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DOE OSTI2025

Dataset for "A Microfluidic Spore Chamber for Long-Term Imaging of Single-Spore Hyphal Development"

Understanding the life cycle of fungal spores is essential for elucidating their roles in pathogenesis, dispersal, and survival. However, studying spore development under controlled, spatially defined conditions remains challenging. Here, we present the Spore Chamber, a custom-built microfluidic platform engineered for parallel trapping and long-term imaging of individual spores under defined media conditions, enabling real-time visualization of hyphal development. Using Aspergillus fumigatus as a model organism, we demonstrate that sparse trapping of individual spores within size-matched trap geometries enables long-term time-lapse imaging of key developmental stages, including germination, polarized hyphal elongation, branching, and conidiophore formation. To assess the device’s capacity to resolve morphogenetic responses to exogenous signals, we introduced lipochitooligosaccharides (LCOs) and short-chain chitooligosaccharides (COs). Rhizobium-derived, non-sulfated LCO (nsLCO) mixtures induced enhanced secondary branching (hyperbranching), a response not previously reported in A. fumigatus under these signal conditions, to our knowledge, whereas sulfated LCOs and CO4 did not significantly alter branching patterns. In addition, long-term confinement and imaging revealed rare developmental morphologies previously described primarily in mutant strains, including split conidiophore formation, elongated phialides, microcyclic conidiation, and chlamydospore development. Together, these results establish the Spore Chamber as a targeted microfluidic platform for single-spore phenotyping and long-term developmental analysis, with applications in fungal biology, chemical signaling studies, and host–microbe interaction research. Videos of the observed phenomena are included in this data set.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Comparative genomic and phenotypic characterization of invasive non-typhoidal Salmonella isolates from Siaya, Kenya

Non-typhoidal Salmonella (NTS) is a major global health concern that often causes bloodstream infections in areas of the world affected by malnutrition and comorbidities such as HIV and malaria. Developing a strategy to control the emergence and spread of highly invasive and antimicrobial resistant NTS isolates requires a comprehensive analysis of epidemiological factors and molecular pathogenesis. Here, we characterize 11 NTS isolates that caused bloodstream infections in pediatric patients in Siaya, Kenya from 2003–2010. Nine isolates were identified as S . Typhimurium sequence type 313 while the other two were S . Enteritidis. Comprehensive genotypic and phenotypic analyses were performed to compare these isolates to those previously identified in sub-Saharan Africa. We identified a S . Typhimurium isolate referred to as UGA14 that displayed novel plasmid, pseudogene and resistance features as compared to other isolates reported from Africa. Notably, UGA14 is able to ferment both lactose and sucrose due to the acquisition of insertion elements on the pKST313 plasmid. These findings show for the first time the co-evolution of plasmid-mediated lactose and sucrose metabolism along with cephalosporin resistance in NTS further elucidating the evolutionary mechanisms of invasive NTS phenotypes. These results further support the use of combined genomic and phenotypic approaches to detect and characterize atypical NTS isolates in order to advance biosurveillance efforts that inform countermeasures aimed at controlling invasive and antimicrobial resistant NTS.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Interaction of amphiphilic lipoarabinomannan with host carrier lipoproteins in tuberculosis patients: Implications for blood-based diagnostics

Lipoarabinomannan (LAM), an amphiphilic lipoglycan of the Mycobacterium tuberculosis cell wall, is a diagnostic target for tuberculosis. Previous work from our laboratory and others suggests that LAM is associated with host serum lipoproteins, which may in turn have implications for diagnostic assays. Our team has developed two serum assays for amphiphile detection: lipoprotein capture and membrane insertion. The lipoprotein capture assay relies on capture of the host lipoproteins, exploiting the biological association of host lipoprotein with microbial amphiphilic biomarkers to “concentrate” LAM. In contrast, the membrane insertion assay is independent of the association between pathogen amphiphiles and host lipoprotein association, and directly captures LAM based on its thermodynamic propensity for association with a supported lipid membrane, which forms the functional surface of an optical biosensor. In this manuscript, we explored the use of these assays for the detection of LAM in sera from adults whose tuberculosis status had been well-characterized using conventional microbiological tests, and endemic controls. Using the lipoprotein capture assay, LAM signal/noise ratios were >1.0 in 29/35 (83%) individuals with culture-confirmed active tuberculosis, 8/13 (62%) individuals with tuberculosis symptoms, but no positive culture for M . tuberculosis , and 0/6 (0%) symptom-free endemic controls. To evaluate serum LAM levels without bias associated with potential differences in circulating host lipoprotein concentrations between individuals, we subsequently processed available samples to liberate LAM from associated host lipoprotein assemblies followed by direct detection of the pathogen biomarker using the membrane insertion approach. Using the membrane insertion assay, signal/noise for detection of serum LAM was greater than that observed using the lipoprotein capture method for culture-confirmed TB patients (6/6), yet remained negative for controls (2/2). Taken together, these results suggest that detection of serum LAM is a promising TB diagnostic approach, but that further work is required to optimize assay performance and to decipher the implications of LAM/host lipoprotein associations for diagnostic assay performance and TB pathogenesis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Association of microbial dynamics with urinary estrogens and estrogen metabolites in patients with endometriosis

Endometriosis is an estrogen dependent gynecological disease associated with altered microbial phenotypes. The association among endogenous estrogen, estrogen metabolites, and microbial dynamics on disease pathogenesis has not been fully investigated. Here, we identified estrogen metabolites as well as microbial phenotypes in non-diseased patients (n = 9) and those with pathologically confirmed endometriosis (P-EOSIS, n = 20), on day of surgery (DOS) and ~1–3 weeks post-surgical intervention (PSI). Then, we examined the effects of surgical intervention with or without hormonal therapy (OCPs) on estrogen and microbial profiles of both study groups. For estrogen metabolism analysis, liquid chromatography/tandem mass spectrometry was used to quantify urinary estrogens. The microbiome data assessment was performed with Next generation sequencing to V4 region of 16S rRNA. Surgical intervention and hormonal therapy altered gastrointestinal (GI), urogenital (UG) microbiomes, urinary estrogen and estrogen metabolite levels in P-EOSIS. At DOS, 17β-estradiol was enhanced in P-EOSIS treated with OCPs. At PSI, 16-keto-17β-estradiol was increased in P-EOSIS not receiving OCPs while 2-hydroxyestradiol and 2-hydroxyestrone were decreased in P-EOSIS receiving OCPs. GI bacterial α-diversity was greater for controls and P-EOSIS that did not receive OCPs. P-EOSIS not utilizing OCPs exhibited a decrease in UG bacterial α-diversity and differences in dominant taxa, while P-EOSIS utilizing OCPs had an increase in UG bacterial α-diversity. P-EOSIS had a strong positive correlation between the GI/UG bacteria species and the concentrations of urinary estrogen and its metabolites. These results indicate an association between microbial dysbiosis and altered urinary estrogens in P-EOSIS, which may impact disease progression.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Biophysical and biochemical evidence for the role of acetate kinases (AckAs) in an acetogenic pathway in pathogenic spirochetes

Unraveling the metabolism of Treponema pallidum is a key component to understanding the pathogenesis of the human disease that it causes, syphilis. For decades, it was assumed that glucose was the sole carbon/energy source for this parasitic spirochete. But the lack of citric-acid-cycle enzymes suggested that alternative sources could be utilized, especially in microaerophilic host environments where glycolysis should not be robust. Recent bioinformatic, biophysical, and biochemical evidence supports the existence of an acetogenic energy-conservation pathway in T . pallidum and related treponemal species. In this hypothetical pathway, exogenous D-lactate can be utilized by the bacterium as an alternative energy source. Herein, we examined the final enzyme in this pathway, acetate kinase (named TP0476), which ostensibly catalyzes the generation of ATP from ADP and acetyl-phosphate. We found that TP0476 was able to carry out this reaction, but the protein was not suitable for biophysical and structural characterization. We thus performed additional studies on the homologous enzyme (75% amino-acid sequence identity) from the oral pathogen Treponema vincentii , TV0924. This protein also exhibited acetate kinase activity, and it was amenable to structural and biophysical studies. We established that the enzyme exists as a dimer in solution, and then determined its crystal structure at a resolution of 1.36 Å, showing that the protein has a similar fold to other known acetate kinases. Mutation of residues in the putative active site drastically altered its enzymatic activity. A second crystal structure of TV0924 in the presence of AMP (at 1.3 Å resolution) provided insight into the binding of one of the enzyme’s substrates. On balance, this evidence strongly supported the roles of TP0476 and TV0924 as acetate kinases, reinforcing the hypothesis of an acetogenic pathway in pathogenic treponemes.

Deka, Ranjit K.↗

DOE OSTI2021

The N-terminus of varicella-zoster virus glycoprotein B has a functional role in fusion

Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae , including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native, full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109 KSQD 112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109 AAAA 112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae .

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

MAVS mediates a protective immune response in the brain to Rift Valley fever virus

Rift Valley fever virus (RVFV) is a highly pathogenic mosquito-borne virus capable of causing hepatitis, encephalitis, blindness, hemorrhagic syndrome, and death in humans and livestock. Upon aerosol infection with RVFV, the brain is a major site of viral replication and tissue damage, yet pathogenesis in this organ has been understudied. Here, we investigated the immune response in the brain of RVFV infected mice. In response to infection, microglia initiated robust transcriptional upregulation of antiviral immune genes, as well as increased levels of activation markers and cytokine secretion that is dependent on mitochondrial antiviral-signaling protein (MAVS) and independent of toll-like receptors 3 and 7. In vivo , Mavs -/- mice displayed enhanced susceptibility to RVFV as determined by increased brain viral burden and higher mortality. Single-cell RNA sequence analysis identified defects in type I interferon and interferon responsive gene expression within microglia in Mavs -/- mice, as well as dysregulated lymphocyte infiltration. The results of this study provide a crucial step towards understanding the precise molecular mechanisms by which RVFV infection is controlled in the brain and will help inform the development of vaccines and antiviral therapies that are effective in preventing encephalitis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

CovS inactivation reduces CovR promoter binding at diverse virulence factor encoding genes in group A Streptococcus

The control of virulence gene regulator (CovR), also called caspsule synthesis regulator (CsrR), is critical to how the major human pathogen group A Streptococcus fine-tunes virulence factor production. CovR phosphorylation (CovR~P) levels are determined by its cognate sensor kinase CovS, and functional abrogating mutations in CovS can occur in invasive GAS isolates leading to hypervirulence. Presently, the mechanism of CovR-DNA binding specificity is unclear, and the impact of CovS inactivation on global CovR binding has not been assessed. Thus, we performed CovR chromatin immunoprecipitation sequencing (ChIP-seq) analysis in the emm1 strain MGAS2221 and its CovS kinase deficient derivative strain 2221-CovS-E281A. We identified that CovR bound in the promoter regions of nearly all virulence factor encoding genes in the CovR regulon. Additionally, direct CovR binding was observed for numerous genes encoding proteins involved in amino acid metabolism, but we found limited direct CovR binding to genes encoding other transcriptional regulators. The consensus sequence AATRANAAAARVABTAAA was present in the promoters of genes directly regulated by CovR, and mutations of highly conserved positions within this motif relieved CovR repression of the hasA and MGAS2221_0187 promoters. Analysis of strain 2221-CovS-E281A revealed that binding of CovR at repressed, but not activated, promoters is highly dependent on CovR~P state. CovR repressed virulence factor encoding genes could be grouped dependent on how CovR~P dependent variation in DNA binding correlated with gene transcript levels. Taken together, the data show that CovR repression of virulence factor encoding genes is primarily direct in nature, involves binding to a newly-identified DNA binding motif, and is relieved by CovS inactivation. These data provide new mechanistic insights into one of the most important bacterial virulence regulators and allow for subsequent focused investigations into how CovR-DNA interaction at directly controlled promoters impacts GAS pathogenesis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

COVID-19 through Adverse Outcome Pathways: Building networks to better understand the disease – 3rd CIAO AOP Design Workshop

On April 28-29, 2021, 50 scientists from different fields of expertise met for the 3rd online CIAO workshop. The CIAO project “Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway (AOP) framework” aims at building a holistic assembly of the available scientific knowledge on COVID-19 using the AOP framework. An individual AOP depicts the disease progression from the initial contact with the SARS-CoV-2 virus through biological key events (KE) toward an adverse outcome such as respiratory distress, anosmia or multiorgan failure. Assembling the individual AOPs into a network highlights shared KEs as central biological nodes involved in multiple outcomes observed in COVID-19 patients. During the workshop, the KEs and AOPs established so far by the CIAO members were presented and positioned on a timeline of the disease course. Modulating factors influencing the progression and severity of the disease were also addressed as well as factors beyond purely biological phenomena. CIAO relies on an interdisciplinary crowdsourcing effort, therefore, approaches to expand the CIAO network by widening the crowd and reaching stakeholders were also discussed. To conclude the workshop, it was decided that the AOPs/KEs will be further consolidated, integrating virus variants and long COVID when relevant, while an outreach campaign will be launched to broaden the CIAO scientific crowd.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Impaired cardiac glycolysis and glycogen depletion are linked to poor myocardial outcomes in juvenile male swine with metabolic syndrome and ischemia

Abstract Obesity continues to rise in the juveniles and obese children are more likely to develop metabolic syndrome (MetS) and related cardiovascular disease. Unfortunately, effective prevention and long‐term treatment options remain limited. We determined the juvenile cardiac response to MetS in a swine model. Juvenile male swine were fed either an obesogenic diet, to induce MetS, or a lean diet, as a control (LD). Myocardial ischemia was induced with surgically placed ameroid constrictor on the left circumflex artery. Physiological data were recorded and at 22 weeks of age the animals underwent a terminal harvest procedure and myocardial tissue was extracted for total metabolic and proteomic LC/MS–MS, RNA‐seq analysis, and data underwent nonnegative matrix factorization for metabolic signatures. Significantly altered in MetS versus. LD were the glycolysis‐related metabolites and enzymes. In MetS compared with LD Glycogen synthase 1 (GYS1)‐glycogen phosphorylases (PYGM/PYGL) expression disbalance resulted in a loss of myocardial glycogen. Our findings are consistent with the concept that transcriptionally driven myocardial changes in glycogen and glucose metabolism‐related enzymes lead to a deficiency of their metabolite products in MetS. This abnormal energy metabolism provides insight into the pathogenesis of the juvenile heart in MetS. This study reveals that MetS and ischemia diminishes ATP availability in the myocardium via altering the glucose‐G6P‐pyruvate axis at the level of metabolites and gene expression of related enzymes. The observed severe glycogen depletion in MetS coincides with disbalance in expression of GYS1 and both PYGM and PYGL. This altered energy substrate metabolism is a potential target of pharmacological agents for improving juvenile myocardial function in MetS and ischemia.

Broadwin, Mark↗

DOE OSTI2023

Association of Serum Bile Acid and Unsaturated Fatty Acid Profiles with the Risk of Diabetic Retinopathy in Type 2 Diabetic Patients

Aim: We aimed to identify the ability of serum bile acids (BAs) and unsaturated fatty acids (UFAs) profiles to predict the development of diabetic retinopathy (DR) in type 2 diabetes mellitus (T2DM) patients. Methods: We first used univariate and multivariate analysis to compare 15 serum BA and 11 UFA levels in healthy control (HC) group (n = 82), T2DM patients with DR (n = 58) and T2DM patients without DR (n = 60). Forty T2DM patients were considered for validation. Then, the receiver operating characteristic curve (ROC) and decision curve analysis were used to assess the diagnostic value and clinical benefit of serum biomarkers alone, clinical variables alone or in combination, and the area under the curve (AUC), integrated discrimination improvement (IDI), and net reclassification improvement (NRI) were used to further assess whether the addition of biomarkers significantly improved the predictive ability of the model. Results: Orthogonal partial least squares-discriminant analysis (OPLS-DA) of serum BAs and UFAs separated the three cohorts including HC, T2DM patients with or without DR. The difference in serum BA and UFA profiles of T2DM patients with or without DR was mainly manifested in the three metabolites of taurolithocholic acid (TLCA), tauroursodeoxycholic acid (TUDCA) and arachidonic acid (AA). Together, they had an AUC of 0.785 (0.918 for validation cohort) for predicting DR in T2DM patients. After adjusting for numerous confounding factors, TLCA, TUDCA, and AA were independent predictors that differentiated T2DM with or without DR. The results of AUC, IDI, and NRI demonstrated that adding these three biomarkers to a model with clinical variables statistically increased their predictive value and were replicated in our independent validation cohort. Conclusion: These findings highlight the association of three metabolites, TLCA, TUDCA and AA, with DR and may indicate their potential value in the pathogenesis of DR.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Altered ß-cell Prohormone Processing and Secretion in Type 1 Diabetes

Analysis of data from clinical cohorts and more recently from human pancreatic tissue, indicate that defects in prohormone processing are an early and persistent component of type 1 diabetes pathogenesis. In this Perspectives article, we review the current state of knowledge of alterations in islet prohormone expression and processing in type 1 diabetes, and consider the clinical impact of these findings. Lingering questions, including pathologic etiologies and consequences of altered prohormone expression and secretion in type 1 diabetes, and the natural history of circulating prohormone production in health and disease are considered. Finally, key steps required to move forward in this area are outlined, including longitudinal testing of relevant clinical populations, studies that probe the genetics of altered prohormone processing, the need for combined functional and histologic testing of human pancreatic tissues, continued interrogation of the intersection between prohormone processing and autoimmunity, and optimal assays or approaches for analysis. Successful resolution of these questions may offer the potential to use altered prohormone processing as a pathogenic anchor that can be used to cluster different endotypes enveloping a large set of variables, and inform therapeutic strategies aimed at personalized intervention in the natural history of type 1 diabetes.

Islets, Insulin, Proinsulin, Islet Amyloid Polypep↗

DOE OSTI2022

Polyunsaturated Fatty Acids Mend Macrophage Transcriptome, Glycome, and Phenotype in the Patients with Neurodegenerative Diseases, Including Alzheimer’s Disease

Macrophages of healthy subjects have a pro-resolution phenotype, upload amyloid-β (Aβ) into endosomes, and degrade Aβ, whereas macrophages of patients with Alzheimer’s disease (AD) generally have a pro-inflammatory phenotype and lack energy for brain clearance of Aβ. To clarify the pathogenesis of sporadic AD and therapeutic effects of polyunsaturated fatty acids (PUFA) with vitamins B and D and antioxidants on monocyte/macrophage (MM) migration in the AD brain, MM transcripts in energy and Aβ degradation, MM glycome, and macrophage clearance of Aβ. We followed for 31.3 months (mean) ten PUFA-supplemented neurodegenerative patients: 3 with subjective cognitive impairment (SCI), 2 with mild cognitive impairment (MCI), 3 MCI/vascular cognitive impairment, 2 with dementia with Lewy bodies, and 7 non-supplemented caregivers. We examined: monocyte migration in the brain and a blood-brain barrier model by immunochemistry and electron microscopy; macrophage transcriptome by RNAseq; macrophage glycome by N-glycan profiling and LTQ-Orbitrap mass spectrometry; and macrophage phenotype and phagocytosis by immunofluorescence. MM invade Aβ plaques, upload but do not degrade Aβ, and release Aβ into vessels, which develop cerebrovascular amyloid angiopathy (CAA); PUFA upregulate energy and Aβ degradation enzyme transcripts in macrophages; PUFA enhance sialylated N-glycans in macrophages; PUFA reduce oxidative stress and increase pro-resolution MM phenotype, mitochondrial membrane potential, and Aβ phagocytosis (p < 0.001). Macrophages of SCI, MCI, and AD patients have interrelated defects in the transcriptome, glycome, Aβ phagocytosis, and Aβ degradation. PUFA mend macrophage transcriptome, enrich glycome, enhance Aβ clearance, and benefit the cognition of early-stage AD patients.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

The multifaceted role of c-di-AMP signaling in the regulation of Porphyromonas gingivalis lipopolysaccharide structure and function

This study unveils the intricate functional association between cyclic di-3’,5’-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLAT n . We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Astronauts Plasma-Derived Exosomes Induced Aberrant EZH2-Mediated H3K27me3 Epigenetic Regulation of the Vitamin D Receptor

There are unique stressors in the spaceflight environment. Exposure to such stressors may be associated with adverse effects on astronauts' health, including increased cancer and cardiovascular disease risks. Small extracellular vesicles (sEVs, i.e., exosomes) play a vital role in intercellular communication and regulate various biological processes contributing to their role in disease pathogenesis. To assess whether spaceflight alters sEVs transcriptome profile, sEVs were isolated from the blood plasma of 3 astronauts at two different time points: 10 days before launch (L-10) and 3 days after return (R+3) from the Shuttle mission. AC16 cells (human cardiomyocyte cell line) were treated with L-10 and R+3 astronauts-derived exosomes for 24 h. Total RNA was isolated and analyzed for gene expression profiling using Affymetrix microarrays. Enrichment analysis was performed using Enrichr. Furthermore, transcription factor (TF) enrichment analysis using the ENCODE/ChEA Consensus TF database identified gene sets related to the polycomb repressive complex 2 (PRC2) and Vitamin D receptor (VDR) in AC16 cells treated with R+3 compared to cells treated with L-10 astronauts-derived exosomes. Further analysis of the histone modifications using datasets from the Roadmap Epigenomics Project confirmed enrichment in gene sets related to the H3K27me3 repressive mark. Interestingly, analysis of previously published H3K27me3–chromatin immunoprecipitation sequencing (ChIP-Seq) ENCODE datasets showed enrichment of H3K27me3 in the VDR promoter. Collectively, our results suggest that astronaut-derived sEVs may epigenetically repress the expression of the VDR in human adult cardiomyocytes by promoting the activation of the PRC2 complex and H3K27me3 levels.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Spaceflight-Associated Changes of snoRNAs in Peripheral Blood Mononuclear Cells and Plasma Exosomes—A Pilot Study

During spaceflight, astronauts are exposed to various physiological and psychological stressors that have been associated with adverse health effects. Therefore, there is an unmet need to develop novel diagnostic tools to predict early alterations in astronauts’ health. Small nucleolar RNA (snoRNA) is a type of short non-coding RNA (60–300 nucleotides) known to guide 2'-O-methylation (Nm) or pseudouridine (ψ) of ribosomal RNA (rRNA), small nuclear RNA (snRNA), or messenger RNA (mRNA). Emerging evidence suggests that dysregulated snoRNAs may be key players in regulating fundamental cellular mechanisms and in the pathogenesis of cancer, heart, and neurological disease. Therefore, we sought to determine whether the spaceflight-induced snoRNA changes in astronaut’s peripheral blood (PB) plasma extracellular vesicles (PB-EV) and peripheral blood mononuclear cells (PBMCs). Using unbiased small RNA sequencing (sRNAseq), we evaluated changes in PB-EV snoRNA content isolated from astronauts (n = 5/group) who underwent median 12-day long Shuttle missions between 1998 and 2001. Using stringent cutoff (fold change > 2 or log 2 -fold change >1, FDR < 0.05), we detected 21 down-and 9—up-regulated snoRNAs in PB-EVs 3 days after return (R + 3) compared to 10 days before launch (L-10). qPCR validation revealed that SNORA74A was significantly down-regulated at R + 3 compared to L-10. We next determined snoRNA expression levels in astronauts’ PBMCs at R + 3 and L-10 (n = 6/group). qPCR analysis further confirmed a significant increase in SNORA19 and SNORA47 in astronauts’ PBMCs at R + 3 compared to L-10. Notably, many downregulated snoRNA-guided rRNA modifications, including four Nms and five ψs. Our findings revealed that spaceflight induced changes in PB-EV and PBMCs snoRNA expression, thus suggesting snoRNAs may serve as potential novel biomarkers for monitoring astronauts’ health.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

Single-cell RNA-Seq reveals changes in immune landscape in post-traumatic osteoarthritis

Osteoarthritis (OA) is the most common joint disease, affecting over 300 million people world-wide. Accumulating evidence attests to the important roles of the immune system in OA pathogenesis. Understanding the role of various immune cells in joint degeneration or joint repair after injury is vital for improving therapeutic strategies for treating OA. Post-traumatic osteoarthritis (PTOA) develops in ~50% of individuals who have experienced an articular trauma like an anterior cruciate ligament (ACL) rupture. Here, using the high resolution of single-cell RNA sequencing, we delineated the temporal dynamics of immune cell accumulation in the mouse knee joint after ACL rupture. Our study identified multiple immune cell types in the joint including neutrophils, monocytes, macrophages, B cells, T cells, NK cells and dendritic cells. Monocytes and macrophage populations showed the most dramatic changes after injury. Further characterization of monocytes and macrophages reveled 9 major subtypes with unique transcriptomics signatures, including a tissue resident Lyve1 hi Folr2 hi macrophage population and Trem2 hi Fcrls + recruited macrophages, both showing enrichment for phagocytic genes and growth factors such as Igf1 , Pdgfa and Pdgfc. We also identified several genes induced or repressed after ACL injury in a cell type-specific manner. This study provides new insight into PTOA-associated changes in the immune microenvironment and highlights macrophage subtypes that may play a role in joint repair after injury.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

In-vivo neuronal dysfunction by Aβ and tau overlaps with brain-wide inflammatory mechanisms in Alzheimer’s disease

The molecular mechanisms underlying neuronal dysfunction in Alzheimer’s disease (AD) remain uncharacterized. Here, we identify genes, molecular pathways and cellular components associated with whole-brain dysregulation caused by amyloid-beta (Aβ) and tau deposits in the living human brain. We obtained in-vivo resting-state functional MRI (rs-fMRI), Aβ- and tau-PET for 47 cognitively unimpaired and 16 AD participants from the Translational Biomarkers in Aging and Dementia cohort. Adverse neuronal activity impacts by Aβ and tau were quantified with personalized dynamical models by fitting pathology-mediated computational signals to the participant’s real rs-fMRIs. Then, we detected robust brain-wide associations between the spatial profiles of Aβ-tau impacts and gene expression in the neurotypical transcriptome (Allen Human Brain Atlas). Within the obtained distinctive signature of in-vivo neuronal dysfunction, several genes have prominent roles in microglial activation and in interactions with Aβ and tau. Moreover, cellular vulnerability estimations revealed strong association of microglial expression patterns with Aβ and tau’s synergistic impact on neuronal activity (q < 0.001). These results further support the central role of the immune system and neuroinflammatory pathways in AD pathogenesis. Neuronal dysregulation by AD pathologies also associated with neurotypical synaptic and developmental processes. In addition, we identified drug candidates from the vast LINCS library to halt or reduce the observed Aβ-tau effects on neuronal activity. Top-ranked pharmacological interventions target inflammatory, cancer and cardiovascular pathways, including specific medications undergoing clinical evaluation in AD. Our findings, based on the examination of molecular-pathological-functional interactions in humans, may accelerate the process of bringing effective therapies into clinical practice.

60 APPLIED LIFE SCIENCES↗