Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “Pathogen”

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 1,351 records · Page 75
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Lessons Learnt From the COVID-19 Pandemic

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has been characterized by unprecedented rates of spatio-temporal spread. Here, we summarize the main events in the pandemic's timeline and evaluate what has been learnt by the public health community. We also discuss the implications for future public health policy and, specifically, the practice of epidemic control. We critically analyze this ongoing pandemic's timeline and contrast it with the 2002–2003 SARS outbreak. We identify specific areas (e.g., pathogen identification and initial reporting) wherein the international community learnt valuable lessons from the SARS outbreak. However, we also identify the key areas where international public health policy failed leading to the exponential spread of the pandemic. We outline a clear agenda for improved pandemic control in the future.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Recalls of tattoo and permanent makeup inks in the United States and a follow-up microbiological survey of inks with a previous recall history

In this study, we collected voluntary recall records of tattoo and permanent makeup ink from the U.S. Food and Drug Administration (US FDA) Enforcement Report Database. The recall records contain information, such as recall date, manufacturer, ink color, reason for recall, and the microorganisms detected from the ink samples. Between 2003 and 2021, a total of 15 voluntary tattoo ink recalls occurred in the U.S. market, involving over 200 tattoo inks marketed by 13 manufacturers and one distributor. Fourteen recalls were due to microbial contamination, and one recall was due to allergic reaction. As follow-up, a microbiological survey of 28 tattoo inks of new batches from seven manufacturers having products that were previously recalled was conducted. Aerobic plate count (APC) and enrichment culture methods based on the FDA’s Bacteriological Analytical Manual (BAM) were used to detect microbial contamination. The results revealed that six out of 28 tattoo inks were contaminated with bacteria and were produced by two manufacturers. The level of microbial contamination was less than 250 CFU/g in three of the tattoo inks and between 1 × 10 3 and 1 × 10 5 CFU/g in the other three inks. Eleven bacterial isolates were identified, including spore-forming Bacillus -related species and potentially pathogenic species. Overall, this study shows that some tattoo ink products produced by manufacturers with a recall history continue to be contaminated with microorganisms. This highlights the need for ongoing monitoring and quality control of such products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Amplification-free nucleic acid detection with a fluorescence-based waveguide biosensor

Early detection of pathogens using nucleic acids in clinical samples often requires sensitivity at the single-copy level, which currently necessitates time-consuming and expensive nucleic acid amplification. Here, we describe 1) a redesigned flow cell in the shape of a trapezoid-subtracted geometric stadium, and 2) modified experimental procedures that allow for the measurement of sub-attomolar analytes in microliter quantities on a fluorescence-based waveguide biosensor. We verified our instrumental sensitivity with a 200-μL sample of a fluorescent streptavidin conjugate at 100 zM (100 zeptomolar, or 100·10 −21 mol L −1 ) and theoretically explored the applicability of this modified sensing platform in a sandwich immunoassay format using a Langmuir adsorption model. We present assays that demonstrate specific detection of synthetic influenza A DNA (in buffer) and RNA (in saliva) oligonucleotides at the single-copy level (200 μL at 10 zM) using a fluorescent molecular beacon. Lastly, we demonstrate detection of isolated genomic influenza A RNA at a clinically relevant concentration. This work constitutes a sensitivity improvement of over twelve orders of magnitude compared to our previous nucleic acid detection work, illustrating the significant enhancements that can be gained with optimized experimental design.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Sustainable production of extracellular polymeric substances and iron or copper complex from glutinous rice processing wastewater

Essential trace minerals play vital roles in maintaining human and animal health. However, an overdose of the existing inorganic trace minerals is prone to induce detrimental effects that outweigh positive benefits. In this study, an extracellular polymeric substances (EPS)-producing bacterium, identified as Bacillus licheniformis CCTCC M2020298, was isolated from marine using glutinous rice processing wastewater as enrichment medium. The EPS yield of Bacillus licheniformis CCTCC M2020298 could reach 8.62 g/L by using glutinous rice-processing wastewater containing medium. Furthermore, the potential of the EPS as a carrier for synthesizing EPS-iron (Fe) and EPS-copper (Cu) complex was explored. The results showed that the optimum condition for the synthesis EPS-Fe were the reaction temperature 70°C, pH 8.5–9.0 and mass ratio of EPS to trisodium citrate 2:1. The iron content of EPS-Fe reached 77.4 mg/g. Under the same condition, the copper content of EPS-Cu reached 90.7 mg/g. The elemental composition, functional groups and valence state of the mineral elements of EPS-Fe and EPS-Cu were well characterized. The EPS-Fe and EPS-Cu exhibited antioxidant activity in scavenging ·OH, DPPH and ·O 2− free radicals, thereby leading to reduced oxidative stress and apoptosis levels in human colonic epithelial cells in vitro . They also inhibited the proliferation of mouse hepatocellular carcinoma H22 and the growth of intestinal pathogens in vitro . This study provided an effective avenue for EPS production from glutinous rice processing wastewater and proved the potential of EPS-Fe and EPS-Cu complexes as a new-type comprehensive essential trace mineral supplement.

Chen, Zhen↗
DOE OSTI2020

Epidemiological Investigation of a Mortality Event in a Translocated Gopher Tortoise (Gopherus polyphemus) Population in Northwest Florida

Nokuse Plantation, a 22,055 ha private conservation preserve in northwest Florida, is a recipient site for gopher tortoises translocated from development sites in Florida. Since 2006, Nokuse has received over 5,000 tortoises from multiple development sites. During 2013–2015, 52 tortoises were found sick (n = 14) or dead (n = 38) in multiple soft-release enclosures in which tortoises consistently exhibited clinical signs, with additional sick (n = 5) and dead (n = 5) tortoises presenting similarly during 2016–2017. When found alive, tortoises behaved abnormally (e.g., frequently out of burrows during cold weather, pacing along enclosure fencing), appeared emaciated, were lethargic, and had developed redness under plastron scutes. Similar numbers of male (n = 28) and female (n = 32) tortoises were recovered along with two of unidentified sex, including mainly adults (n = 59) and three subadults. Physical examination, blood analysis, and other diagnostics were indicative of starvation and dehydration. Most sick tortoises provided with supportive care recovered. Necropsy findings generally confirmed starvation, with no evidence of infectious pathogens or contaminants. There were no apparent differences in quality of habitat, plant community, or soil or water among affected and unaffected enclosures. Botanical surveys indicated adequate forage quality and quantity, with no poisonous exotic or native plants detected. No land management practices changed prior to this event. Analysis of epidemiological data and demographic factors from before and during this mortality event identified initial density of tortoises in the enclosures as exerting the strongest influence on detection of tortoise morbidity and mortality. We believe that the stress associated with mixing tortoises from different populations and at higher densities during translocation impacted an individual tortoise’s ability to obtain or absorb adequate nutrients from foraging, ultimately leading to a wasting condition consistent with starvation. Based on our findings, we recommend a maximum of 3 gopher tortoises per ha in soft-release enclosures for translocation, but further research is warranted to investigate the complexity of stress and social pressures associated with translocation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Duration of Contagion of Foot-And-Mouth Disease Virus in Infected Live Pigs and Carcasses

Data-driven modeling of incursions of high-consequence, transboundary pathogens of animals is a critical component of veterinary preparedness. However, simplifying assumptions and excessive use of proxy measures to compensate for gaps in available data may compromise modeled outcomes. The current investigation was prospectively designed to address two major gaps in current knowledge of foot-and-mouth disease virus (FMDV) pathogenesis in pigs: the end (duration) of the infectious period and the viability of FMDV in decaying carcasses. By serial exposure of sentinel groups of pigs to the same group of donor pigs infected by FMDV A24 Cruzeiro, it was demonstrated that infected pigs transmitted disease at 10 days post infection (dpi), but not at 15 dpi. Assuming a latent period of 1 day, this would result in a conservative estimate of an infectious duration of 9 days, which is considerably longer than suggested by a previous report from an experiment performed in cattle. Airborne contagion was diminished within two days of removal of infected pigs from isolation rooms. FMDV in muscle was inactivated within 7 days in carcasses stored at 4°C. By contrast, FMDV infectivity in vesicle epithelium harvested from intact carcasses stored under similar conditions remained remarkably high until the study termination at 11 weeks post mortem. The output from this study consists of experimentally determined data on contagion associated with FMDV-infected pigs. This information may be utilized to update parameterization of models used for foot-and-mouth disease outbreak simulations involving areas of substantial pig production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Leptospira borgpetersenii serovar Hardjo and Leptospira santarosai serogroup Pyrogenes isolated from bovine dairy herds in Puerto Rico

Leptospirosis is one of the most common zoonotic diseases in the world and endemic in the Caribbean Islands. Bovine leptospirosis is an important reproductive disease. Globally, cattle are recognized as a reservoir host for L. borgpetersenii serovar Hardjo, which is transmitted via urine, semen, and uterine discharges, and can result in abortion and poor reproductive performance. The dairy industry in Puerto Rico comprises up to 25% of agriculture-related income and is historically the most financially important agricultural commodity on the island. In this study, we report the isolation of two different pathogenic Leptospira species, from two different serogroups, from urine samples collected from dairy cows in Puerto Rico: L. borgpetersenii serogroup Sejroe serovar Hardjo and L. santarosai serogroup Pyrogenes. Recovered isolates were classified using whole-genome sequencing, serotyping with reference antisera and monoclonal antibodies, and immunoblotting. These results demonstrate that dairy herds in Puerto Rico can be concurrently infected with more than one species and serovar of Leptospira, and that bacterin vaccines and serologic diagnostics should account for this when applying intervention and diagnostic strategies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Assessing complexity and dynamics in epidemics: geographical barriers and facilitators of foot-and-mouth disease dissemination

Introduction: Physical and non-physical processes that occur in nature may influence biological processes, such as dissemination of infectious diseases. However, such processes may be hard to detect when they are complex systems. Because complexity is a dynamic and non-linear interaction among numerous elements and structural levels in which specific effects are not necessarily linked to any one specific element, cause-effect connections are rarely or poorly observed. Methods: To test this hypothesis, the complex and dynamic properties of geo-biological data were explored with high-resolution epidemiological data collected in the 2001 Uruguayan foot-and-mouth disease (FMD) epizootic that mainly affected cattle. County-level data on cases, farm density, road density, river density, and the ratio of road (or river) length/county perimeter were analyzed with an open-ended procedure that identified geographical clustering in the first 11 epidemic weeks. Two questions were asked: (i) do geo-referenced epidemiologic data display complex properties? and (ii) can such properties facilitate or prevent disease dissemination? Results: Emergent patterns were detected when complex data structures were analyzed, which were not observed when variables were assessed individually. Complex properties–including data circularity–were demonstrated. The emergent patterns helped identify 11 counties as ‘disseminators’ or ‘facilitators’ (F) and 264 counties as ‘barriers’ (B) of epidemic spread. In the early epidemic phase, F and B counties differed in terms of road density and FMD case density. Focusing on non-biological, geographical data, a second analysis indicated that complex relationships may identify B-like counties even before epidemics occur. Discussion: Geographical barriers and/or promoters of disease dispersal may precede the introduction of emerging pathogens. If corroborated, the analysis of geo-referenced complexity may support anticipatory epidemiological policies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Overexpression of the Mas1 gene mitigated LPS-induced inflammatory injury in mammary epithelial cells by inhibiting the NF-κB/MAPKs signaling pathways

Breast infection is the primary etiology of mastitis in dairy cows, leading to a reduction in the quality of dairy products and resulting in substantial economic losses for animal husbandry. Although antibiotic treatment can eliminate the pathogenic microorganisms that induce mastitis, it cannot repair the inflammatory damage of mammary epithelial cells and blood milk barrier. Mas1 is a G protein-coupled receptor, and its role in lipopolysaccharide (LPS) -induced inflammatory injury to mammary epithelial cells has not been studied. LPS treatment of EpH4 EV cells led to a significant downregulation of Mas1 transcript levels, which attracted our great interest, suggesting that Mas1 may be an important target for the treatment of mastitis. Therefore, this study intends to verify the role of Mas1 in the inflammatory injury of EpH4 EV cells by gene overexpression technology and gene silencing technology. The findings demonstrated that the overexpression of the Mas1 gene effectively reversed the activation of the nuclear factor-κB/mitogen-activated protein kinase (NF-κB/MAPK) signaling pathways induced by LPS, while also suppressing the upregulation of pro-inflammatory mediators. Furthermore, overexpression of the Mas1 gene reversed the downregulation of zonula occludens 1 (ZO-1), Occludin, and Claudin-3 caused by LPS, suggesting that Mas1 could promote to repair the blood-milk barrier. However, the silencing of the Mas1 gene using siRNA resulted in a contrasting effect. These results indicated that Mas1 alleviated the inflammatory injury of mammary epithelial cells induced by LPS.

Yan, Shuping↗
DOE OSTI2023

classLog: Logistic regression for the classification of genetic sequences

Introduction Sequencing and phylogenetic classification have become a common task in human and animal diagnostic laboratories. It is routine to sequence pathogens to identify genetic variations of diagnostic significance and to use these data in realtime genomic contact tracing and surveillance. Under this paradigm, unprecedented volumes of data are generated that require rapid analysis to provide meaningful inference. Methods We present a machine learning logistic regression pipeline that can assign classifications to genetic sequence data. The pipeline implements an intuitive and customizable approach to developing a trained prediction model that runs in linear time complexity, generating accurate output rapidly, even with incomplete data. Our approach was benchmarked against porcine respiratory and reproductive syndrome virus (PRRSv) and swine H1 influenza A virus (IAV) datasets. Trained classifiers were tested against sequences and simulated datasets that artificially degraded sequence quality at 0, 10, 20, 30, and 40%. Results When applied to a poor-quality sequence data, the classifier achieved between >85% to 95% accuracy for the PRRSv and the swine H1 IAV HA dataset and this increased to near perfect accuracy when using the full dataset. The model also identifies amino acid positions used to determine genetic clade identity through a feature selection ranking within the model. These positions can be mapped onto a maximum-likelihood phylogenetic tree, allowing for the inference of clade defining mutations. Discussion Our approach is implemented as a python package with code available at https://github.com/flu-crew/classLog .

Zeller, Michael A.↗
DOE OSTI2021

How to Evaluate Non-Growing Cells—Current Strategies for Determining Antimicrobial Resistance of VBNC Bacteria

Thanks to the achievements in sanitation, hygiene practices, and antibiotics, we have considerably improved in our ongoing battle against pathogenic bacteria. However, with our increasing knowledge about the complex bacterial lifestyles and cycles and their plethora of defense mechanisms, it is clear that the fight is far from over. One of these resistance mechanisms that has received increasing attention is the ability to enter a dormancy state termed viable but non-culturable (VBNC). Bacteria that enter the VBNC state, either through unfavorable environmental conditions or through potentially lethal stress, lose their ability to grow on standard enrichment media, but show a drastically increased tolerance against antimicrobials including antibiotics. The inability to utilize traditional culture-based methods represents a considerable experimental hurdle to investigate their increased antimicrobial resistance and impedes the development and evaluation of effective treatments or interventions against bacteria in the VBNC state. Although experimental approaches were developed to detect and quantify VBNCs, only a few have been utilized for antimicrobial resistance screening and this review aims to provide an overview of possible methodological approaches.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Sulfonamidoboronic Acids as “Cross-Class” Inhibitors of an Expanded-Spectrum Class C Cephalosporinase, ADC-33, and a Class D Carbapenemase, OXA-24/40: Strategic Compound Design to Combat Resistance in Acinetobacter baumannii

Acinetobacter baumannii is a Gram-negative organism listed as an urgent threat pathogen by the World Health Organization (WHO). Carbapenem-resistant A. baumannii (CRAB), especially, present therapeutic challenges due to complex mechanisms of resistance to β-lactams. One of the most important mechanisms is the production of β-lactamase enzymes capable of hydrolyzing β-lactam antibiotics. Co-expression of multiple classes of β-lactamases is present in CRAB; therefore, the design and synthesis of “cross-class” inhibitors is an important strategy to preserve the efficacy of currently available antibiotics. To identify new, nonclassical β-lactamase inhibitors, we previously identified a sulfonamidomethaneboronic acid CR167 active against Acinetobacter-derived class C β-lactamases (ADC-7). The compound demonstrated affinity for ADC-7 with a K i = 160 nM and proved to be able to decrease MIC values of ceftazidime and cefotaxime in different bacterial strains. Herein, we describe the activity of CR167 against other β-lactamases in A. baumannii: the cefepime-hydrolysing class C extended-spectrum β-lactamase (ESAC) ADC-33 and the carbapenem-hydrolyzing OXA-24/40 (class D). These investigations demonstrate CR167 as a valuable cross-class (C and D) inhibitor, and the paper describes our attempts to further improve its activity. Five chiral analogues of CR167 were rationally designed and synthesized. The structures of OXA-24/40 and ADC-33 in complex with CR167 and select chiral analogues were obtained. The structure activity relationships (SARs) are highlighted, offering insights into the main determinants for cross-class C/D inhibitors and impetus for novel drug design.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

The Missing Pieces: The Role of Secretion Systems in Campylobacter jejuni Virulence

Campylobacter jejuni is likely the most common bacterial cause of gastroenteritis worldwide, responsible for millions of cases of inflammatory diarrhea characterized by severe abdominal cramps and blood in the stool. Further, C. jejuni infections are associated with post-infection sequelae in developed countries and malnutrition and growth-stunting in low- and middle-income countries. Despite the increasing prevalence of the disease, campylobacteriosis, and the recognition that this pathogen is a serious health threat, our understanding of C. jejuni pathogenesis remains incomplete. In this review, we focus on the Campylobacter secretion systems proposed to contribute to host-cell interactions and survival in the host. Moreover, we have applied a genomics approach to defining the structural and mechanistic features of C. jejuni type III, IV, and VI secretion systems. Special attention is focused on the flagellar type III secretion system and the prediction of putative effectors, given that the proteins exported via this system are essential for host cell invasion and the inflammatory response. We conclude that C. jejuni does not possess a type IV secretion system and relies on the type III and type VI secretion systems to establish a niche and potentiate disease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Piperitone ( p -Menth-1-En-3-One): A New Repellent for Tea Shot Hole Borer (Coleoptera: Curculionidae) in Florida Avocado Groves

The tea shot hole borer, Euwallacea perbrevis, has been recently established in Florida, USA, where it vectors fungal pathogens that cause Fusarium dieback in avocado. Pest monitoring uses a two-component lure containing quercivorol and α-copaene. Incorporation of a repellent into IPM programs may reduce the incidence of dieback in avocado groves, particularly if combined with lures in a push–pull system. This study evaluated piperitone and α-farnesene as potential repellents for E. perbrevis, comparing their efficacy to that of verbenone. Replicate 12-week field tests were conducted in commercial avocado groves. Each test compared beetle captures in traps baited with two-component lures versus captures in traps containing lures plus repellent. To complement field trials, Super-Q collections followed by GC analyses were performed to quantify emissions from repellent dispensers field-aged for 12 weeks. Electroantennography (EAG) was also used to measure beetle olfactory response to each repellent. Results indicated that α-farnesene was ineffective; however, piperitone and verbenone were comparable in repellency, achieving 50–70% reduction in captures, with longevity of 10–12 weeks. EAG responses to piperitone and verbenone were equivalent, and significantly greater than response to α-farnesene. Since piperitone is less expensive than verbenone, this study identifies a potential new E. perbrevis repellent.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Early Immune Modulation in European Seabass (Dicentrarchus labrax) Juveniles in Response to Betanodavirus Infection

The early host–pathogen interaction between European seabass (Dicentrarchus labrax) and Betanodavirus was examined by using juvenile fish infected intramuscularly with RGNNV (red-spotted grouper nervous necrosis virus). The time course selected for sampling (0–144 h post-infection (hpi)) covered the early stages of infection, with hematological, antioxidant and immunological responses examined. Early activation of the host’s immune system was seen in the first few hours post-infection (6 to 9 hpi), as evidenced by an increase in tnfα, cd28 and c3 expression in the head kidney of infected fish. Most hematological parameters that were examined showed significant differences between sampling times, including differences in the number of thrombocytes and various leukocyte populations. The plasma lysozyme concentration decreased significantly over the course of the trial, and most antioxidant parameters examined in the liver showed significant differences over the infection period. At 144 hpi, peak expression of tnfα and il-1β coincided with the appearance of disease symptoms, peak levels of virus in the brain and high levels of fish mortality. The results of the study show the importance of analyzing the early interactions between European seabass and Betanodavirus to establish early indicators of infection to prevent more severe outcomes of the infection from occurring.

Vaz, Mariana↗
DOE OSTI2020

Genotypic and Phenotypic Characterization of Incompatibility Group FIB Positive Salmonella enterica Serovar Typhimurium Isolates from Food Animal Sources

Salmonella enterica is one of the most common bacterial foodborne pathogens in the United States, causing illnesses that range from self-limiting gastroenteritis to more severe, life threatening invasive disease. Many Salmonella strains contain plasmids that carry virulence, antimicrobial resistance, and/or transfer genes which allow them to adapt to diverse environments, and these can include incompatibility group (Inc) FIB plasmids. This study was undertaken to evaluate the genomic and phenotypic characteristics of IncFIB-positive Salmonella enterica serovar Typhimurium isolates from food animal sources, to identify their plasmid content, assess antimicrobial resistance and virulence properties, and compare their genotypic isolates with more recently isolated S. Typhimurium isolates from food animal sources. Methods: We identified 71 S. Typhimurium isolates that carried IncFIB plasmids. These isolates were subjected to whole genome sequencing and evaluated for bacteriocin production, antimicrobial susceptibility, the ability to transfer resistance plasmids, and a subset was evaluated for their ability to invade and persist in intestinal human epithelial cells. Results: Approximately 30% of isolates (n = 21) displayed bacteriocin inhibition of Escherichia coli strain J53. Bioinformatic analyses using PlasmidFinder software confirmed that all isolates contained IncFIB plasmids along with multiple other plasmid replicon types. Comparative analyses showed that all strains carried multiple antimicrobial resistance genes and virulence factors including iron acquisition genes, such as iucABCD (75%), iutA (94%), sitABCD (76%) and sitAB (100%). In 17 cases (71%), IncFIB plasmids, along with other plasmid replicon types, were able to conjugally transfer antimicrobial resistance and virulence genes to the susceptible recipient strain. For ten strains, persistence cell counts (27%) were noted to be significantly higher than invasion bacterial cell counts. When the genome sequences of the study isolates collected from 1998–2003 were compared to those published from subsequent years (2005–2018), overlapping genotypes were found, indicating the perseverance of IncFIB positive strains in food animal populations. This study confirms that IncFIB plasmids can play a potential role in disseminating antimicrobial resistance and virulence genes amongst bacteria from several food animal species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Target Affinity and Structural Analysis for a Selection of Norovirus Aptamers

Aptamers, single-stranded oligonucleotides that specifically bind a molecule with high affinity, are used as ligands in analytical and therapeutic applications. For the foodborne pathogen norovirus, multiple aptamers exist but have not been thoroughly characterized. Consequently, there is little research on aptamer-mediated assay development. This study characterized seven previously described norovirus aptamers for target affinity, structure, and potential use in extraction and detection assays. Norovirus-aptamer affinities were determined by filter retention assays using norovirus genotype (G) I.1, GI.7, GII.3, GII.4 New Orleans and GII.4 Sydney virus-like particles. Of the seven aptamers characterized, equilibrium dissociation constants for GI.7, GII.3, GII.4 New Orleans and GII.4 Sydney ranged from 71 ± 38 to 1777 ± 1021 nM. Four aptamers exhibited affinity to norovirus GII.4 strains; three aptamers additionally exhibited affinity toward GII.3 and GI.7. Aptamer affinity towards GI.1 was not observed. Aptamer structure analysis by circular dichroism (CD) spectroscopy showed that six aptamers exhibit B-DNA structure, and one aptamer displays parallel/antiparallel G-quadruplex hybrid structure. CD studies also showed that biotinylated aptamer structures were unchanged from non-biotinylated aptamers. Finally, norovirus aptamer assay feasibility was demonstrated in dot-blot and pull-down assays. This characterization of existing aptamers provides a knowledge base for future aptamer-based norovirus detection and extraction assay development and aptamer modification.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structure and Function of the Zinc Binding Protein ZrgA from Vibrio cholerae

ATP binding cassette (ABC) transporters are the primary means by which bacteria acquire trace elements from the environment. They rely on solute binding proteins (SBPs) to bind the relevant substrate and deliver it to the integral membrane permease for ATP-powered import into the cytoplasm. SBPs of cluster A-I are known to facilitate the transport of essential metals zinc, manganese, and iron, and many have been characterized to date. A group of ABC transporter operons dubbed zinc-regulated genes (zrg) have recently been shown to transport zinc with putative SBPs (zrgA) bearing no homology to the classical cluster A-I family, and a recent crystal structure of a representative protein from Pseudomonas aeruginosa shows no structural similarity to classical SBPs. Thus, the ZrgA proteins appear to represent a newly discovered family of zinc SBPs widespread among Gram-negative bacteria, including human pathogens. Here, we have determined the crystal structure of ZrgA from Vibrio cholerae and characterized its zinc binding in vitro and function in vivo. We also assessed the role of a histidine-rich sequence that appears to be a hallmark of ZrgA proteins that is particularly long in V. cholerae ZrgA. The results show that the zrgA gene is critical to the function of the operon, consistent with a function as an SBP in this system. Further, the His-rich region is not essential to the function of ZrgA, but it does provide additional zinc binding sites in vitro. The structure and zinc binding data for ZrgA reveal interesting differences between it and its homologue from P. aeruginosa, illustrating diversity within this little-studied protein family.

59 BASIC BIOLOGICAL SCIENCES↗