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At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Spray‐induced gene silencing to identify powdery mildew gene targets and processes for powdery mildew control

Abstract Spray‐induced gene silencing (SIGS) is an emerging tool for crop pest protection. It utilizes exogenously applied double‐stranded RNA to specifically reduce pest target gene expression using endogenous RNA interference machinery. In this study, SIGS methods were developed and optimized for powdery mildew fungi, which are widespread obligate biotrophic fungi that infect agricultural crops, using the known azole‐fungicide target cytochrome P450 51 (CYP51) in the Golovinomyces orontii–Arabidopsis thaliana pathosystem. Additional screening resulted in the identification of conserved gene targets and processes important to powdery mildew proliferation: apoptosis‐antagonizing transcription factor in essential cellular metabolism and stress response; lipid catabolism genes lipase a , lipase 1 , and acetyl‐CoA oxidase in energy production ; and genes involved in manipulation of the plant host via abscisic acid metabolism ( 9‐cis‐epoxycarotenoid dioxygenase , xanthoxin dehydrogenase , and a putative abscisic acid G‐protein coupled receptor ) and secretion of the effector protein, effector candidate 2 . Powdery mildew is the dominant disease impacting grapes and extensive powdery mildew resistance to applied fungicides has been reported. We therefore developed SIGS for the Erysiphe necator–Vitis vinifera system and tested six successful targets identified using the G. orontii–A. thaliana system. For all targets tested, a similar reduction in powdery mildew disease was observed between systems. This indicates screening of broadly conserved targets in the G. orontii–A. thaliana pathosystem identifies targets and processes for the successful control of other powdery mildew fungi. The efficacy of SIGS on powdery mildew fungi makes SIGS an exciting prospect for commercial powdery mildew control.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Proteome-wide characterization of PTMs reveals host cell responses to viral infection and identifies putative antiviral drug targets

Post-translational modifications (PTMs) are biochemical modifications that can significantly alter protein structure, function, stability, localization, and interactions with other molecules, thereby activating or inactivating intracellular processes. A growing body of research has begun to highlight the role of PTMs, including phosphorylation, ubiquitination, acetylation, and redox modifications, during virus-host interactions. Collectively, these PTMs regulate key steps in mounting the host immune response and control critical host pathways required for productive viral replication. This has led to the conception of antiviral therapeutics that focus on controlling host protein PTMs, potentially offering pathogen-agnostic treatment options and revolutionizing our capacity to prevent virus transmission. On the other hand, viruses can hijack the host cellular PTM machinery to modify viral proteins in promoting viral replication and evading immune surveillance. PTM regulation during virus-host interactions is complex and poorly mapped, and the development of effective PTM-targeted antiviral drugs will require a more comprehensive understanding of the cellular pathways essential for virus replication. In this review, we discuss the roles of PTMs in virus infection and how technological advances in mass spectrometry-based proteomics can capture systems-level PTM changes during viral infection. Additionally, we explore how such knowledge is leveraged to identify PTM-targeted candidates for developing antiviral drugs. Looking ahead, studies focusing on the discovery and functional elucidation of PTMs, either on the host or viral proteins, will not only deepen our understanding of molecular pathology but also pave the way for developing better drugs to fight emerging viruses.

Immunology↗
DOE OSTI2021

Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons

Enteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC’s. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen.

99 GENERAL AND MISCELLANEOUS↗
DOE OSTI2021

Structures of full-length VanR from Streptomyces coelicolor in both the inactive and activated states

Vancomycin has historically been used as a last-resort treatment for serious bacterial infections. However, vancomycin resistance has become widespread in certain pathogens, presenting a serious threat to public health. Resistance to vancomycin is conferred by a suite of resistance genes, the expression of which is controlled by the VanR–VanS two-component system. VanR is the response regulator in this system; in the presence of vancomycin, VanR accepts a phosphoryl group from VanS, thereby activating VanR as a transcription factor and inducing expression of the resistance genes. This paper presents the X-ray crystal structures of full-length VanR from Streptomyces coelicolor in both the inactive and activated states at resolutions of 2.3 and 2.0 Å, respectively. Comparison of the two structures illustrates that phosphorylation of VanR is accompanied by a disorder-to-order transition of helix 4, which lies within the receiver domain of the protein. This transition generates an interface that promotes dimerization of the receiver domain; dimerization in solution was verified using analytical ultracentrifugation. The inactive conformation of the protein does not appear intrinsically unable to bind DNA; rather, it is proposed that in the activated form DNA binding is enhanced by an avidity effect contributed by the receiver-domain dimerization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structure and Fc-Effector Function of Rhesusized Variants of Human Anti-HIV-1 IgG1s

Passive transfer of monoclonal antibodies (mAbs) of human origin into Non-Human Primates (NHPs), especially those which function predominantly by a Fc-effector mechanism, requires an a priori preparation step, in which the human mAb is reengineered to an equivalent NHP IgG subclass. This can be achieved by changing both the Fc and Fab sequence while simultaneously maintaining the epitope specificity of the parent antibody. This Ab reengineering process, referred to as rhesusization, can be challenging because the simple grafting of the complementarity determining regions (CDRs) into an NHP IgG subclass may impact the functionality of the mAb. Here we describe the successful rhesusization of a set of human mAbs targeting HIV-1 envelope (Env) epitopes involved in potent Fc-effector function against the virus. This set includes a mAb targeting a linear gp120 V1V2 epitope isolated from a RV144 vaccinee, a gp120 conformational epitope within the Cluster A region isolated from a RV305 vaccinated individual, and a linear gp41 epitope within the immunodominant Cys-loop region commonly targeted by most HIV-1 infected individuals. Structural analyses confirm that the rhesusized variants bind their respective Env antigens with almost identical specificity preserving epitope footprints and most antigen-Fab atomic contacts with constant regions folded as in control RM IgG1s. In addition, functional analyses confirm preservation of the Fc effector function of the rhesusized mAbs including the ability to mediate Antibody Dependent Cell-mediated Cytotoxicity (ADCC) and antibody dependent cellular phagocytosis by monocytes (ADCP) and neutrophils (ADNP) with potencies comparable to native macaque antibodies of similar specificity. While the antibodies chosen here are relevant for the examination of the correlates of protection in HIV-1 vaccine trials, the methods used are generally applicable to antibodies for other purposes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

A tyrosine phosphoregulatory system controls exopolysaccharide biosynthesis and biofilm formation in Vibrio cholerae

Production of an extracellular matrix is essential for biofilm formation, as this matrix both secures and protects the cells it encases. Mechanisms underlying production and assembly of matrices are poorly understood. Vibrio cholerae, relies heavily on biofilm formation for survival, infectivity, and transmission. Biofilm formation requires Vibrio polysaccharide (VPS), which is produced by vps gene-products, yet the function of these products remains unknown. Here, we demonstrate that the vps gene-products vpsO and vpsU encode respectively for a tyrosine kinase and a cognate tyrosine phosphatase. Collectively, VpsO and VpsU act as a tyrosine phosphoregulatory system to modulate VPS production. We present structures of VpsU and the kinase domain of VpsO, and we report observed autocatalytic tyrosine phosphorylation of the VpsO C-terminal tail. The position and amount of tyrosine phosphorylation in the VpsO C-terminal tail represses VPS production and biofilm formation through a mechanism involving the modulation of VpsO oligomerization. We found that tyrosine phosphorylation enhances stability of VpsO. Regulation of VpsO phosphorylation by the phosphatase VpsU is vital for maintaining native VPS levels. This study provides new insights into the mechanism and regulation of VPS production and establishes general principles of biofilm matrix production and its inhibition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Detection of Apparent Early Rabies Infection by LN34 Pan-Lyssavirus Real-Time RT-PCR Assay in Pennsylvania

The Pennsylvania Department of Health Bureau of Laboratories (PABOL) tested 6855 animal samples for rabies using both the direct fluorescent antibody test (DFA) and LN34 pan-lyssavirus reverse transcriptase quantitative PCR (RT-qPCR) during 2017–2019. Only two samples (0.03%) were initially DFA negative but positive by LN34 RT-qPCR. Both cases were confirmed positive upon re-testing at PABOL and confirmatory testing at the Centers for Disease Control and Prevention by LN34 RT-qPCR and DFA. Rabies virus sequences from one sample were distinct from all positive samples processed at PABOL within two weeks, ruling out cross-contamination. Levels of rabies virus antigen and RNA were low in all brain structures tested, but were higher in brain stem and rostral spinal cord than in cerebellum, hippocampus or cortex. Taken together, the low level of rabies virus combined with higher abundance in more caudal brain structures suggest early infection. These cases highlight the increased sensitivity and ease of interpretation of LN34 RT-qPCR for low positive cases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Gut microbiome associations with outcome following co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in pigs immunized with a PRRS modified live virus vaccine

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most significant pathogens affecting swine. Co-infections are common and result in respiratory disease and reduced weight gain in growing pigs. Although PRRS modified live virus (MLV) vaccines are widely used to decrease PRRS-associated losses, they are generally considered inadequate for disease control. The gut microbiome provides an alternative strategy to enhance vaccine efficacy and improve PRRS control. The objective of this work was to identify gut microbiome characteristics associated with improved outcome in pigs immunized with a PRRS MLV and co-challenged with PRRSV and PCV2b. Twenty-eight days after vaccination and prior to co-challenge, fecal samples were collected from an experimental population of 50 nursery pigs. At 42 days post-challenge, 20 pigs were retrospectively identified as having high or low growth outcomes during the post-challenge period. Gut microbiomes of the two outcome groups were compared using the Lawrence Livermore Microbial Detection Array (LLMDA) and 16S rDNA sequencing. High growth outcomes were associated with several gut microbiome characteristics, such as increased bacterial diversity, increased Bacteroides pectinophilus, decreased Mycoplasmataceae species diversity, higher Firmicutes:Bacteroidetes ratios, increased relative abundance of the phylum Spirochaetes, reduced relative abundance of the family Lachnospiraceae, and increased Lachnospiraceae species C6A11 and P6B14. Overall, this study identifies gut microbiomes associated with improved outcomes in PRRS vaccinated pigs following a polymicrobial respiratory challenge and provides evidence towards the gut microbiome playing a role in PRRS vaccine efficacy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Autocrine and paracrine interferon signalling as ‘ring vaccination’ and ‘contact tracing’ strategies to suppress virus infection in a host

The innate immune response, particularly the interferon response, represents a first line of defence against viral infections. The interferon molecules produced from infected cells act through autocrine and paracrine signalling to turn host cells into an antiviral state. Although the molecular mechanisms of IFN signalling have been well characterized, how the interferon response collectively contribute to the regulation of host cells to stop or suppress viral infection during early infection remain unclear. Here, we use mathematical models to delineate the roles of the autocrine and the paracrine signalling, and show that their impacts on viral spread are dependent on how infection proceeds. In particular, we found that when infection is well-mixed, the paracrine signalling is not as effective; by contrast, when infection spreads in a spatial manner, a likely scenario during initial infection in tissue, the paracrine signalling can impede the spread of infection by decreasing the number of susceptible cells close to the site of infection. Furthermore, we argue that the interferon response can be seen as a parallel to population-level epidemic prevention strategies such as ‘contact tracing’ or ‘ring vaccination’. Thus, our results here may have implications for the outbreak control at the population scale more broadly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Antimicrobial mechanisms of g‐C 3 N 4 @ ZnO against oomycetes Phytophthora capsici: from its metabolism, membrane structures and growth

Abstract BACKGROUND Phytophthora capsici , a refractory and model oomycete plant pathogen, especially threatens multiple vegetable crops. A limited number of chemical pesticides play a vital role in controlling oomycete plant diseases. However, this approach often leads to excessive use of chemical agent, exacerbates environmental issues and more and more drug‐resistant strains of oomycete. Therefore, it is imperative to devise innovative solutions that can effectively address the infection of oomycete while maintaining high levels of environmental sustainability and low toxicity. RESULTS In this study, g‐C 3 N 4 @ZnO heterostructure was synthesized and characterized. The g‐C 3 N 4 @ZnO showed higher toxicity on Phytophthora capsici than graphitic carbon nitride (g‐C 3 N 4 ) nanosheets and zinc oxide (ZnO) nanoparticles in vitro and in vivo . Except the hyphal growth of Phytophthora capsici , their germination rate of spores, sporangium formation and number of spores were all suppressed by g‐C 3 N 4 @ZnO heterostructure. Furthermore, we found that this g‐C 3 N 4 @ZnO heterostructure has higher photocatalytic activity under visible light, which potentially enhanced the reactive oxygen species (ROS) mediated stress on Phytophthora capsici . Ultrastructural morphology, global changes of gene expression and weighted gene co‐expression network analysis all supported that the anti‐oomycete activity of g‐C 3 N 4 @ZnO was manifested in the destruction of membrane system and inhibition of multiple metabolisms of Phytophthora capsici under visible irradiation, which also could be attributed to the ROS and zinc ion (Zn 2+ ) mediated stress. CONCLUSION This works offers a novel oomycete disease management strategy by using g‐C 3 N 4 @ZnO, which were attributed to the ROS stress, destruction of membrane system and inhibition of multiple metabolisms. © 2023 Society of Chemical Industry.

Cai, Lin↗
DOE OSTI2021

Symbiont-Mediated Protection of Acromyrmex Leaf-Cutter Ants from the Entomopathogenic Fungus Metarhizium anisopliae

Many fungus-growing ants engage in a defensive symbiosis with antibiotic-producing ectosymbiotic bacteria in the genus Pseudonocardia, which help protect the ants’ fungal mutualist from a specialized mycoparasite, Escovopsis. Here, using germfree ant rearing and experimental pathogen infection treatments, we evaluate if Acromyrmex ants derive higher immunity to the entomopathogenic fungus Metarhizium anisopliae from their Pseudonocardia symbionts. We further examine the ecological dynamics and defensive capacities of Pseudonocardia against M. anisopliae across seven different Acromyrmex species by controlling Pseudonocardia acquisition using ant-nonnative Pseudonocardia switches, in vitro challenges, and in situ mass spectrometry imaging (MSI). We show that Pseudonocardia protects the ants against M. anisopliae across different Acromyrmex species and appears to afford higher protection than metapleural gland (MG) secretions. Although Acromyrmex echinatior ants with nonnative Pseudonocardia symbionts receive protection from M. anisopliae regardless of the strain acquired compared with Pseudonocardia-free conditions, we find significant variation in the degree of protection conferred by different Pseudonocardia strains. Additionally, when ants were reared in Pseudonocardia-free conditions, some species exhibit more susceptibility to M. anisopliae than others, indicating that some ant species depend more on defensive symbionts than others. In vitro challenge experiments indicate that Pseudonocardia reduces Metarhizium conidiospore germination area. Our chemometric analysis using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) reveals that Pseudonocardia-carrying ants produce more chemical signals than Pseudonocardia-free treatments, indicating that Pseudonocardia produces bioactive metabolites on the Acromyrmex cuticle. Our results indicate that Pseudonocardia can serve as a dual-purpose defensive symbiont, conferring increased immunity for both the obligate fungal mutualist and the ants themselves.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Deletion of E184L, a Putative DIVA Target from the Pandemic Strain of African Swine Fever Virus, Produces a Reduction in Virulence and Protection against Virulent Challenge

African swine fever (ASF) is currently causing a major pandemic affecting the swine industry and protein availability from Central Europe to East and South Asia. No commercial vaccines are available, making disease control dependent on the elimination of affected animals. Here, we show that the deletion of the African swine fever virus (ASFV) E184L gene from the highly virulent ASFV Georgia 2010 (ASFV-G) isolate produces a reduction in virus virulence during the infection in swine. Of domestic pigs intramuscularly inoculated with a recombinant virus lacking the E184L gene (ASFV-GDE184L), 40% experienced a significantly (5 days) delayed presentation of clinical disease and, overall, had a 60% rate of survival compared to animals inoculated with the virulent parental ASFV-G. Importantly, all animals surviving ASFV-G-DE184L infection developed a strong antibody response and were protected when challenged with ASFV-G. As expected, a pool of sera from ASFV-G-DE184L-inoculated animals lacked any detectable antibody response to peptides partially representing the E184L protein, while sera from animals inoculated with an efficacious vaccine candidate, ASFV-GDMGF, strongly recognize the same set of peptides. These results support the potential use of the E184L deletion for the development of vaccines able to differentiate infected from vaccinated animals (DIVA). Therefore, it is shown here that the E184L gene is a novel ASFV determinant of virulence that can potentially be used to increase safety in preexisting vaccine candidates, as well as to provide them with DIVA capabilities. To our knowledge, E184L is the first ASFV gene product experimentally shown to be a functional DIVA antigenic marker.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Dynamical model for social distancing in the U.S. during the COVID-19 epidemic

Social distancing has led to a “flattening of the curve” in many states across the U.S. This is part of a novel, massive, global social experiment which has served to mitigate the COVID-19 pandemic in the absence of a vaccine or effective anti-viral drugs. Hence it is important to be able to forecast hospitalizations reasonably accurately. We propose on phenomenological grounds a random walk/generalized diffusion equation which incorporates the effect of social distancing to describe the temporal evolution of the probability of having a given number of hospitalizations. The probability density function is log-normal in the number of hospitalizations, which is useful in describing pandemics where the number of hospitalizations is very high. We used this insight and data to make forecasts for states using Monte Carlo methods. Back testing validates our approach, which yields good results about a week into the future. States are beginning to reopen at the time of submission of this paper and our forecasts indicate possible precursors of increased hospitalizations. However, the trends we forecast for hospitalizations as well as infections thus far show moderate growth. Additionally we studied the reproducibility in New York (Italian strain) and California (Wuhan strain). We find that even if there is a difference in the transmission of the two strains, social distancing has been able to control the progression of COVID 19.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2025

Feed status and skin injury modulate immunopathology, global gene expression, and survival in channel catfish during virulent Aeromonas hydrophila infection

Introduction VirulentAeromonas hydrophilais a major pathogen in channel catfish (Ictalurus punctatus), that causes motileAeromonassepticemia and significant economic losses. We investigated the effect of feeding status and skin integrity on the host immune response, disease survival, and gastrointestinal pathology following a vAh challenge. Methods Using a bath immersion model, channel catfish were divided into four treatment groups: fin clipped and fed (FCF), fin clipped but not fed (FCN), not fin clipped but fed (NCF), and not fin clipped nor fed (NCN) alongside non-challenged control groups The FCF and NCF groups were fed 2 h prior to the challenge, but the FCN and NCN groups were not. Survival analysis, histopathological assessment, and RNA sequencing were conducted across groups at different time intervals throughout the vAh challenge. Results Survival rates were lowest in the FCF and FCN groups (30% and 23% survival, respectively), suggesting that both feeding and skin damage contributed to disease severity. Histopathological analyses revealed more severe intestinal and gastric lesions in fed groups, characterized by epithelial necrosis, hemorrhage, and edema. Transcriptomic analysis among the groups identified significant differentially expressed genes associated with inflammation, apoptosis, and metabolic stress, with notable upregulation of interleukin 1-beta (il-1β), and complement C3 (c3). Gene ontology enrichment highlighted distinct immune activation patterns between fed and unfed groups, with enhanced pathogen recognition and pro-inflammatory responses in unfed fish. Discussion These findings suggest feeding prior to infection may exacerbate disease pathology, potentially by creating a physiological state conducive to facilitate pathogen proliferation and dampened early immune responses, whereas short-term fasting appears to promote early immune activation. This study provides novel insights into the complex interplay between feed status, physical injury, and immune response to vAh infection.

Immunology↗
DOE OSTI2025

Patchy burn severity explains heterogeneous soil viral and prokaryotic responses to fire in a mixed conifer forest

ABSTRACT Effects of fire on soil viruses and virus–host dynamics are largely unexplored, despite known microbial contributions to biogeochemical processes and ecosystem recovery. Here, we assessed how viral and prokaryotic communities responded to a prescribed burn in a mixed conifer forest. We sequenced 91 viral-size fraction metagenomes (viromes) and 115 16S rRNA gene amplicon libraries from 120 samples: four samples at five timepoints (two before fire and three after fire) at six sites (four treatment, two control). We hypothesized that compositional differences would be most significant between burned and unburned soils, but instead, plot location best distinguished viral communities, more than treatment (burned or not), depth (0–3 or 3–6 cm), or timepoint. For both viruses and prokaryotes, some burned communities resembled unburned controls, while others were significantly different, revealing heterogeneous responses to fire. These patterns were explained by burn severity, here defined by soil chemistry. Viral but not prokaryotic richness decreased significantly with burn severity, and low viromic DNA yields indicated substantial loss of viral biomass at higher severity. The relative abundances of Firmicutes, Actinobacteriota, and the viruses predicted to infect them increased significantly with burn severity, suggesting survival and viral infection of these fire-responsive and potentially spore-forming taxa. The degree of burn severity experienced by each patch of soil, rather than burn status alone, differed over mere meters in the same fire. Therefore, our analyses highlight the importance of high-resolution, paired biogeochemical data to explain soil community responses to fire. IMPORTANCE The impact of fire on the soil microbiome, particularly on understudied soil viral communities, warrants investigation, given known microbial contributions to biogeochemical processes and ecosystem recovery. Here, we collected 120 soil samples before and after a prescribed burn in a mixed conifer forest to assess the impacts of this disturbance on soil viral and prokaryotic communities. We show that simple categorical comparisons of burned and unburned areas were insufficient to reveal the underlying community response patterns. The patchy nature of the fire (indicated by soil chemistry data) led to significant changes in viral and prokaryotic community composition in areas of high burn severity, while communities that experienced lower burn severity were indistinguishable from those in unburned controls. Our results highlight the importance of considering highly resolved burn severity and biogeochemical measurements, even in nearby soils after the same fire, in order to understand soil microbial responses to prescribed burns.

Microbiology↗
DOE OSTI2023

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. Furthermore, by activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein–protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Evaluation of Three RNA Extraction Kits with a 2019-nCoV Assay

The following characteristics of three RNA extraction kits (Norgen Biotek Total RNA Purification Kit, #17200; Bioneer AccuPrep Viral RNA Extraction Kit, #K-3033; and Promega Maxwell HT Viral TNA Kit, #AX2340) were evaluated: Limits of detection for quantitative synthetic RNA using the Centers for Disease Control and Prevention’s (CDC) 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel and the Applied Biosystems 7500 Fast Dx system; Limits of detection for positive clinical specimen using the Centers for Disease Control and Prevention’s (CDC) 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel and the Applied Biosystems 7500 Fast Dx system; Ability of the lysis buffers in each kit to inactivate the virus as measured by a cell-based infectivity assay.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Metal-induced oxidative stress and human plasma protein oxidation after SARS-CoV-2 infection

Pathogenesis of COVID-19 by SARS-CoV-2 resulted in a global pandemic and public health emergency in 2020. Viral infection can induce oxidative stress through reactive oxygen species (ROS). Inflammation and environmental stress are major sources of oxidative stress after infection. Micronutrients such as iron, copper, zinc, and manganese play various roles in human tissues and their imbalance in blood can impact immune responses against pathogens including SARS CoV-2. We hypothesized that alteration of free metal ions during infection and metal-catalyzed oxidation plays a critical role towards pathogenesis after infection. We analyzed convalescent and hospitalized COVID-19 patient plasma using orthogonal analytical techniques to determine redox active metal concentrations, overall protein oxidation, oxidative modifications, and protein levels via proteomics to understand the consequences of metal-induced oxidative stress in COVID-19 plasma proteins. Metal analysis using ICP-MS showed significantly greater concentrations of copper in COVID-19 plasma compared to healthy controls. We demonstrate significantly greater total protein carbonylation, other oxidative modifications, and deamidation of plasma proteins in COVID-19 plasma compared to healthy controls. Proteomics analysis showed that levels of redox active proteins including hemoglobulin were elevated in COVID-19 plasma. Molecular modeling concurred with potential interactions between iron binding proteins and SARS CoV-2 surface proteins. Overall, increased levels of redox active metals and protein oxidation indicate that oxidative stress-induced protein oxidation in COVID-19 may be a consequence of the interactions of SARS-CoV-2 proteins with host cell metal binding proteins resulting in altered cellular homeostasis.

60 APPLIED LIFE SCIENCES↗