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At least 703 records · Page 39
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Protein nanoparticle vaccine based on flagellin carrier fused to influenza conserved epitopes confers full protection against influenza A virus challenge

Currently marketed influenza vaccines only confer protection against matching influenza virus strains. The influenza A composition of these vaccines needs to be annually updated. Vaccines that target conserved epitopes of influenza viruses would in principle offer broad cross-protection against influenza A viruses. In our study, we investigated the specific immune responses and protective efficacy of protein nanoparticles based on fusion proteins of flagellin carrier linked to conserved influenza epitopes. We designed fusion proteins by replacing the hyperimmunogenic region of flagellin (FliC) with four tandem copies of the ectodomain of matrix protein 2 (f4M2e), H1 HA2 domain (fHApr8) or H3 HA2 domain (fHAaichi). Protein nanoparticles fabricated from these fusion proteins by using DTSSP crosslinking retained Toll-like receptor 5 agonist activity of FliC. Intranasal immunization with f4M2e, f4M2e/fHApr8 or f4M2e/fHAaichi nanoparticles induced vaccine antigen-specific humoral immune responses. It was also found that the incorporation of the H1 HA2 domain into f4M2e/fHApr8 nanoparticles boosted M2e specific antibody responses. Immunized mice were fully protected against lethal doses of virus challenge. - Highlights: • Stable protein nanoparticles can be fabricated using DTSSP crosslinker. • Protein nanoparticles induce strong antibody responses against M2e and HA stalk domains in mice. • Vaccinated mice are fully protected against lethal dose of influenza A virus challenges.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Mesenchymal stem cell exosomes promote immunosuppression of regulatory T cells in asthma

Highlights: • MSC exosomes promote Tregs proliferation by upregulating IL-10 and TGF-β1 in asthma. • MSC exosomes enhance immunosuppression capacity of Tregs from asthmatic patient. • The effect of MSC exosomes on Tregs may be mediated by APCs. Mesenchymal stem cells (MSCs) and regulatory T cells (Tregs) are both potent immune-modulators. The aberrant proliferation and function of Tregs plays an important role in the development of asthma. Our previous studies have demonstrated the role of MSCs in promoting proliferation and immune-modulating of Tregs, as well as alleviating airway inflammation of asthmatic mice. In the present study, we isolated exosomes secreted by MSCs and investigated their immunomodulation effect on peripheral blood mononuclear cells (PBMCs) of asthmatic patient. We found that MSC exosomes upregulated IL-10 and TGF-β1 from PBMCs, thus promoting proliferation and immune-suppression capacity of Tregs. Furthermore, antigen presenting cells (APCs) but not CD4+ T cells-dependent pathway was shown to be possible mechanism involved in MSC exosome-mediated regulation. Our data elucidated the key role of exosomes in immune-modulation of MSCs, and suggested the therapeutic potential of MSC exosomes for asthma.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Comprehensive analysis of differentially expressed mRNA and circRNA in Ankylosing spondylitis patients’ platelets

Highlights: • 4996 mRNAs and 2942 circRNAs were identified differentially expressed in platelets of AS patients. • Several platelet-derived immune mediators were upregulated to regulate the interaction between platelets and immune cells. • Two downregulated circRNA were identified, and the corresponding circRNA-miRNA-mRNA regulatory network was constructed. • mRNAs and circRNAs found herein could be used as diagnostic biomarkers or new therapeutic targets. Ankylosing spondylitis (AS) is a chronic inflammatory disease significantly decreasing the quality of life. Platelets play an important and active role in the development of AS. Accumulating evidence demonstrated platelets contain diverse RNA repository inherited from megakaryocytes or microvesicles. Platelet RNAs are dynamically affected by pathological conditions and could be used as diagnostic or prognostic biomarkers. However, the role of the platelet RNAs in AS is elusive. In this study, we compared mRNA and circRNA profiles in platelets between AS patients and healthy controls using RNA sequencing and bioinformatic analysis, and found 4996 mRNAs and 2942 circRNAs were differently expressed. The significantly over-expressed mRNAs in AS patients are involved in platelet activity, gap junction, focal adhesion, rap1 and toll and Imd signaling pathway. The previous identified platelet-derived immune mediators such as P2Y1, P2Y12, PF4, GPIbα, CD40L, ICAM2, CCL5 (RANTES), TGF-β (TGF-β1 and TGF-β2) and PDGF (PDGFB and PDGFA) are also included in these over expressed mRNAs, implying these factors may trigger inflammatory cascades and promote the development of AS. Additionally, we found two down-regulated circRNA (circPTPN22 and circFCHSD2) from the intersection analyses of platelets and spinal ligament tissues of AS patients. The circRNA-miRNA-mRNA regulatory network of these two circRNAs was constructed, and the target mRNAs were enriched in Th17 cell differentiation, inflammatory bowel disease, cell adhesion molecules, cytokine-cytokine receptor interaction, Jak-STAT and Wnt signaling pathway, all these pathways participate in the bone remodeling and pro/anti-inflammatory immune regulation in AS. Then, qRT-PCR was performed to validate the expression of selected key mRNAs and circRNAs and the results demonstrated that the expression levels of P2Y12, GPIbα, circPTPN22 and circFCHSD2 were consistent with the sequencing analysis. In addition, the high expression of five predicted miRNAs interacting with circPTPN22 and circFCHSD2 were also detected in AS by qRT-PCR. Taken together, our study presents a comprehensive overview of mRNAs and circRNAs in platelets in AS patients and offers new insight into the mechanisms of platelet involving in the pathogenesis of AS. The mRNAs and circRNAs identified in this study may serve as candidates for diagnosis and targeted treatment of AS.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Defense against phytopathogens relies on efficient antimicrobial protein secretion mediated by the microtubule-binding protein TGNap1

Plant immunity depends on the secretion of antimicrobial proteins, which occurs through yet-largely unknown mechanisms. The trans-Golgi network (TGN), a hub for intracellular and extracellular trafficking pathways, and the cytoskeleton, which is required for antimicrobial protein secretion, are emerging as pathogen targets to dampen plant immunity. In this work, we demonstrate that tgnap1-2, a loss-of-function mutant of Arabidopsis TGNap1, a TGN-associated and microtubule (MT)-binding protein, is susceptible to Pseudomonas syringae (Pst DC3000). Pst DC3000 infected tgnap1-2 is capable of mobilizing defense pathways, accumulating salicylic acid (SA), and expressing antimicrobial proteins. The susceptibility of tgnap1-2 is due to a failure to efficiently transport antimicrobial proteins to the apoplast in a partially MT-dependent pathway but independent from SA and is additive to the pathogen-antagonizing MIN7, a TGN-associated ARF-GEF protein. Therefore, our data demonstrate that plant immunity relies on TGNap1 for secretion of antimicrobial proteins, and that TGNap1 is a key immunity element that functionally links secretion and cytoskeleton in SA-independent pathogen responses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structural basis for influenza virus NS1 protein block of mRNA nuclear export

Influenza viruses antagonize key immune defence mechanisms via the virulence factor non-structural protein 1 (NS1). A key mechanism of virulence by NS1 is blocking nuclear export of host messenger RNAs, including those encoding immune factors; however, the direct cellular target of NS1 and the mechanism of host mRNA export inhibition are not known. Here, we identify the target of NS1 as the mRNA export receptor complex, nuclear RNA export factor 1–nuclear transport factor 2-related export protein 1 (NXF1–NXT1), which is the principal receptor mediating docking and translocation of mRNAs through the nuclear pore complex via interactions with nucleoporins. We determined the crystal structure of NS1 in complex with NXF1–NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1–NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. We demonstrate that a mutant influenza virus deficient in binding NXF1–NXT1 does not block host mRNA export and is attenuated. Here, this attenuation is marked by the release of mRNAs encoding immune factors from the nucleus. In sum, our study uncovers the molecular basis of a major nuclear function of influenza NS1 protein that causes potent blockage of host gene expression and contributes to inhibition of host immunity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The CRISPR effector Cam1 mediates membrane depolarization for phage defence

Prokaryotic type III CRISPR–Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors. Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain. Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF–transmembrane helix fusion proteins facilitate the type III CRISPR–Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Underground azelaic acid-conferred resistance to Pseudomonas syringae in Arabidopsis

Local interactions between individual plant organs and diverse microorganisms can lead to whole plant immunity via the mobilization of defense signals. One such signal is the plastid lipid-derived oxylipin azelaic acid (AZA). Arabidopsis lacking AZI1 or EARLI1, related lipid transfer family proteins, exhibit reduced AZA transport among leaves and cannot mount systemic immunity. AZA has been detected in roots as well as leaves. Thus, the present study addresses the effects on plants of AZA application to roots. AZA, but not the structurally related suberic acid, inhibits root growth when directly in contact with roots. Treatment of roots with AZA also induces resistance to Pseudomonas syringae in aerial tissues. Additionally, these effects of AZA on root growth and disease resistance depend at least partially on AZI1 and EARLI1. AZI1 in roots localizes to plastids, similar to its known location in leaves. Interestingly, kinases previously shown to modify AZI1 in vitro, MPK3/6, are also needed for AZA-induced root growth inhibition and above ground immunity. Finally, [2H]-AZA applied to the roots does not move to aerial tissues. Therefore, AZA application to roots triggers systemic immunity through an AZI1/EARLI1/MPK3/MPK6-dependent pathway and AZA’s effects may involve an additional mobile signal(s).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Progress in multiple genetically modified minipigs for xenotransplantation in China

Abstract Pig‐to‐human organ transplantation provides an alternative for critical shortage of human organs worldwide. Genetically modified pigs are promising donors for xenotransplantation as they show many anatomical and physiological similarities to humans. However, immunological rejection including hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), immune cell–mediated rejection, and other barriers associated with xenotransplantation must be overcome with various strategies for the genetic modification of pigs. In this review, we summarize the outcomes of genetically modified and cloned pigs achieved by Chinese scientists to resolve the above‐mentioned problems in xenotransplantation. It is now possible to knockout several porcine genes associated with the expression of sugar residues, antigens for (naturally) existing antibodies in humans, including GGTA1, CMAH, and β4GalNT2, and thereby preventing the antigen‐antibody response. Moreover, insertion of human complement‐ and coagulation‐regulatory transgenes, such as CD46, CD55, CD59, and hTBM, can further overcome effects of the humoral immune response and coagulation dysfunction, while expression of regulatory factors of immune responses can inhibit the adaptive immune rejection. Furthermore, transgenic strategies have been developed by Chinese scientists to reduce the potential risk of infections by endogenous porcine retroviruses (PERVs). Breeding of multi‐gene low‐immunogenicity pigs in China is also presented in this review. Lastly, we will briefly mention the preclinical studies on pig‐to‐non‐human primate xenotransplantation conducted in several centers in China.

Pan, Dengke↗
DOE OSTI2019

Aggressive Mammary Cancers Lacking Lymphocytic Infiltration Arise in Irradiated Mice and Can Be Prevented by Dietary Intervention

Because the incidence of breast cancer increases decades after ionizing radiation exposure, aging has been implicated in the evolution of the tumor microenvironment and tumor progression. In this paper, we investigated radiation-induced carcinogenesis using a model in which the mammary glands of 10-month-old BALB/c mice were transplanted with Trp53-null mammary tissue 3 days after exposure to low doses of sparsely ionizing γ-radiation or densely ionizing particle radiation. Mammary transplants in aged, irradiated hosts gave rise to significantly more tumors that grew more rapidly than those in sham-irradiated mice, with the most pronounced effects seen in mice irradiated with densely ionizing particle radiation. Tumor transcriptomes identified a characteristic immune signature of these aggressive cancers. Consistent with this, fast-growing tumors exhibited an immunosuppressive tumor microenvironment with few infiltrating lymphocytes, abundant immunosuppressive myeloid cells, and high COX-2 and TGFβ. Only irradiated hosts gave rise to tumors lacking cytotoxic CD8 + lymphocytes (defined here as immune desert), which also occurred in younger irradiated hosts. These data suggest that host irradiation may promote immunosuppression. To test this, young chimera mice were fed chow containing a honeybee-derived compound with anti-inflammatory and immunomodulatory properties, caffeic acid phenethyl ester (CAPE). CAPE prevented the detrimental effects of host irradiation on tumor growth rate, immune signature, and immunosuppression. These data indicated that low-dose radiation, particularly densely ionizing exposure of aged mice, promoted more aggressive cancers by suppressing antitumor immunity. Dietary intervention with a nontoxic immunomodulatory agent could prevent systemic effects of radiation that fuel carcinogenesis, supporting the potential of this strategy for cancer prevention.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Transient knockdown of Anopheles stephensi LRIM1 using RNAi increases Plasmodium falciparum sporozoite salivary gland infections

Abstract Background Plasmodium falciparum (Pf) sporozoites (PfSPZ) can be administered as a highly protective vaccine conferring the highest protection seen to date. Sanaria® PfSPZ vaccines are produced using aseptically reared Anopheles stephensi mosquitoes. The bionomics of sporogonic development of P. falciparum in A. stephensi to fully mature salivary gland PfSPZ is thought to be modulated by several components of the mosquito innate immune system. In order to increase salivary gland PfSPZ infections in A. stephensi and thereby increase vaccine production efficiency, a gene knock down approach was used to investigate the activity of the immune deficiency (IMD) signaling pathway downstream effector leucine-rich repeat immune molecule 1 ( LRIM1 ), an antagonist to Plasmodium development. Methods Expression of LRIM1 in A. stephensi was reduced following injection of double stranded (ds) RNA into mosquitoes. By combining the Gal4/UAS bipartite system with in vivo expression of short hairpin (sh) RNA coding for LRIM1 reduced expression of LRIM1 was targeted in the midgut, fat body, and salivary glands. RT-qPCR was used to demonstrate fold-changes in gene expression in three transgenic crosses and the effects on P. falciparum infections determined in mosquitoes showing the greatest reduction in LRIM1 expression. Results LRIM1 expression could be reduced, but not completely silenced, by expression of LRIM1 dsRNA. Infections of P. falciparum oocysts and PfSPZ were consistently and significantly higher in transgenic mosquitoes than wild type controls, with increases in PfSPZ ranging from 2.5- to tenfold. Conclusions Plasmodium falciparum infections in A. stephensi can be increased following reduced expression of LRIM1. These data provide the springboard for more precise knockout of LRIM1 for the eventual incorporation of immune-compromised A. stephensi into manufacturing of Sanaria’s PfSPZ products.

Billingsley, Peter F. (ORCID:0000000311424813)↗
DOE OSTI2021

A vision of immuno-oncology: the Siena think tank of the Italian Network for Tumor Biotherapy (NIBIT) foundation

The yearly Think Tank Meeting of the Italian Network for Tumor Biotherapy (NIBIT) Foundation, brings together in Siena, Tuscany (Italy), experts in immuno-oncology to review the learnings from current immunotherapy treatments, and to propose new pre-clinical and clinical investigations in selected research areas. While immunotherapies in non-small cell lung cancer and melanoma led to practice changing therapies, the same therapies had only modest benefit for patients with other malignancies, such as mesothelioma and glioblastoma. One way to improve on current immunotherapies is to alter the sequence of each combination agent. Matching the immunotherapy to the host’s immune response may thus improve the activity of the current treatments. A second approach is to combine current immunotherapies with novel agents targeting complementary mechanisms. Identifying the appropriate novel agents may require different approaches than the traditional laboratory-based discovery work. For example, artificial intelligence-based research may help focusing the search for innovative and most promising combination partners. Novel immunotherapies are needed in cancer patients with resistance to or relapse after current immunotherapeutic drugs. Such new treatments may include targeted agents or monoclonal antibodies to overcome the immune-suppressive tumor microenvironment. The mode of combining the novel treatments, including vaccines, needs to be matched to the patient’s immune status for achieving the maximum benefit. In this scenario, specific attention should be also paid nowadays to the immune intersection between COVID-19 and cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Identification, cloning and characterization of SpEX exotoxin produced by Staphylococcus pseudintermedius

Staphylococci have evolved numerous strategies to evade their hosts’ immune systems. Some staphylococcal toxins target essential components of host innate immunity, one of the two main branches of the immune system. Analysis of the Staphylococcus pseudintermedius secretome using liquid chromatography mass spectrometry guided by genomic data, was used to identify an S. pseudintermedius exotoxin provisionally named SpEX. This exoprotein has low overall amino acid identity with the Staphylococcus aureus group of proteins named staphylococcal superantigen like proteins (SSLs) and staphylococcal enterotoxin- like toxin X (SEIX), but predictive modeling showed that it shares similar folds and domain architecture to these important virulence factors. In this study, we found SpEX binds to complement component C5, prevents complement mediated lysis of sensitized bovine red blood cells, kills polymorphonuclear leukocytes and monocytes and inhibits neutrophil migration at sub-lethal concentrations. A mutant version of SpEX, produced through amino acid substitution at selected positions, had diminished cytotoxicity. Anti-SpEX produced in dogs reduced the inhibitory effect of native SpEX on canine neutrophil migration and protected immune cells from the toxic effects of the native recombinant protein. These results suggest that SpEX likely plays an important role in S. pseudintermedius virulence and that attenuated SpEX may be an important candidate for inclusion in a vaccine against S. pseudintermedius infections.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Single-nuclei transcriptome analysis of channel catfish spleen provides insight into the immunome of an aquaculture-relevant species

The catfish industry is the largest sector of U.S. aquaculture production. Given its role in food production, the catfish immune response to industry-relevant pathogens has been extensively studied and has provided crucial information on innate and adaptive immune function during disease progression. To further examine the channel catfish immune system, we performed single-cell RNA sequencing on nuclei isolated from whole spleens, a major lymphoid organ in teleost fish. Libraries were prepared using the 10X Genomics Chromium X with the Next GEM Single Cell 3’ reagents and sequenced on an Illumina sequencer. Each demultiplexed sample was aligned to the Coco_2.0 channel catfish reference assembly, filtered, and counted to generate feature-barcode matrices. From whole spleen samples, outputs were analyzed both individually and as an integrated dataset. The three splenic transcriptome libraries generated an average of 278,717,872 reads from a mean 8,157 cells. The integrated data included 19,613 cells, counts for 20,121 genes, with a median 665 genes/cell. Cluster analysis of all cells identified 17 clusters which were classified as erythroid, hematopoietic stem cells, B cells, T cells, myeloid cells, and endothelial cells. Subcluster analysis was carried out on the immune cell populations. Here, distinct subclusters such as immature B cells, mature B cells, plasma cells, γδ T cells, dendritic cells, and macrophages were further identified. Differential gene expression analyses allowed for the identification of the most highly expressed genes for each cluster and subcluster. This dataset is a rich cellular gene expression resource for investigation of the channel catfish and teleost splenic immunome.

Science & Technology - Other Topics↗
DOE OSTI2020

Dynamic metabolic reprogramming in dendritic cells: An early response to influenza infection that is essential for effector function

Infection with the influenza virus triggers an innate immune response that initiates the adaptive response to halt viral replication and spread. However, the metabolic response fueling the molecular mechanisms underlying changes in innate immune cell homeostasis remain undefined. Although influenza increases parasitized cell metabolism, it does not productively replicate in dendritic cells. To dissect these mechanisms, we compared the metabolism of dendritic cells to that of those infected with active and inactive influenza A virus and those treated with toll-like receptor agonists. Using quantitative mass spectrometry, pulse chase substrate utilization assays and metabolic flux measurements, we found global metabolic changes in dendritic cells 17 hours post infection, including significant changes in carbon commitment via glycolysis and glutaminolysis, as well as mitochondrial respiration. Influenza infection of dendritic cells led to a metabolic phenotype distinct from that induced by TLR agonists, with significant resilience in terms of metabolic plasticity. We identified c-Myc as one transcription factor modulating this response. Restriction of c-Myc activity or mitochondrial substrates significantly changed the immune functions of dendritic cells, such as reducing motility and T cell activation. Transcriptome analysis of inflammatory dendritic cells isolated following influenza infection showed similar metabolic reprogramming occurs in vivo. Thus, early in the infection process, dendritic cells respond with global metabolic restructuring, that is present in inflammatory lung dendritic cells after infection, and this is important for effector function. These findings suggest metabolic switching in dendritic cells plays a vital role in initiating the immune response to influenza infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Shotgun Immunoproteomic Approach for the Discovery of Linear B-Cell Epitopes in Biothreat Agents Francisella tularensis and Burkholderia pseudomallei

Peptide-based subunit vaccines are coming to the forefront of current vaccine approaches, with safety and cost-effective production among their top advantages. Peptide vaccine formulations consist of multiple synthetic linear epitopes that together trigger desired immune responses that can result in robust immune memory. The advantages of linear compared to conformational epitopes are their simple structure, ease of synthesis, and ability to stimulate immune responses by means that do not require complex 3D conformation. Prediction of linear epitopes through use of computational tools is fast and cost-effective, but typically of low accuracy, necessitating extensive experimentation to verify results. On the other hand, identification of linear epitopes through experimental screening has been an inefficient process that requires thorough characterization of previously identified full-length protein antigens, or laborious techniques involving genetic manipulation of organisms. In this study, we apply a newly developed generalizable screening method that enables efficient identification of B-cell epitopes in the proteomes of pathogenic bacteria. As a test case, we used this method to identify epitopes in the proteome of Francisella tularensis (Ft), a Select Agent with a well-characterized immunoproteome. Our screen identified many peptides that map to known antigens, including verified and predicted outer membrane proteins and extracellular proteins, validating the utility of this approach. We then used the method to identify seroreactive peptides in the less characterized immunoproteome of Select Agent Burkholderia pseudomallei (Bp). This screen revealed known Bp antigens as well as proteins that have not been previously identified as antigens. Although B-cell epitope prediction tools Bepipred 2.0 and iBCE-EL classified many of our seroreactive peptides as epitopes, they did not score them significantly higher than the non-reactive tryptic peptides in our study, nor did they assign higher scores to seroreactive peptides from known Ft or Bp antigens, highlighting the need for experimental data instead of relying on computational epitope predictions alone. The present workflow is easily adaptable to detecting peptide targets relevant to the immune systems of other mammalian species, including humans (depending upon the availability of convalescent sera from patients), and could aid in accelerating the discovery of B-cell epitopes and development of vaccines to counter emerging biological threats.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Single-nuclei transcriptome analysis of IgM+ cells isolated from channel catfish (Ictalurus punctatus) spleen

Catfish production is the primary aquaculture sector in the United States, and the key cultured species is channel catfish (Ictalurus punctatus). The major causes of production losses are pathogenic diseases, and the spleen, an important site of adaptive immunity, is implicated in these diseases. To examine the channel catfish immune system, single-nuclei transcriptomes of sorted and captured IgM + cells were produced from adult channel catfish. Three channel catfish (~1 kg) were euthanized, the spleen dissected, and the tissue dissociated. The lymphocytes were isolated using a Ficoll gradient and IgM + cells were then sorted with flow cytometry. The IgM + cells were lysed and single-nuclei libraries generated using a Chromium Next GEM Single Cell 3’ GEM Kit and the Chromium X Instrument (10x Genomics) and sequenced with the Illumina NovaSeq X Plus sequencer. The reads were aligned to theI. punctatusreference assembly (Coco_2.0) using Cell Ranger, and normalization, cluster analysis, and differential gene expression analysis were carried out with Seurat. Across the three samples, approximately 753.5 million reads were generated for 18,686 cells. After filtering, 10,637 cells remained for the cluster analysis. The cluster analysis identified 16 clusters which were classified as B cells (10,276), natural killer-like (NK-like) cells (178), T cells or natural killer cells (45), hematopoietic stem and progenitor cells (HSPC)/megakaryocytes (MK) (66), myeloid/epithelial cells (40), and plasma cells (32). The B cell clusters were further defined as different populations of mature B cells, cycling B cells, and plasma cells. The plasma cells highly expressedighmand we demonstrated that the secreted form of the transcript was largely being expressed by these cells. This atlas provides insight into the gene expression of IgM + immune cells in channel catfish. The atlas is publicly available and could be used garner more important information regarding the gene expression of splenic immune cells.

Immunology↗
DOE OSTI2023

Deoptimization of FMDV P1 Region Results in Robust Serotype-Independent Viral Attenuation

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is a highly contagious disease of cloven-hoofed livestock that can have severe economic impacts. Control and prevention strategies, including the development of improved vaccines, are urgently needed to effectively control FMD outbreaks in endemic settings. Previously, we employed two distinct strategies (codon pair bias deoptimization (CPD) and codon bias deoptimization (CD)) to deoptimize various regions of the FMDV serotype A subtype A12 genome, which resulted in the development of an attenuated virus in vitro and in vivo, inducing varying levels of humoral responses. In the current study, we examined the versatility of the system by using CPD applied to the P1 capsid coding region of FMDV serotype A subtype, A24, and another serotype, Asia1. Viruses carrying recoded P1 (A24-P1Deopt or Asia1-P1Deopt) exhibited different degrees of attenuation (i.e., delayed viral growth kinetics and replication) in cultured cells. Studies in vivo using a mouse model of FMD demonstrated that inoculation with the A24-P1Deopt and Asia1-P1Deopt strains elicited a strong humoral immune response capable of offering protection against challenge with homologous wildtype (WT) viruses. However, different results were obtained in pigs. While clear attenuation was detected for both the A24-P1Deopt and Asia1-P1Deopt strains, only a limited induction of adaptive immunity and protection against challenge was detected, depending on the inoculated dose and serotype deoptimized. Our work demonstrates that while CPD of the P1 coding region attenuates viral strains of multiple FMDV serotypes/subtypes, a thorough assessment of virulence and induction of adaptive immunity in the natural host is required in each case in order to finely adjust the degree of deoptimization required for attenuation without affecting the induction of protective adaptive immune responses.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1973

Actuator digital interface unit (AIU)

Alternate versions of the actuator interface unit are presented. One alternate is a dual-failure immune configuration which feeds a look-and-switch dual-failure immune hydraulic system. The other alternate is a single-failure immune configuration which feeds a majority voting hydraulic system. Both systems communicate with the data bus through data terminals dedicated to each user subsystem. Both operational control data and configuration control information are processed in and out of the subsystem via the data terminal which yields the actuator interface subsystem, self-managing within its failure immunity capability.

Source record↗