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Results for “Biological Assay”

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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At least 361 records · Page 20
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Structure of the divergent human astrovirus MLB capsid spike

Despite their worldwide prevalence and association with human disease, the molecular bases of human astrovirus (HAstV) infection and evolution remain poorly characterized. Here, we report the structure of the capsid protein spike of the divergent HAstV MLB clade (HAstV MLB). While the structure shares a similar folding topology with that of classical-clade HAstV spikes, it is otherwise strikingly different. We find no evidence of a conserved receptor-binding site between the MLB and classical HAstV spikes, suggesting that MLB and classical HAstVs utilize different receptors for host-cell attachment. We provide evidence for this hypothesis using a novel HAstV infection competition assay. Comparisons of the HAstV MLB spike structure with structures predicted from its sequence reveal poor matches, but template-based predictions were surprisingly accurate relative to machine-learning-based predictions. Our data provide a foundation for understanding the mechanisms of infection by diverse HAstVs and can support structure determination in similarly unstudied systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides

The use of non-standard toxicity models is a hurdle in the early development of antimicrobial peptides towards clinical applications. Herein we report an extensive in vitro and in vivo toxicity study of a library of 24 peptide-based antimicrobials with narrow spectrum activity towards veterinary pathogens. The haemolytic activity of the compounds was evaluated against four different species and the relative sensitivity against the compounds was highest for canine erythrocytes, intermediate for rat and human cells and lowest for bovine cells. Selected peptides were additionally evaluated against HeLa, HaCaT and HepG2 cells which showed increased stability towards the peptides. Therapeutic indexes of 50–500 suggest significant cellular selectivity in comparison to bacterial cells. Three peptides were administered to rats in intravenous acute dose toxicity studies up to 2– 8 × MIC. None of the injected compounds induced any systemic toxic effects in vivo at the concentrations employed illustrating that the correlation between the different assays is not obvious. This work sheds light on the in vitro and in vivo toxicity of this class of promising compounds and provides insights into the relationship between the different toxicity models often employed in different manners to evaluate the toxicity of novel bioactive compounds in general.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Chromosome-Level Genome Assembly of Mentha longifolia L. Reveals Gene Organization Underlying Disease Resistance and Essential Oil Traits

Mentha longifolia (L.) Huds., a wild, diploid mint species, has been developed as a model for mint genetic and genomic research to aid breeding efforts that target Verticillium wilt disease resistance and essential oil monoterpene composition. Here, we present a near-complete, chromosome-scale mint genome assembly for M. longifolia USDA accession CMEN 585. This new assembly is an update of a previously published genome draft, with dramatic improvements. A total of 42,107 protein-coding genes were annotated and placed on 12 chromosomal scaffolds. One hundred fifty-three genes contained conserved sequence domains consistent with nucleotide binding site-leucine-rich-repeat (NBS-LRR) plant disease resistance genes. Homologs of genes implicated in Verticillium wilt resistance in other plant species were also identified. Multiple paralogs of genes putatively involved in p-menthane monoterpenoid biosynthesis were identified and several cases of gene clustering documented. Heterologous expression of candidate genes, purification of recombinant target proteins, and subsequent enzyme assays allowed us to identify the genes underlying the pathway that leads to the most abundant monoterpenoid volatiles. Here, the bioinformatic and functional analyses presented here are laying the groundwork for using marker-assisted selection in improving disease resistance and essential oil traits in mints.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Ni( II )-binding affinity of Cc NikZ-II and its homologs: the role of the HH -prong and variable loop revealed by structural and mutational studies

Extracytoplasmic Ni(II)-binding proteins (NiBPs) are molecular shuttles involved in cellular nickel uptake. Here, we determined the crystal structure of apo CcNikZ-II at 2.38 Å, which revealed a Ni(II)-binding site comprised of the double His (HH-)prong (His511, His512) and a short variable (v-)loop nearby (Thr59-Thr64, TEDKYT). Mutagenesis of the site identified Glu60 and His511 as critical for high affinity Ni(II)-binding. Phylogenetic analysis showed 15 protein clusters with two groups containing the HH-prong. Metal-binding assays with 11 purified NiBPs containing this feature yielded higher Ni(II)-binding affinities. Replacement of the wild type v-loop with those from other NiBPs improved the affinity by up to an order of magnitude. This work provides molecular insights into the determinants for Ni(II) affinity and paves way for NiBP engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Rational design of photosynthetic reaction center protein maquettes

New technologies for efficient solar-to-fuel energy conversion will help facilitate a global shift from dependence on fossil fuels to renewable energy. Nature uses photosynthetic reaction centers to convert photon energy into a cascade of electron-transfer reactions that eventually produce chemical fuel. The design of new reaction centers de novo deepens our understanding of photosynthetic charge separation and may one day allow production of biofuels with higher thermodynamic efficiency than natural photosystems. Recently, we described the multi-step electron-transfer activity of a designed reaction center maquette protein (the RC maquette), which can assemble metal ions, tyrosine, a Zn tetrapyrrole, and heme into an electron-transport chain. Here, we detail our modular strategy for rational protein design and show that the intended RC maquette design agrees with crystal structures in various states of assembly. A flexible, dynamic apo-state collapses by design into a more ordered holo-state upon cofactor binding. Crystal structures illustrate the structural transitions upon binding of different cofactors. Spectroscopic assays demonstrate that the RC maquette binds various electron donors, pigments, and electron acceptors with high affinity. We close with a critique of the present RC maquette design and use electron-tunneling theory to envision a path toward a designed RC with a substantially higher thermodynamic efficiency than natural photosystems.

09 BIOMASS FUELS↗
DOE OSTI2020

Lateral flow assay for detection of Yesinia pestis

The goal for this project is to create an accurate, deployable, and selective diagnostic tool for Yersinia pestis. Yersinia pestis is the causative agent of bubonic plague. One aspect of the pathogenic nature of Yersinia pestis comes from the F1 antigen. A major concern with Yersinia pestis is the ability to develop antibiotic resistance and become a deadly biothreat. A diagnostic tool for Yersinia pestis is imperative for national safety.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Chronic Cardiotoxicity Assays Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs)

Cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs) are increasingly used in cardiac safety assessment, disease modeling and regenerative medicine. A vast majority of cardiotoxicity studies in the past have tested acute effects of compounds and drugs; however, these studies lack information on the morphological or physiological responses that may occur after prolonged exposure to a cardiotoxic compound. In this review, we focus on recent advances in chronic cardiotoxicity assays using hiPSC-CMs. We summarize recently published literature on hiPSC-CMs assays applied to chronic cardiotoxicity induced by anticancer agents, as well as non-cancer classes of drugs, including antibiotics, anti-hepatitis C virus (HCV) and antidiabetic drugs. We then review publications on the implementation of hiPSC-CMs-based assays to investigate the effects of non-pharmaceutical cardiotoxicants, such as environmental chemicals or chronic alcohol consumption. We also highlight studies demonstrating the chronic effects of smoking and implementation of hiPSC-CMs to perform genomic screens and metabolomics-based biomarker assay development. The acceptance and wide implementation of hiPSC-CMs-based assays for chronic cardiotoxicity assessment will require multi-site standardization of assay protocols, chronic cardiac maturity marker reproducibility, time points optimization, minimal cellular variation (commercial vs. lab reprogrammed), stringent and matched controls and close clinical setting resemblance. A comprehensive investigation of long-term repeated exposure-induced effects on both the structure and function of cardiomyocytes can provide mechanistic insights and recapitulate drug and environmental cardiotoxicity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

PagMYB128 regulates secondary cell wall formation by direct activation of cell wall biosynthetic genes during wood formation in poplar

The biosynthesis of cellulose, lignin, and hemicelluloses in plant secondary cell walls (SCWs) is regulated by a hierarchical transcriptional regulatory network. Here, this network features orthologous transcription factors shared between poplar and Arabidopsis, highlighting a foundational similarity in their genetic regulation. However, knowledge on the discrepant behavior of the transcriptional-level molecular regulatory mechanisms between poplar and Arabidopsis remains limited. In this study, we investigated the function of PagMYB128 during wood formation and found it had broader impacts on SCW formation compared to its Arabidopsis ortholog, AtMYB103. Transgenic poplar trees overexpressing PagMYB128 exhibited significantly enhanced xylem development, with fiber cells and vessels displaying thicker walls, and an increase in the levels of cellulose, lignin, and hemicelluloses in the wood. In contrast, plants with dominant repression of PagMYB128 demonstrated the opposite phenotypes. RNA sequencing and reverse transcription – quantitative polymerase chain reaction showed that PagMYB128 could activate SCW biosynthetic gene expression, and chromatin immunoprecipitation along with yeast one-hybrid, and effector–reporter assays showed this regulation was direct. Further analysis revealed that PagSND1 (SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN1) directly regulates PagMYB128 but not cell wall metabolic genes, highlighting the pivotal role of PagMYB128 in the SND1-driven regulatory network for wood development, thereby creating a feedforward loop in SCW biosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Linking the Salmonella enterica 1,2-Propanediol Utilization Bacterial Microcompartment Shell to the Enzymatic Core via the Shell Protein PduB

Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here, we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is critical for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy, we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and system-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis.

1, 2-propanediol utilization↗
DOE OSTI2025

An elastin-like polymer targeting vascular endothelial growth factor receptor-1 reduces survival in serum-starved endothelial cells

Peptides often exhibit biological activity that depends on the context in which they are displayed and delivered. Understanding and controlling these contextual effects on peptide function is critical for designing targeted and responsive peptide-based biomaterials and therapeutics. Genetically engineered protein polymers such as elastin-like polypeptides (ELPs) can incorporate bioactive peptide motifs and are attractive candidates for biomaterials used in tissue engineering and targeted drug delivery. They also present an opportunity for investigating and modulating cell signaling pathways by presenting a peptide ligand in various defined chemical and physical environments. Vascular endothelial growth factor receptor-1 (VEGFR1) signaling plays important and complex roles in cell survival and angiogenesis, but polymeric materials that interact with this signaling axis are scarce. In this study, a novel genetically engineered elastin-like polymer that targets VEGFR1 is characterized. This polymer, termed R1B-ELP, binds to human endothelial cells in a manner dependent on its VEGFR1-targeting motif and, based on cell proliferation and cytotoxicity assays, demonstrates activity consistent with disrupting pro-survival signaling necessary for endothelial cell function under conditions of environmental stress. Notably, these findings indicate that ELP fusion alters the functional behavior of the targeting peptide. Modulators of VEGFR1 signaling have potential applications in basic studies of angiogenesis as well as in therapeutic applications targeting vascular or inflammatory diseases.

36 MATERIALS SCIENCE↗
DOE OSTI2023

An extensible vector toolkit and parts library for advanced engineering of plant genomes

Abstract Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T‐DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site‐specific recombinases. Finally, we use a series of dual‐luciferase assays to measure contributions to expression from promoters, terminators, and from cross‐cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

42 ENGINEERING↗
DOE OSTI2021

Epistatic Net allows the sparse spectral regularization of deep neural networks for inferring fitness functions

Abstract Despite recent advances in high-throughput combinatorial mutagenesis assays, the number of labeled sequences available to predict molecular functions has remained small for the vastness of the sequence space combined with the ruggedness of many fitness functions. While deep neural networks (DNNs) can capture high-order epistatic interactions among the mutational sites, they tend to overfit to the small number of labeled sequences available for training. Here, we developed Epistatic Net (EN), a method for spectral regularization of DNNs that exploits evidence that epistatic interactions in many fitness functions are sparse. We built a scalable extension of EN, usable for larger sequences, which enables spectral regularization using fast sparse recovery algorithms informed by coding theory. Results on several biological landscapes show that EN consistently improves the prediction accuracy of DNNs and enables them to outperform competing models which assume other priors. EN estimates the higher-order epistatic interactions of DNNs trained on massive sequence spaces-a computational problem that otherwise takes years to solve.

Aghazadeh, Amirali (ORCID:0000000302230873)↗
DOE OSTI2024

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg 2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg 2+ -dependent 3'–5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

High-throughput screening of environmental polysaccharide-degrading bacteria using biomass containment and complex insoluble substrates

Abstract Carbohydrate degradation by microbes plays an important role in global nutrient cycling, human nutrition, and biotechnological applications. Studies that focus on the degradation of complex recalcitrant polysaccharides are challenging because of the insolubility of these substrates as found in their natural contexts. Specifically, current methods to examine carbohydrate-based biomass degradation using bacterial strains or purified enzymes are not compatible with high-throughput screening using complex insoluble materials. In this report, we developed a small 3D printed filter device that fits inside a microplate well that allows for the free movement of bacterial cells, media, and enzymes while containing insoluble biomass. These devices do not interfere with standard microplate readers and can be used for both short- (24–48 h) and long-duration (> 100 h) experiments using complex insoluble substrates. These devices were used to quantitatively screen in a high-throughput manner environmental isolates for their ability to grow using lignocellulose or rice grains as a sole nutrient source. Additionally, we determined that the microplate-based containment devices are compatible with existing enzymatic assays to measure activity against insoluble biomass. Overall, these microplate containment devices provide a platform to study the degradation of complex insoluble materials in a high-throughput manner and have the potential to help uncover ecologically important aspects of bacterial metabolism as well as to accelerate biotechnological innovation.

3D printing↗
DOE OSTI2021

Modeling temporal and hormonal regulation of plant transcriptional response to wounding

Plants respond to wounding stress by changing gene expression patterns and inducing the production of hormones including jasmonic acid. This wounding transcriptional response activates specialized metabolism pathways such as the glucosinolate pathways in Arabidopsis thaliana. While the regulatory factors and sequences controlling a subset of wound-response genes are known, it remains unclear how wound response is regulated globally. Here, we how these responses are regulated by incorporating putative cis-regulatory elements, known transcription factor binding sites, in vitro DNA affinity purification sequencing, and DNase I hypersensitive sites to predict genes with different wound-response patterns using machine learning. We observed that regulatory sites and regions of open chromatin differed between genes upregulated at early and late wounding time-points as well as between genes induced by jasmonic acid and those not induced. Expanding on what we currently know, we identified cis-elements that improved model predictions of expression clusters over known binding sites. Using a combination of genome editing, in vitro DNA-binding assays, and transient expression assays using native and mutated cis-regulatory elements, we experimentally validated four of the predicted elements, three of which were not previously known to function in wound-response regulation. Our study provides a global model predictive of wound response and identifies new regulatory sequences important for wounding without requiring prior knowledge of the transcriptional regulators.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Development of a CNS-permeable reactivator for nerve agent exposure: an iterative, multi-disciplinary approach

Nerve agents have experienced a resurgence in recent times with their use against civilian targets during the attacks in Syria (2012), the poisoning of Sergei and Yulia Skripal in the United Kingdom (2018) and Alexei Navalny in Russia (2020), strongly renewing the importance of antidote development against these lethal substances. The current standard treatment against their effects relies on the use of small molecule-based oximes that can efficiently restore acetylcholinesterase (AChE) activity. Despite their efficacy in reactivating AChE, the action of drugs like 2-pralidoxime (2-PAM) is primarily limited to the peripheral nervous system (PNS) and, thus, provides no significant protection to the central nervous system (CNS). This lack of action in the CNS stems from their ionic nature that, on one end makes them very powerful reactivators and on the other renders them ineffective at crossing the Blood Brain Barrier (BBB) to reach the CNS. In this report, we describe the use of an iterative approach composed of parallel chemical and in silico syntheses, computational modeling, and a battery of detailed in vitro and in vivo assays that resulted in the identification of a promising, novel CNS-permeable oxime reactivator. Additional experiments to determine acute and chronic toxicity are ongoing.

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

The fluoride permeation pathway and anion recognition in Fluc family fluoride channels

Fluc family fluoride channels protect microbes against ambient environmental fluoride by undermining the cytoplasmic accumulation of this toxic halide. These proteins are structurally idiosyncratic, and thus the permeation pathway and mechanism have no analogy in other known ion channels. Although fluoride-binding sites were identified in previous structural studies, it was not evident how these ions access aqueous solution, and the molecular determinants of anion recognition and selectivity have not been elucidated. Using x-ray crystallography, planar bilayer electrophysiology, and liposome-based assays, we identified additional binding sites along the permeation pathway. We used this information to develop an oriented system for planar lipid bilayer electrophysiology and observed anion block at one of these sites, revealing insights into the mechanism of anion recognition. We propose a permeation mechanism involving alternating occupancy of anion-binding sites that are fully assembled only as the substrate approaches.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Rigid monoclonal antibodies improve detection of SARS-CoV-2 nucleocapsid protein

Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small-angle X-ray scattering and transmission electron microscopy. Our solution-based results distinguished the mAbs’ flexibility and how this flexibility affects the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the antigen-binding fragments is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich enzyme-linked immunosorbent assay, we show that rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices, which are critical for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.

60 APPLIED LIFE SCIENCES↗