Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “assays”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 577 records · Page 32
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Peripheral positions encode transport specificity in the small multidrug resistance exporters

In secondary active transporters, a relatively limited set of protein folds have evolved diverse solute transport functions. Because of the conformational changes inherent to transport, altering substrate specificity typically involves remodeling the entire structural landscape, limiting our understanding of how novel substrate specificities evolve. In the current work, we examine a structurally minimalist family of model transport proteins, the small multidrug resistance (SMR) transporters, to understand the molecular basis for the emergence of a novel substrate specificity. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to the activity of a clade of multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this engineered activity. Using X-ray crystallography, solid-supported membrane electrophysiology, binding assays, and a proteoliposome-based quaternary ammonium antiseptic transport assay that we developed, we dissect the mechanistic contributions of these residues to substrate polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate but through mutations peripheral to the binding pocket. Our work provides molecular insight into substrate promiscuity among the SMRs and can be applied to understand multidrug export and the evolution of novel transport functions more generally.

Science & Technology - Other Topics↗
DOE OSTI2023

A compact dication source for Ba 2+ tagging and heavy metal ion sensor development

Here, we present a tunable metal ion beam that delivers controllable ion currents in the picoamp range for testing of dry-phase ion sensors. Ion beams are formed by sequential atomic evaporation and single or multiple electron impact ionization, followed by acceleration into a sensing region. Controllability of the ionic charge state is achieved through tuning of electrode potentials that influence the retention time in the ionization region. Barium, lead, and cadmium samples have been used to test the system, with ion currents identified and quantified using a quadrupole mass analyzer. Realization of a clean Ba 2+ ion beam within a bench-top system represents an important technical advance toward the development and characterization of barium tagging systems for neutrinoless double beta decay searches in xenon gas. This system also provides a testbed for investigation of novel ion sensing methodologies for environmental assay applications, with dication beams of Pb 2+ and Cd 2+ also demonstrated for this purpose.

47 OTHER INSTRUMENTATION↗
DOE OSTI2023

Metagenomic Methods for Addressing NASA's Planetary Protection Policy Requirements on Future Missions: A Workshop Report

Molecular biology methods and technologies have advanced substantially over the past decade. These new molecular methods should be incorporated among the standard tools of planetary protection (PP) and could be validated for incorporation by 2026. To address the feasibility of applying modern molecular techniques to such an application, NASA conducted a technology workshop with private industry partners, academics, and government agency stakeholders, along with NASA staff and contractors. The technical discussions and presentations of the Multi-Mission Metagenomics Technology Development Workshop focused on modernizing and supplementing the current PP assays. The goals of the workshop were to assess the state of metagenomics and other advanced molecular techniques in the context of providing a validated framework to supplement the bacterial endospore-based NASA Standard Assay and to identify knowledge and technology gaps. In particular, workshop participants were tasked with discussing metagenomics as a stand-alone technology to provide rapid and comprehensive analysis of total nucleic acids and viable microorganisms on spacecraft surfaces, thereby allowing for the development of tailored and cost-effective microbial reduction plans for each hardware item on a spacecraft. Workshop participants recommended metagenomics approaches as the only data source that can adequately feed into quantitative microbial risk assessment models for evaluating the risk of forward (exploring extraterrestrial planet) and back (Earth harmful biological) contamination. Participants were unanimous that a metagenomics workflow, in tandem with rapid targeted quantitative (digital) PCR, represents a revolutionary advance over existing methods for the assessment of microbial bioburden on spacecraft surfaces. The workshop highlighted low biomass sampling, reagent contamination, and inconsistent bioinformatics data analysis as key areas for technology development. Finally, it was concluded that implementing metagenomics as an additional workflow for addressing concerns of NASA's robotic mission will represent a dramatic improvement in technology advancement for PP and will benefit future missions where mission success is affected by backward and forward contamination.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Computational and Systems Biology Advances to Enable Bioagent Agnostic Signatures

Enumerated threat agent lists have long driven biodefense priorities. The global SARS-CoV-2 pandemic demonstrated the limitations of searching for known threat agents as compared to a more agnostic approach. Recent technological advances are enabling agent-agnostic biodefense, especially through the integration of multi-modal observations of host-pathogen interactions directed by a human immunological model. Although well-developed technical assays exist for many aspects of human-pathogen interaction, the analytic methods and pipelines to combine and holistically interpret the results of such assays are immature and require further investments to exploit new technologies. In this manuscript, we discuss potential immunologically based bioagent-agnostic approaches and the computational tool gaps the community should prioritize filling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Increased SUMOylation of TCF21 improves its stability and function in human endometriotic stromal cells

Abstract Endometriosis is an estrogen-dependent disease. Our previous study demonstrated that elevated levels of transcription factor 21 (TCF21) in endometriotic tissues enhanced steroidogenic factor-1 (SF-1) and estrogen receptor β (ERβ) expression by forming a heterodimer with upstream stimulatory factor 2 (USF2), allowing these TCF21/USF2 complexes to bind to the promoters of SF-1 and ERβ. Furthermore, TCF21 contributed to the increased proliferation of endometriotic stromal cells (ESCs), suggesting that TCF21 may play a vital role in the pathogenesis of endometriosis. SUMOylation is a posttranslational modification that has emerged as a crucial molecular regulatory mechanism. However, the mechanism regulating TCF21 SUMOylation in endometriosis is incompletely characterized. Thus, this study aimed to explore the effect of TCF21 SUMOylation on its expression and regulation in ovarian endometriosis. We found that the levels of SUMOylated TCF21 were increased in endometriotic tissues and stromal cells compared with eutopic endometrial tissues and stromal cells and enhanced by estrogen. Treatment with the SUMOylation inhibitor ginkgolic acid (GA) and the results of a protein half-life assay demonstrated that SUMOylation can stabilize the TCF21 protein. A coimmunoprecipitation (Co-IP) assay showed that SUMOylation probably increased its interaction with USF2. Further analyses elucidated that SUMOylation of TCF21 significantly increased the binding activity of USF2 to the SF-1 and ERβ promoters. Moreover, the SUMOylation motifs in TCF21 affected the proliferation ability of ESCs. The results of this study suggest that SUMOylation plays a critical role in mediating the high expression of TCF21 in ESCs and may participate in the development of endometriosis.

Zhu, Jingwen↗
DOE OSTI2021

A novel polypeptide vaccine and adjuvant formulation of EV71

ABSTRACT Hand foot and mouth disease (HFMD) is an infectious disease mainly caused by Enterovirus 71 (EV 71). However, the effective treatment is limited currently. The aim of this study was to investigate the activity of the vaccine including the EV71 polypeptides mixed with a novel adjuvant containing CpG oligodeoxynucleotides (CpG ODNs). After collecting mouse sera, we determined the antibody concentration in serum by enzyme-linked immunosorbent assays (ELISA). Then, CD19+CD27+ B cells in the spleen were analysed by flow cytometry. The assay revealed that a substantial increase in antibody titers was achieved. This indicates a high level of immunogenicity for peptide vaccine and the good stability of adjuvant, also suggests that the combination of vaccine and adjuvant can stimulate the production of high-level antibodies and CD19+CD27+ B lymphocytes in mice. Furthermore, the antibody could effectively identify EV71 inactivated virus. The results demonstrated that the autonomous construction of EV71 polypeptide vaccine had a good immunogenicity. Moreover, the peptide vaccine injection with a novel adjuvant, which is easy to prepare, could cause a high antibody level of EV71 and shown a good application prospect.

Liu, Zhiang↗
DOE OSTI2020

Occurrence of NDM-1-producing Morganella morganii and Proteus mirabilis in a single patient in Portugal: probable in vivo transfer by conjugation

Abstract Objectives To decipher the genetics of acquisition of carbapenemase-encoding genes identified in two carbapenem-resistant Enterobacteriaceae recovered from a single patient in Portugal. Methods Carbapenemase genes were searched by PCR assays and mating-out assays were performed to further characterize the plasmid support of the carbapenemase genes. Genetic characterization of the plasmid supports was performed by whole-plasmid sequencing using the Illumina technology. Results We identified here two NDM-1-producing isolates, namely a Morganella morganii and a Proteus mirabilis, sharing the same blaNDM-1-positive plasmid. This 154 kb plasmid belonged to the IncA/C2 type, recently renamed IncC, and co-harboured two AmpC β-lactamase genes, namely blaCMY-4 and blaDHA-1, in addition to the 16S rRNA methylase gene armA encoding high-level resistance to aminoglycosides. In addition, the M. morganii isolate produced the CTX-M-33 extended-spectrum β-lactamase possessing weak carbapenemase activity, encoded by another plasmid. Conclusions We showed here that, in addition to KPC-type and OXA-181 carbapenemases, which have been identified as widespread in this country, another concern is the emergence of NDM-1-producing enterobacterial isolates in Portugal. We demonstrated here the in vivo plasmid transfer of a blaNDM-1-positive plasmid leading to dissemination of this carbapenemase gene within different enterobacterial species in a single patient.

Aires-de-Sousa, Marta↗
DOE OSTI2022

Optimization of sporulation and purification methods for sporicidal efficacy assessment on Bacillus spores

Abstract Validating the efficacy of sporicidal agents is a critical step in current good manufacturing practices for disinfection requirements. A limitation is that the poor quality of spores can lead to false positive sporicidal results. The aim of this study was to explore optimal sporulation and purification methods in Bacillus spores. Spores of 7 Bacillus strains were produced in 5 different sporulation media. After density centrifugation, spore yields were measured by phase-contrast microscopy and enumeration assays. Effects of purification methods including heat, sonication and lysozyme, and maturation on spore qualities were determined by sodium hypochlorite sporicidal assay. Difco sporulation media was identified as the preferred sporulation medium for 4 out of 7 tested Bacillus strains. Sporulation rates in B. cereus, B. sphaericus, and B. thuringiensis were higher at 30°C than the rates at 37°C at a difference of 5%, 65%, and 20%, respectively. Bacillus licheniformis favored Mn2+-amended 10% Columbia Broth at 37°C for sporulation with 40–72% higher sporulation rates than other media. The maximum sporulation rates of B. cereus and B. thuringiensis were observed on double-strength Schaeffer's-glucose broth. All studied purification methods improved the spore purity with strain variations. However, intense heat (80°C for 20 min) and lysozyme (100 μg/mL) treatment impaired the spore quality of specific Bacillus strains by sensitizing them against sodium hypochlorite. The length of the maturation period had an impact on the spore resistance, and the most optimal maturation periods ranged from 7 to 21 days in Bacillus strains. The results of this study will pave the way for further evaluation of the sporicidal activity of disinfectants.

Biotechnology & Applied Microbiology↗
DOE OSTI2020

Pectin methylesterase selectively softens the onion epidermal wall yet reduces acid-induced creep

De-esterification of homogalacturonan (HG) is thought to stiffen pectin gels and primary cell walls by increasing calcium cross-linking between HG chains. Contrary to this idea, recent studies found that HG de-esterification correlated with reduced stiffness of living tissues, measured by surface indentation. The physical basis of such apparent wall softening is unclear, but possibly involves complex biological responses to HG modification. To assess the direct physical consequences of HG de-esterification on wall mechanics without such complications, we treated isolated onion (Allium cepa) epidermal walls with pectin methylesterase (PME) and assessed wall biomechanics with indentation and tensile tests. In nanoindentation assays, PME action softened the wall (reduced the indentation modulus). In tensile force/extension assays, PME increased plasticity, but not elasticity. These softening effects are attributed, at least in part, to increased electrostatic repulsion and swelling of the wall after PME treatment. Despite softening and swelling upon HG de-esterification, PME treatment alone failed to induce cell wall creep. Instead, acid-induced creep, mediated by endogenous α-expansin, was reduced. We conclude that HG de-esterification physically softens the onion wall, yet reduces expansin-mediated wall extensibility.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Functional characterization of a cellulose synthase, CtCESA1, from the marine red alga Calliarthron tuberculosum (Corallinales)

In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. Furthermore, the relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evaluation of overtime phenotypic variation of yeasts in chronic vulvovaginal candidosis cases

Abstract Chronic vulvovaginal candidosis results either from reinfection or from the ability of Candida spp. to persist in the vulva and/or vagina. Persistence is usually associated with increased antifungal (mainly azoles) resistance rates, which can explain treatment failure, and/or increased expression of virulence factors by Candida spp. The aim of this study was to assess the mechanisms leading to Candida spp persistence, by studying sequential isolates from women with chronic vulvovaginal candidosis, focusing on strains genotypes, azole resistance, and ability to form biofilms along the period of clinical evaluation. The strains were identified at species level by automated analysis of biochemical profiles and molecular typing evaluated by polymorphic DNA analysis. The capacity to form biofilm was assessed with a microtiter plate assay. Fluconazole susceptibility was determined by the microdilution broth assay at both pH 7 (following the recommended guideline) and pH 4.5 (as representative of vaginal pH). We studied samples from 17 clinically recurrent cases. In 53% of the chronic cases there were two or more isolates that had a phylogenetic relationship while the remaining (47%) were caused by different species. In those cases where related strains were involved in recurrence, we verified an increase in MIC at pH 7 and also an increased capacity to form biofilms over time. Significant correlation between these two parameters was observed only in cases caused by C. glabrata, evidencing the importance of these two factors to enhance persistence in the vaginal mucosa for this particular species.

Faria-Gonçalves, Paula↗
DOE OSTI2024

Structure of the E. coli nucleoid-associated protein YejK reveals a novel DNA binding clamp

Abstract Nucleoid-associated proteins (NAPs) play central roles in bacterial chromosome organization and DNA processes. The Escherichia coli YejK protein is a highly abundant, yet poorly understood NAP. YejK proteins are conserved among Gram-negative bacteria but show no homology to any previously characterized DNA-binding protein. Hence, how YejK binds DNA is unknown. To gain insight into YejK structure and its DNA binding mechanism we performed biochemical and structural analyses on the E. coli YejK protein. Biochemical assays demonstrate that, unlike many NAPs, YejK does not show a preference for AT-rich DNA and binds non-sequence specifically. A crystal structure revealed YejK adopts a novel fold comprised of two domains. Strikingly, each of the domains harbors an extended arm that mediates dimerization, creating an asymmetric clamp with a 30 Å diameter pore. The lining of the pore is electropositive and mutagenesis combined with fluorescence polarization assays support DNA binding within the pore. Finally, our biochemical analyses on truncated YejK proteins suggest a mechanism for YejK clamp loading. Thus, these data reveal YejK contains a newly described DNA-binding motif that functions as a novel clamp.

Biochemistry & Molecular Biology↗
DOE OSTI2025

Directed evolution expands CRISPR–Cas12a genome-editing capacity

CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture, and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a with expanded PAM recognition. The resulting Cas12a variants use a range of noncanonical PAMs while retaining recognition of the canonical 5'-TTTV-3' PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5'-NYHV-3' PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.

Ma, Enbo↗
DOE OSTI2021

Knocksideways in plants: an inducible system for in planta visualization of protein interactions

Understanding protein-protein interactions (PPIs) is key to unlocking the secrets of various cellular processes. While many tools have been developed to detect PPIs, such as the yeast two-hybrid assay, bimolecular fluorescence complementation, and affinity purification, there is great demand for additional methods to complement existing ones. As all approaches have limitations, evidence from at least two independent assays is required to validate PPIs. In a Breakthrough Report, Joanna Winkler and colleagues present here a promising method, named “knocksideways in plants” (KSP), for inducible visualization of PPIs in planta.

59 BASIC BIOLOGICAL SCIENCES↗
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

GTP binding by Arabidopsis extra-large G protein 2 is not essential for its functions

The extra-large guanosine-5'-triphosphate (GTP)-binding protein 2, XLG2, is an unconventional Gα subunit of the Arabidopsis (Arabidopsis thaliana) heterotrimeric GTP-binding protein complex with a major role in plant defense. In vitro biochemical analyses and molecular dynamic simulations show that affinity of XLG2 for GTP is two orders of magnitude lower than that of the conventional Gα, AtGPA1. Here we tested the physiological relevance of GTP binding by XLG2. We generated an XLG2(T476N) variant with abolished GTP binding, as confirmed by in vitro GTPγS binding assay. Yeast three-hybrid, bimolecular fluorescence complementation, and split firefly-luciferase complementation assays revealed that the nucleotide-depleted XLG2(T476N) retained wild-type XLG2-like interactions with the Gβγ dimer and defense-related receptor-like kinases. Both wild-type and nucleotide-depleted XLG2(T476N) restored the defense responses against Fusarium oxysporum and Pseudomonas syringae compromised in the xlg2 xlg3 double mutant. Additionally, XLG2(T476N) was fully functional restoring stomatal density, root growth, and sensitivity to NaCl, but failed to complement impaired germination and vernalization-induced flowering. We conclude that XLG2 is able to function in a GTP-independent manner and discuss its possible mechanisms of action.

Plant Sciences↗
DOE OSTI2023

SPOTTED-LEAF7 targets the gene encoding β-galactosidase9, which functions in rice growth and stress responses

Abstract β-Galactosidases (Bgals) remove terminal β-D-galactosyl residues from the nonreducing ends of β-D-galactosidases and oligosaccharides. Bgals are present in bacteria, fungi, animals, and plants and have various functions. Despite the many studies on the evolution of BGALs in plants, their functions remain obscure. Here, we identified rice (Oryza sativa) β-galactosidase9 (OsBGAL9) as a direct target of the heat stress-induced transcription factor SPOTTED-LEAF7 (OsSPL7), as demonstrated by protoplast transactivation analysis and yeast 1-hybrid and electrophoretic mobility shift assays. Knockout plants for OsBGAL9 (Osbgal9) showed short stature and growth retardation. Histochemical β-glucuronidase (GUS) analysis of transgenic lines harboring an OsBGAL9pro:GUS reporter construct revealed that OsBGAL9 is mainly expressed in internodes at the mature stage. OsBGAL9 expression was barely detectable in seedlings under normal conditions but increased in response to biotic and abiotic stresses. Ectopic expression of OsBGAL9 enhanced resistance to the rice pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae, as well as tolerance to cold and heat stress, while Osbgal9 mutant plants showed the opposite phenotypes. OsBGAL9 localized to the cell wall, suggesting that OsBGAL9 and its plant putative orthologs likely evolved functions distinct from those of its closely related animal enzymes. Enzyme activity assays and analysis of the cell wall composition of OsBGAL9 overexpression and mutant plants indicated that OsBGAL9 has activity toward galactose residues of arabinogalactan proteins (AGPs). Our study clearly demonstrates a role for a member of the BGAL family in AGP processing during plant development and stress responses.

54 ENVIRONMENTAL SCIENCES↗