, which is essential for its function. Additionally, we observe how these dynamic structural features may be linked to pathological mutations associated with amyotrophic lateral sclerosis (ALS). By advancing our understanding of hSOD1’s mechanistic intricacies and the influence of water coordination, this study offers valuable insights for developing therapeutic strategies targeting ALS. Our structure’s unique conformations and active site interactions illuminate new facets of hSOD1 function, underscoring the critical role of structural dynamics in enzyme catalysis. Moreover, we conducted a molecular docking analysis using SOD1 for potential radical scavengers and Abelson non-receptor tyrosine kinase (c-Abl, Abl1) inhibitors targeting misfolded SOD1 aggregation along with oxidative stress and apoptosis, respectively. The results showed that CHEMBL1075867, a free radical scavenger derivative, showed the most promising docking results and interactions at the binding site of hSOD1, highlighting its promising role for further studies against SOD1-mediated ALS.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

MAL33 drives natural variation in maltose metabolism in Saccharomyces eubayanus

Maltose is one of the most abundant sugars in brewer’s wort, and its efficient utilization is critical for successful fermentation. However, maltose consumption varies naturally among Saccharomyces eubayanus strains isolated from different host trees, such as Quercus and Nothofagus. To identify the genetic determinants underlying these phenotypic differences, we performed bulk segregant analysis (BSA) and quantitative trait loci (QTL) mapping using an F 2 offspring derived from QC18 (Quercus-associated) and CL467.1 (Nothofagus-associated) strains. QTL mapping identified two significant genomic regions on subtelomeric loci of chromosomes V-R and XVI-L, each containing complete MAL loci composed of MAL32 (encoding maltase), MAL31 (transporter), and MAL33 (transcriptional activator) genes. Comparative polymorphism analyses identified mutations in MAL32 and MAL33 of QC18, including frameshift mutations resulting in premature stop codons. Functional validation demonstrated that the heterologous expression of MAL33 ChrV from CL467.1 fully restored maltose utilization in QC18, indicating the functional presence of MAL33 cis-regulatory sequences and MAL32 and MAL31 genes in QC18. While structural protein predictions identified truncation and impaired functionality in the maltose-responsive activation domain of Mal33p from QC18, overexpression of QC18’s own MAL33 ChrV allele also improved maltose metabolism, suggesting dosage-dependent transcriptional limitations rather than complete functional loss. These results indicate that allelic variations in the maltose-responsive activation domain of Mal33p result in differences in maltose consumption between strains. Here, we hypothesized that reduced maltose metabolism in QC18 is an adaptive response to the distinct sugar composition in Quercus robur bark, contrasting with the starch-rich environment of Nothofagus pumilio. These findings highlight subtelomeric MAL gene diversity as a reservoir of genetic variation, representing a key evolutionary mechanism that influences maltose adaptation among natural Saccharomyces isolates.

evolutionary plasticity↗
DOE OSTI2023

Mass Spectrometry–Based Proteogenomics: New Therapeutic Opportunities for Precision Medicine

Proteogenomics refers to the integration of comprehensive genomic, transcriptomic, and proteomic measurements from the same samples with the goal of fully understanding the regulatory processes converting genotypes to phenotypes, often with an emphasis on gaining a deeper understanding of disease processes. Although specific genetic mutations have long been known to drive the development of multiple cancers, gene mutations alone do not always predict prognosis or response to targeted therapy. The benefit of proteogenomics research is that information obtained from proteins and their corresponding pathways provides insight into therapeutic targets that can complement genomic information by providing an additional dimension regarding the underlying mechanisms and pathophysiology of tumors. This review describes the novel insights into tumor biology and drug resistance derived from proteogenomic analysis while highlighting the clinical potential of proteogenomic observations and advances in technique and analysis tools.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2004

A Study of Penalty Function Methods for Constraint Handling with Genetic Algorithm

COMETBOARDS (Comparative Evaluation Testbed of Optimization and Analysis Routines for Design of Structures) is a design optimization test bed that can evaluate the performance of several different optimization algorithms. A few of these optimization algorithms are the sequence of unconstrained minimization techniques (SUMT), sequential linear programming (SLP) and the sequential quadratic programming techniques (SQP). A genetic algorithm (GA) is a search technique that is based on the principles of natural selection or "survival of the fittest". Instead of using gradient information, the GA uses the objective function directly in the search. The GA searches the solution space by maintaining a population of potential solutions. Then, using evolving operations such as recombination, mutation and selection, the GA creates successive generations of solutions that will evolve and take on the positive characteristics of their parents and thus gradually approach optimal or near-optimal solutions. By using the objective function directly in the search, genetic algorithms can be effectively applied in non-convex, highly nonlinear, complex problems. The genetic algorithm is not guaranteed to find the global optimum, but it is less likely to get trapped at a local optimum than traditional gradient-based search methods when the objective function is not smooth and generally well behaved. The purpose of this research is to assist in the integration of genetic algorithm (GA) into COMETBOARDS. COMETBOARDS cast the design of structures as a constrained nonlinear optimization problem. One method used to solve constrained optimization problem with a GA to convert the constrained optimization problem into an unconstrained optimization problem by developing a penalty function that penalizes infeasible solutions. There have been several suggested penalty function in the literature each with there own strengths and weaknesses. A statistical analysis of some suggested penalty functions is performed in this study. Also, a response surface approach to robust design is used to develop a new penalty function approach. This new penalty function approach is then compared with the other existing penalty functions.

Ortiz, Francisco↗
DOE OSTI2022

Artificial intelligence based analysis of nanoindentation load–displacement data using a genetic algorithm

In this work, we developed an automated tool, Nanoindentation Neo package for the analysis of nanoindentation load–displacement curves using a Genetic Algorithm (GA) applied to the Oliver-Pharr method (Oliver et al.,1992). For some materials, such as polycrystalline isotropic graphites, Least Squares Fitting (LSF) of the unload curve can produce unrealistic fit parameters. These graphites exhibit sharply peaked unloading curves not easily fit using the LSF, which tends to overestimate the indenter tip geometry parameter. To tackle this problem, we extended our general materials characterization tool Neo for EXAFS analysis (Terry et al., 2021) to fit nanoindentation data. Nanoindentation Neo automatically processes and analyzes nanoindentation data with minimal user input while producing meaningful fit parameters. GA, a robust metaheuristic method, begins with a population of temporary solutions using model parameters called chromosomes; from these we evaluate a fitness value for each solution, and select the best solutions to mix with random solutions producing the next generation. A mutation operator then modifies existing solutions by random perturbations, and the optimal solution is selected. We tested the GA method using Silica and Al reference standards. We fit samples of graphite and a high entropy alloy (HEA) consisting of BCC and FCC phases.

42 ENGINEERING↗
NASA NTRS1992

Biodiversity: molecular biological domains, symbiosis and kingdom origins

The number of extant species of organisms is estimated to be from fewer than 3 to more than 30 x 10(6) (May, 1992). Molecular biology, comparative genetics and ultrastructural analyses provide new insights into evolutionary relationships between these species, including increasingly precise ideas of how species and higher taxa have evolved from common ancestors. Accumulation of random mutations and large macromolecular sequence change in all organisms since the Proterozoic Eon has been importantly supplemented by acquisition of inherited genomes ('symbiogenesis'). Karyotypic alterations (polyploidization and karyotypic fissioning) have been added to these other mechanisms of species origin in plants and animals during the Phanerozoic Eon. The new evolution concepts (coupled with current rapid rates of species extinction and ignorance of the extent of biodiversity) prompted this analysis of the field of systematic biology and its role in the reorganization of extant species into higher taxa. Two superkingdoms (= Domains: Prokaryotae and Eukaryotae) and five kingdoms (Monera = Procaryotae or Bacteria; Protoctista: algae, amoebae, ciliates, foraminifera, oomycetes, slime molds, etc.; Mychota: 'true' fungi; Plantae: one phylum (division) of bryophytes and nine phyla of tracheophytes; and Animalia) are recognized. Two subkingdoms comprise the monera: the great diverse lineages are Archaebacteria and Eubacteria. The criteria for classification using molecular, ultrastructural and genetic data for this scheme are mentioned. For the first time since the nineteenth century, logical, technical definitions for each group are given with their time of appearance as inferred from the fossil record in the primary scientific literature. This classification scheme, which most closely reflects the evolutionary history, molecular biology, genetics and ultrastructure of extant life, requires changes in social organization of biologists, many of whom as botanists and zoologists, still behave as if there were only two important kingdoms (plants and animals).

Non-NASA Center↗
DOE OSTI2021

Predicting Antimicrobial Resistance Using Partial Genome Alignments

Antimicrobial resistance (AMR) is an important global health threat that impacts millions of people worldwide each year. Developing methods that can detect and predict AMR phenotypes can help to mitigate the spread of AMR by informing clinical decision making and appropriate mitigation strategies. Many bioinformatic methods have been developed for predicting AMR phenotypes from whole-genome sequences and AMR genes, but recent studies have indicated that predictions can be made from incomplete genome sequence data. In order to more systematically understand this, we built random forest-based machine learning classifiers for predicting susceptible and resistant phenotypes for Klebsiella pneumoniae (1,640 strains), Mycobacterium tuberculosis (2,497 strains), and Salmonella enterica (1,981 strains). We started by building models from alignments that were based on a reference chromosome for each species. We then subsampled each chromosomal alignment and built models for the resulting subalignments, finding that very small regions, representing approximately 0.1 to 0.2% of the chromosome, are predictive. In K. pneumoniae, M. tuberculosis, and S. enterica, the subalignments are able to predict multiple AMR phenotypes with at least 70% accuracy, even though most do not encode an AMR-related function. We used these models to identify regions of the chromosome with high and low predictive signals. Finally, subalignments that retain high accuracy across larger phylogenetic distances were examined in greater detail, revealing genes and intergenic regions with potential links to AMR, virulence, transport, and survival under stress conditions. IMPORTANCE Antimicrobial resistance causes thousands of deaths annually worldwide. Understanding the regions of the genome that are involved in antimicrobial resistance is important for developing mitigation strategies and preventing transmission. Machine learning models are capable of predicting antimicrobial resistance phenotypes from bacterial genome sequence data by identifying resistance genes, mutations, and other correlated features. They are also capable of implicating regions of the genome that have not been previously characterized as being involved in resistance. In this study, we generated global chromosomal alignments for Klebsiella pneumoniae, Mycobacterium tuberculosis, and Salmonella enterica and systematically searched them for small conserved regions of the genome that enable the prediction of antimicrobial resistance phenotypes. In addition to known antimicrobial resistance genes, this analysis identified genes involved in virulence and transport functions, as well as many genes with no previous implication in antimicrobial resistance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Comparative Uptake Patterns of Radioactive Iodine and [18F]-Fluorodeoxyglucose (FDG) in Metastatic Differentiated Thyroid Cancers

Background: Metastatic differentiated thyroid cancer (DTC) represents a molecularly heterogeneous group of cancers with varying radioactive iodine (RAI) and [ 18 F]-fluorodeoxyglucose (FDG) uptake patterns potentially correlated with the degree of de-differentiation through the so-called “flip-flop” phenomenon. However, it is unknown if RAI and FDG uptake patterns correlate with molecular status or metastatic site. Materials and Methods: A retrospective analysis of metastatic DTC patients (n = 46) with radioactive 131-iodine whole body scan (WBS) and FDG-PET imaging between 2008 and 2022 was performed. The inclusion criteria included accessible FDG-PET and WBS studies within 1 year of each other. Studies were interpreted by two blinded radiologists for iodine or FDG uptake in extrathyroidal sites including lungs, lymph nodes, and bone. Cases were stratified by BRAF V600E mutation status, histology, and a combination of tumor genotype and histology. The data were analyzed by McNemar’s Chi-square test. Results: Lung metastasis FDG uptake was significantly more common than iodine uptake (WBS: 52%, FDG: 84%, p = 0.04), but no significant differences were found for lymph or bone metastases. Lung metastasis FDG uptake was significantly more prevalent in the papillary pattern sub-cohort (WBS: 37%, FDG: 89%, p = 0.02) than the follicular pattern sub-cohort (WBS: 75%, FDG: 75%, p = 1.00). Similarly, BRAF V600E+ tumors with lung metastases also demonstrated a preponderance of FDG uptake (WBS: 29%, FDG: 93%, p = 0.02) than BRAF V600E- tumors (WBS: 83%, FDG: 83%, p = 1.00) with lung metastases. Papillary histology featured higher FDG uptake in lung metastasis (WBS: 39%, FDG: 89%, p = 0.03) compared with follicular histology (WBS: 69%, FDG: 77%, p = 1.00). Patients with papillary pattern disease, BRAF V600E+ mutation, or papillary histology had reduced agreement between both modalities in uptake at all metastatic sites compared with those with follicular pattern disease, BRAF V600E- mutation, or follicular histology. Low agreement in lymph node uptake was observed in all patients irrespective of molecular status or histology. Conclusions: The pattern of FDG-PET and radioiodine uptake is dependent on molecular status and metastatic site, with those with papillary histology or BRAF V600E+ mutation featuring increased FDG uptake in distant metastasis. Further study with an expanded cohort may identify which patients may benefit from specific imaging modalities to recognize and surveil metastases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Ligand dependent interaction between PC-TP and PPARδ mitigates diet-induced hepatic steatosis in male mice

Phosphatidylcholine transfer protein (PC-TP; synonym StarD2) is a soluble lipid-binding protein that transports phosphatidylcholine (PC) between cellular membranes. To better understand the protective metabolic effects associated with hepatic PC-TP, we generated a hepatocyte-specific PC-TP knockdown (L-Pctp -/- ) in male mice, which gains less weight and accumulates less liver fat compared to wild-type mice when challenged with a high-fat diet. Hepatic deletion of PC-TP also reduced adipose tissue mass and decreases levels of triglycerides and phospholipids in skeletal muscle, liver and plasma. Gene expression analysis suggest that the observed metabolic changes are related to transcriptional activity of peroxisome proliferative activating receptor (PPAR) family members. An in-cell protein complementation screen between lipid transfer proteins and PPARs uncovered a direct interaction between PC-TP and PPARδ that was not observed for other PPARs. We confirmed the PC-TP– PPARδ interaction in Huh7 hepatocytes, where it was found to repress PPARδ-mediated transactivation. Mutations of PC-TP residues implicated in PC binding and transfer reduce the PC-TP-PPARδ interaction and relieve PC-TP-mediated PPARδ repression. Reduction of exogenously supplied methionine and choline reduces the interaction while serum starvation enhances the interaction in cultured hepatocytes. Together our data points to a ligand sensitive PC-TP– PPARδ interaction that suppresses PPAR activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

A Fluorescence‐Based Transient Expression Assay for the Analysis of Upstream Open Reading Frames in Plants

Upstream open reading frames (uORFs) are regulatory elements present in the 5′ leaders of mRNA that can significantly impact downstream gene expression in eukaryotes. In crop engineering, editing of uORFs can provide an avenue to upregulate expression of native genes without the need to add persistent transgenic copies. Even with genome-wide methods to identify translated uORFs such as ribosome profiling, their functional characterization depends on validation through reporter gene assays and mutagenesis studies. Current screening methods for plants use luciferases or protoplasts to measure differential gene expression between wild-type and mutated transcript leaders, which requires tissue processing and/or substrate addition. Here, we present a time- and cost-efficient alternative to investigate transcript leaders by co-expression of two fluorescent proteins in Nicotiana benthamiana leaf tissue and test our assay on genes involved in photoprotection, editing of which could provide a pathway to increase CO 2 assimilation during sun–shade transitions.

Nicotiana benthamiana↗
DOE OSTI2024

Structural and biochemical basis for regiospecificity of the flavonoid glycosyltransferase UGT95A1

Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases

Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH’s global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Analysis of the ARTIC Version 3 and Version 4 SARS-CoV-2 Primers and Their Impact on the Detection of the G142D Amino Acid Substitution in the Spike Protein

The ARTIC Network provides a common resource of PCR primer sequences and recommendations for amplifying SARS-CoV-2 genomes. The initial tiling strategy was developed with the reference genome Wuhan-01, and subsequent iterations have addressed areas of low amplification and sequence drop out. Recently, a new version (V4) was released, based on new variant genome sequences, in response to the realization that some V3 primers were located in regions with key mutations. Herein, we compare the performance of the ARTIC V3 and V4 primer sets with a matched set of 663 SARS-CoV-2 clinical samples sequenced with an Illumina NovaSeq 6000 instrument. We observe general improvements in sequencing depth and quality, and improved resolution of the SNP causing the D950N variation in the spike protein. Importantly, we also find nearly universal presence of spike protein substitution G142D in Delta-lineage samples. Due to the prior release and widespread use of the ARTIC V3 primers during the initial surge of the Delta variant, it is likely that the G142D amino acid substitution is substantially underrepresented among early Delta variant genomes deposited in public repositories. In addition to the improved performance of the ARTIC V4 primer set, this study also illustrates the importance of the primer scheme in downstream analyses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Tracking ebolavirus genomic drift with a resequencing microarray

Filoviruses are emerging pathogens that cause acute fever with high fatality rate and present a global public health threat. During the 2013–2016 Ebola virus outbreak, genome sequencing allowed the study of virus evolution, mutations affecting pathogenicity and infectivity, and tracing the viral spread. In 2018, early sequence identification of the Ebolavirus as EBOV in the Democratic Republic of the Congo supported the use of an Ebola virus vaccine. However, field-deployable sequencing methods are needed to enable a rapid public health response. Resequencing microarrays (RMA) are a targeted method to obtain genomic sequence on clinical specimens rapidly, and sensitively, overcoming the need for extensive bioinformatic analysis. This study presents the design and initial evaluation of an ebolavirus resequencing microarray (Ebolavirus-RMA) system for sequencing the major genomic regions of four Ebolaviruses that cause disease in humans. The design of the Ebolavirus-RMA system is described and evaluated by sequencing repository samples of three Ebolaviruses and two EBOV variants. The ability of the system to identify genetic drift in a replicating virus was achieved by sequencing the ebolavirus glycoprotein gene in a recombinant virus cultured under pressure from a neutralizing antibody. Comparison of the Ebolavirus-RMA results to the Genbank database sequence file with the accession number given for the source RNA and Ebolavirus-RMA results compared to Next Generation Sequence results of the same RNA samples showed up to 99% agreement.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Lasofoxifene as a potential treatment for therapy-resistant ER-positive metastatic breast cancer

Background: Endocrine therapy remains the mainstay of treatment for estrogen receptor-positive (ER+) breast cancer. Constitutively active mutations in the ligand binding domain of ERα render tumors resistant to endocrine agents. Breast cancers with the two most common ERα mutations, Y537S and D538G, have low sensitivity to fulvestrant inhibition, a typical second-line endocrine therapy. Lasofoxifene is a selective estrogen receptor modulator with benefits on bone health and breast cancer prevention potential. This study investigated the anti-tumor activity of lasofoxifene in breast cancer xenografts expressing Y537S and D538G ERα mutants. The combination of lasofoxifene with palbociclib, a CDK4/6 inhibitor, was also evaluated. Methods: Luciferase-GFP tagged MCF7 cells bearing wild-type, Y537S, or D538G ERα were injected into the mammary ducts of NSG mice (MIND model), which were subsequently treated with lasofoxifene or fulvestrant as single agents or in combination with palbociclib. Tumor growth and metastasis were monitored with in vivo and ex vivo luminescence imaging, terminal tumor weight measurements, and histological analysis. Results: As a monotherapy, lasofoxifene was more effective than fulvestrant at inhibiting primary tumor growth and reducing metastases. Adding palbociclib improved the effectiveness of both lasofoxifene and fulvestrant for tumor suppression and metastasis prevention at four distal sites (lung, liver, bone, and brain), with the combination of lasofoxifene/palbociclib being generally more potent than that of fulvestrant/palbociclib. X-ray crystallography of the ERα ligand binding domain (LBD) shows that lasofoxifene stabilizes an antagonist conformation of both wild-type and Y537S LBD. The ability of lasofoxifene to promote an antagonist conformation of Y537S, combined with its long half-life and bioavailability, likely contributes to the observed potent inhibition of primary tumor growth and metastasis of MCF7 Y537S cells. Conclusions: We report for the first time the anti-tumor activity of lasofoxifene in mouse models of endocrine therapy-resistant breast cancer. The results demonstrate the potential of using lasofoxifene as an effective therapy for women with advanced or metastatic ER+ breast cancers expressing the most common constitutively active ERα mutations.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Identification of ancestral gnathostome Gli3 enhancers with activity in mammals

Abnormal expression of the transcriptional regulator and hedgehog (Hh) signaling pathway effector Gli3 is known to trigger congenital disease, most frequently affecting the central nervous system (CNS) and the limbs. Accurate delineation of the genomic cis-regulatory landscape controlling Gli3 transcription during embryonic development is critical for the interpretation of noncoding variants associated with congenital defects. Here, we employed a comparative genomic analysis on fish species with a slow rate of molecular evolution to identify seven previously unknown conserved noncoding elements (CNEs) in Gli3 intronic intervals (CNE15–21). Transgenic assays in zebrafish revealed that most of these elements drive activities in Gli3 expressing tissues, predominantly the fins, CNS, and the heart. Intersection of these CNEs with human disease associated SNPs identified CNE15 as a putative mammalian craniofacial enhancer, with conserved activity in vertebrates and potentially affected by mutation associated with human craniofacial morphology. Finally, comparative functional dissection of an appendage-specific CNE conserved in slowly evolving fish (elephant shark), but not in teleost (CNE14/hs1586) indicates co-option of limb specificity from other tissues prior to the divergence of amniotes and lobe-finned fish. In conclusion, these results uncover a novel subset of intronic Gli3 enhancers that arose in the common ancestor of gnathostomes and whose sequence components were likely gradually modified in other species during the process of evolutionary diversification.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1999

The response of lazy-2 tomato seedlings to curvature-inducing magnetic gradients is modulated by light

Shoots of the lazy-2 mutant of tomato (Lycopersicon esculentum Mill., cv. Ailsa Craig) exhibit negative gravitropism in the dark, but respond positively gravitropically in (red) light. In order to test whether high-gradient magnetic fields (HGMFs) exert only ponderomotive effects on amyloplasts or affect other physiological processes, we induced magnetophoretic curvature in wild-type (WT) and lazy-2 mutant seedlings. Straight hypocotyls of 4-d-old plants were selected and the tips of their hooks were placed in an HGMF near the edge of a magnetized ferromagnetic wedge [grad (H2/2) approximately 10(9)-10(10) Oe2/cm] and mounted on a 1-rpm clinostat. After 4 h in the dark, 85% of WT hypocotyls and 67% of mutant hypocotyls curved toward the wedge. When the seedlings were exposed to red light for 1 h prior to and during the application of the HGMF, 78% of the WT seedlings curved toward the magnetic gradient, but the majority of the lazy-2 seedlings (75%) curved away from the stronger field area. Intracellular amyloplast displacement in the HGMF was similar for both varieties and resembled the displacement after horizontal reorientation. The WT showed a distinct graviresponse pattern depending on the orientation of the hook, even after excision of the apex. Application of HGMFs to decapitated hypocotyls resulted in curvature consistent with that obtained after horizontal reorientation. After light exposure, decapitated lazy-2 seedlings did not respond positively gravitropically. The data imply that the lazy-2 mutants perceive the displacement of amyloplasts in a similar manner to the WT and that the HGMF does not affect the graviresponse mechanism. The study demonstrates that ponderomotive forces due to HGMFs are useful for the analysis of the gravity-sensing mechanism in plants.

NASA Discipline Plant Biology↗
DOE OSTI2023

Structure of Rhizobium sp. 4-9 histamine dehydrogenase and analysis of the electron transfer pathway to an abiological electron acceptor

Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. Here, in this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe 4 S 4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators.

59 BASIC BIOLOGICAL SCIENCES↗