bims-traimu Biomed News
on Trained immunity
Issue of 2024–05–12
fourteen papers selected by
Yantong Wan, Southern Medical University
  1. Navigating the crossroads: asthma, trained immunity, and infection susceptibility.
  2. BCG activation of trained immunity is associated with induction of cross reactive COVID-19 antibodies in a BCG vaccinated population.
  3. Inflammatory Memory in Chronic Skin Disease.
  4. Modulation of innate immune response to mRNA vaccination after SARS-CoV-2 infection or sequential vaccination in humans.
  5. Genetic variation in IL-4 activated tissue resident macrophages alters the epigenetic state to determine strain specific synergistic responses to LPS.
  6. Maintenance of homeostasis by TLR4 ligands.
  7. GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.
  8. MiR-106a-5p targets PFKFB3 and improves sepsis through regulating macrophage pyroptosis and inflammatory response.
  9. Itaconate protects ferroptotic neurons by alkylating GPx4 post stroke.
  10. A comprehensive genetic map of cytokine responses in Lyme borreliosis.
  11. E4BP4 in macrophages induces an anti-inflammatory phenotype that ameliorates the severity of colitis.
  12. NLRP6 negatively regulates host defense against polymicrobial sepsis.
  13. The immune landscape of sepsis and using immune clusters for identifying sepsis endotypes.
  14. Itaconic acid inhibits nontuberculous mycobacterial growth in pH dependent manner while 4-octyl-itaconic acid enhances THP-1 clearance of nontuberculous mycobacteria in vitro.
  1. Expert Rev Clin Immunol. 2024 May 09. 1-3
    Navigating the crossroads: asthma, trained immunity, and infection susceptibility.
    Ahmad Z Al Meslamani, Eman Merghani Ali.
      
    Keywords:  Asthma; infections; microbiome; precision medicine; trained immunity
    DOI:  https://doi.org/10.1080/1744666X.2024.2353743
  2. PLoS One. 2024 ;19(5): e0302722
    BCG activation of trained immunity is associated with induction of cross reactive COVID-19 antibodies in a BCG vaccinated population.
    Najeeha Talat Iqbal, Kumail Ahmed, Tehniat Sattar, Fatima Aziz, Rabia Hussain.
       BACKGROUND: Pakistan is endemic to a diverse set of parasitic, mycobacterial and viral diseases. The recognition of BCG Trained Immunity (TI) led us to postulate that the continued presence of BCG-TI may play a protective role, previously reported for both infectious and noninfectious conditions. Most of the previous studies have addressed the issue of BCG-TI in the paediatric populations. This study addressed the key issue of maintenance of BCG-TI in a wider age range (adolescent and adults) to identify the strength and quality of the immune responses.
    OBJECTIVE: To assess the BCG-induced recall responses in healthy individuals by cytokines secreted from the TI network and its potential role in providing cross-protection against COVID-19 and other viral infections.
    STUDY DESIGN: In this cross-sectional study, healthy young adults and adolescents (n = 20) were recruited from 16-40 years of age, with no prior history of TB treatment, autoimmune, or chronic inflammatory condition.
    METHODS: BCG-induced cytokine responses were assessed using prototypic markers for cells of the TI network [macrophages [M1 (TNFα, IFNγ), M2 (IL10)], NK (IL2), Gamma delta (γδ) T (IL17, IL4)] and SARS CoV2 IgG antibodies against RBD using short-term (12 hrs.) cultures assay.
    RESULTS: Significant differences were observed in the magnitude of recall responses to BCG with macrophage cytokines showing the highest mean levels of TNFα (9148 pg/ml) followed by IL10 (488 pg/ml) and IFNγ (355 pg/ml). The ratio of unstimulated vs.BCG-stimulated cytokines was 132 fold higher for TNFα, 40 fold fo r IL10, and 27 fold for IFNγ. Furthermore, SARS-CoV-2 antibodies were also detected in unstimulated plasma which showed cross reactivity with BCG.
    CONCLUSION: The presence of cross reactive antibodies to SARS-CoV-2 and the relative ratio of pro- and anti-inflammatory cytokines secreted by activated TI cellular network may play a pivotal role in protection in the early stages of infection as observed during the COVID-19 pandemic in the younger age groups resulting in lower morbidity and mortality.
    DOI:  https://doi.org/10.1371/journal.pone.0302722
  3. JID Innov. 2024 May;4(3): 100277
    Inflammatory Memory in Chronic Skin Disease.
    Joseph A Daccache, Shruti Naik.
      Inflammation is a hallmark of remitting-relapsing dermatological diseases. Although a large emphasis has been placed on adaptive immune cells as mediators of relapse, evidence in epithelial and innate immune biology suggests that disease memory is widespread. In this study, we bring to the fore the concept of inflammatory memory or nonspecific training of long-lived cells in the skin, highlighting the epigenetic and other mechanisms that propagate memory at the cellular level. We place these findings in the context of psoriasis, a prototypic flaring disease known to have localized memory, and underscore the importance of targeting memory to limit disease flares.
    Keywords:  Epigenetics; Inflammatory memory; Psoriasis
    DOI:  https://doi.org/10.1016/j.xjidi.2024.100277
  4. JCI Insight. 2024 May 08. pii: e175401. [Epub ahead of print]9(9):
    Modulation of innate immune response to mRNA vaccination after SARS-CoV-2 infection or sequential vaccination in humans.
    Fredrika Hellgren, Anja Rosdahl, Rodrigo Arcoverde Cerveira, Klara Lenart, Sebastian Ols, Yong-Dae Gwon, Seta Kurt, Anna Maria Delis, Gustav Joas, Magnus Evander, Johan Normark, Clas Ahlm, Mattias Ne Forsell, Sara Cajander, Karin Loré.
      mRNA vaccines are likely to become widely used for the prevention of infectious diseases in the future. Nevertheless, a notable gap exists in mechanistic data, particularly concerning the potential effects of sequential mRNA immunization or preexisting immunity on the early innate immune response triggered by vaccination. In this study, healthy adults, with or without documented prior SARS-CoV-2 infection, were vaccinated with the BNT162b2/Comirnaty mRNA vaccine. Prior infection conferred significantly stronger induction of proinflammatory and type I IFN-related gene signatures, serum cytokines, and monocyte expansion after the prime vaccination. The response to the second vaccination further increased the magnitude of the early innate response in both study groups. The third vaccination did not further increase vaccine-induced inflammation. In vitro stimulation of PBMCs with TLR ligands showed no difference in cytokine responses between groups, or before or after prime vaccination, indicating absence of a trained immunity effect. We observed that levels of preexisting antigen-specific CD4 T cells, antibody, and memory B cells correlated with elements of the early innate response to the first vaccination. Our data thereby indicate that preexisting memory formed by infection may augment the innate immune activation induced by mRNA vaccines.
    Keywords:  Adaptive immunity; Immunology; Innate immunity; Vaccines
    DOI:  https://doi.org/10.1172/jci.insight.175401
  5. Res Sq. 2024 Jan 18. pii: rs.3.rs-3759654. [Epub ahead of print]
    Genetic variation in IL-4 activated tissue resident macrophages alters the epigenetic state to determine strain specific synergistic responses to LPS.
    Png Loke, Mingming Zhao, Dragana Jankovic, Katherine Hornick, Verena Link, Camila Oliveira Silva Souza, Yasmine Belkaid, Justin Lack.
      How macrophages in the tissue environment integrate multiple stimuli will depend on the genetic background of the host, but this is poorly understood. Here, we investigated C57BL/6 and BALB/c strain specific in vivo IL-4 activation of tissue-resident macrophages (TRMs) from the peritoneal cavity. C57BL/6 TRMs are more transcriptionally responsive to IL-4 stimulation, with a greater association of induced genes with super enhancers, induced enhancers, and topologically associating domains (TAD) boundaries. IL-4-directed epigenomic remodeling revealed BL/6 specific enrichment of NF-κB, IRF, and STAT motifs. Additionally, IL-4-activated BL/6 TRMs demonstrated an augmented synergistic response upon in vitro lipopolysaccharide (LPS) exposure compared to BALB/c TRMs, despite naïve BALB/c TRMs displaying a more robust transcriptional response to LPS than naïve BL/6 TRMs. Single-cell RNA sequencing (scRNA-seq) analysis of mixed bone marrow chimeric mice indicated that transcriptional differences between BL/6 and BALB/c TRMs, and synergy between IL-4 and LPS, are cell intrinsic within the same tissue environment. Hence, genetic variation alters IL-4-induced cell intrinsic epigenetic reprogramming resulting in strain specific synergistic responses to LPS exposure.
    DOI:  https://doi.org/10.21203/rs.3.rs-3759654/v1
  6. Front Immunol. 2024 ;15 1286270
    Maintenance of homeostasis by TLR4 ligands.
    Masataka Oda, Hirofumi Yamamoto, Takashige Kawakami.
      Immunotherapy is renowned for its capacity to elicit anti-infective and anti-cancer effects by harnessing immune responses to microbial components and bolstering innate healing mechanisms through a cascade of immunological reactions. Specifically, mammalian Toll-like receptors (TLRs) have been identified as key receptors responsible for detecting microbial components. The discovery of these mammalian Toll-like receptors has clarified antigen recognition by the innate immune system. It has furnished a molecular foundation for comprehending the interplay between innate immunity and its anti-tumor or anti-infective capabilities. Moreover, accumulating evidence highlights the crucial role of TLRs in maintaining tissue homeostasis. It has also become evident that TLR-expressing macrophages play a central role in immunity by participating in the clearance of foreign substances, tissue repair, and the establishment of new tissue. This macrophage network, centered on macrophages, significantly contributes to innate healing. This review will primarily delve into innate immunity, specifically focusing on substances targeting TLR4.
    Keywords:  TLR4 ligands; innate immunity; macrophage; macrophage network; self-healing ability
    DOI:  https://doi.org/10.3389/fimmu.2024.1286270
  7. Respir Res. 2024 May 09. 25(1): 201
    GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.
    Shasha Lu, Ranran Li, Yunxin Deng, Ju Bai, Bangqi Ji, Yufeng Chu, Yan Xu, Hongping Qu, Xiaosun Guo, Pibao Li, Mei Meng.
      Growth differentiation factor 15 (GDF15) as a stress response cytokine is involved in the development and progression of several diseases associated with metabolic disorders. However, the regulatory role and the underlying mechanisms of GDF15 in sepsis remain poorly defined. Our study analyzed the levels of GDF15 and its correlations with the clinical prognosis of patients with sepsis. In vivo and in vitro models of sepsis were applied to elucidate the role and mechanisms of GDF15 in sepsis-associated lung injury. We observed strong correlations of plasma GDF15 levels with the levels of C-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), and lactate as well as Sequential Organ Failure Assessment (SOFA) scores in patients with sepsis. In the mouse model of lipopolysaccharide-induced sepsis, recombinant GDF15 inhibited the proinflammatory responses and alleviated lung tissue injury. In addition, GDF15 decreased the levels of cytokines produced by alveolar macrophages (AMs). The anti-inflammatory effect of glycolysis inhibitor 2-DG on AMs during sepsis was mediated by GDF15 via inducing the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) and the expression of activating transcription factor 4 (ATF4). Furthermore, we explored the mechanism underlying the beneficial effects of GDF15 and found that GDF15 inhibited glycolysis and mitogen-activated protein kinases (MAPK)/nuclear factor-κB (NF-κB) signaling via promoting AMPK phosphorylation. This study demonstrated that GDF15 inhibited glycolysis and NF-κB/MAPKs signaling via activating AMP-activated protein kinase (AMPK), thereby alleviating the inflammatory responses of AMs and sepsis-associated lung injury. Our findings provided new insights into novel therapeutic strategies for treating sepsis.
    Keywords:  Alveolar macrophage; Glycolysis; Growth differentiation factor 15(GDF15); Inflammation; sepsis
    DOI:  https://doi.org/10.1186/s12931-024-02824-z
  8. J Biol Chem. 2024 May 03. pii: S0021-9258(24)01835-0. [Epub ahead of print] 107334
    MiR-106a-5p targets PFKFB3 and improves sepsis through regulating macrophage pyroptosis and inflammatory response.
    Yixin Chen, Ping Zhang, Fangwei Han, Yanying Zhou, Juexian Wei, Cailing Wang, Mingchuan Song, Shaopeng Lin, Yiming Xu, Xiaohui Chen.
      The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) is a critical regulator of glycolysis and plays a key role in modulating the inflammatory response, thereby contributing to the development of inflammatory diseases such as sepsis. Despite its importance, the development of strategies to target PFKFB3 in the context of sepsis remains challenging. In this study, we employed a microRNA-based approach to decrease PFKFB3 expression. Through multiple meta-analyses, we observed a downregulation of miR-106a-5p expression and an upregulation of PFKFB3 expression in clinical sepsis samples. These changes were also confirmed in blood monocytes from patients with early sepsis and from a mouse model of lipopolysaccharide (LPS)-induced sepsis. Overexpression of miR-106a-5p significantly decreased the LPS-induced increase in glycolytic capacity, inflammatory response, and pyroptosis in macrophages. Mechanistically, we identified PFKFB3 as a direct target protein of miR-106a-5p and demonstrated its essential role in LPS-induced pyroptosis and inflammatory response in macrophages. Furthermore, treatment with agomir-miR-106a-5p conferred a protective effect in an LPS mouse model of sepsis, but this effect was attenuated in myeloid-specific Pfkfb3 knockout mice. These findings indicate that miR-106a-5p inhibits macrophage pyroptosis and inflammatory response in sepsis by regulating PFKFB3-mediated glucose metabolism, representing a potential therapeutic option for the treatment of sepsis.
    Keywords:  PFKFB3; glycolysis; inflammation; macrophage; miRNA-106a-5p; pyroptosis; sepsis
    DOI:  https://doi.org/10.1016/j.jbc.2024.107334
  9. Cell Death Differ. 2024 May 08.
    Itaconate protects ferroptotic neurons by alkylating GPx4 post stroke.
    Chao Wei, Zhongnan Xiao, Yanling Zhang, Zhaoli Luo, Dongyang Liu, Liye Hu, Danmin Shen, Meng Liu, Lei Shi, Xiaotong Wang, Ting Lan, Qingqing Dai, Jing Liu, Wen Chen, Yurui Zhang, Qingyu Sun, Weihua Wu, Peipei Wang, Chenguang Zhang, Junchi Hu, Chu Wang, Fei Yang, Qian Li.
      Neuronal ferroptosis plays a key role in neurologic deficits post intracerebral hemorrhage (ICH). However, the endogenous regulation of rescuing ferroptotic neurons is largely unexplored. Here, we analyzed the integrated alteration of metabolomic landscape after ICH using LC-MS and MALDI-TOF/TOF MS, and demonstrated that aconitate decarboxylase 1 (Irg1) and its product itaconate, a derivative of the tricarboxylic acid cycle, were protectively upregulated. Deficiency of Irg1 or depletion of neuronal Irg1 in striatal neurons was shown to exaggerate neuronal loss and behavioral dysfunction in an ICH mouse model using transgenic mice. Administration of 4-Octyl itaconate (4-OI), a cell-permeable itaconate derivative, and neuronal Irg1 overexpression protected neurons in vivo. In addition, itaconate inhibited ferroptosis in cortical neurons derived from mouse and human induced pluripotent stem cells in vitro. Mechanistically, we demonstrated that itaconate alkylated glutathione peroxidase 4 (GPx4) on its cysteine 66 and the modification allosterically enhanced GPx4's enzymatic activity by using a bioorthogonal probe, itaconate-alkyne (ITalk), and a GPx4 activity assay using phosphatidylcholine hydroperoxide. Altogether, our research suggested that Irg1/itaconate-GPx4 axis may be a future therapeutic strategy for protecting neurons from ferroptosis post ICH.
    DOI:  https://doi.org/10.1038/s41418-024-01303-8
  10. Nat Commun. 2024 May 07. 15(1): 3795
    A comprehensive genetic map of cytokine responses in Lyme borreliosis.
    Javier Botey-Bataller, Hedwig D Vrijmoeth, Jeanine Ursinus, Bart-Jan Kullberg, Cees C van den Wijngaard, Hadewych Ter Hofstede, Ahmed Alaswad, Manoj K Gupta, Lennart M Roesner, Jochen Huehn, Thomas Werfel, Thomas F Schulz, Cheng-Jian Xu, Mihai G Netea, Joppe W Hovius, Leo A B Joosten, Yang Li.
      The incidence of Lyme borreliosis has risen, accompanied by persistent symptoms. The innate immune system and related cytokines are crucial in the host response and symptom development. We characterized cytokine production capacity before and after antibiotic treatment in 1,060 Lyme borreliosis patients. We observed a negative correlation between antibody production and IL-10 responses, as well as increased IL-1Ra responses in patients with disseminated disease. Genome-wide mapping the cytokine production allowed us to identify 34 cytokine quantitative trait loci (cQTLs), with 31 novel ones. We pinpointed the causal variant at the TLR1-6-10 locus and validated the regulation of IL-1Ra responses at transcritpome level using an independent cohort. We found that cQTLs contribute to Lyme borreliosis susceptibility and are relevant to other immune-mediated diseases. Our findings improve the understanding of cytokine responses in Lyme borreliosis and provide a genetic map of immune function as an expanded resource.
    DOI:  https://doi.org/10.1038/s41467-024-47505-z
  11. Commun Biol. 2024 May 07. 7(1): 527
    E4BP4 in macrophages induces an anti-inflammatory phenotype that ameliorates the severity of colitis.
    Yasuko Kajimura, Akihiko Taguchi, Yuko Nagao, Kaoru Yamamoto, Konosuke Masuda, Kensuke Shibata, Yoichi Asaoka, Makoto Furutani-Seiki, Yukio Tanizawa, Yasuharu Ohta.
      Macrophages are versatile cells of the innate immune system that work by altering their pro- or anti-inflammatory features. Their dysregulation leads to inflammatory disorders such as inflammatory bowel disease. We show that macrophage-specific upregulation of the clock output gene and transcription factor E4BP4 reduces the severity of colitis in mice. RNA-sequencing and single-cell analyses of macrophages revealed that increased expression of E4BP4 leads to an overall increase in expression of anti-inflammatory genes including Il4ra with a concomitant reduction in pro-inflammatory gene expression. In contrast, knockout of E4BP4 in macrophages leads to increased proinflammatory gene expression and decreased expression of anti-inflammatory genes. ChIP-seq and ATAC-seq analyses further identified Il4ra as a target of E4BP4, which drives anti-inflammatory polarization in macrophages. Together, these results reveal a critical role for E4BP4 in regulating macrophage inflammatory phenotypes and resolving inflammatory bowel diseases.
    DOI:  https://doi.org/10.1038/s42003-024-06099-4
  12. Front Immunol. 2024 ;15 1248907
    NLRP6 negatively regulates host defense against polymicrobial sepsis.
    Laxman Ghimire, Sagar Paudel, John Le, Liliang Jin, Shanshan Cai, Dinesh Bhattarai, Samithamby Jeyaseelan.
       Introduction: Sepsis remains a major cause of death in Intensive Care Units. Sepsis is a life-threatening multi-organ dysfunction caused by a dysregulated systemic inflammatory response. Pattern recognition receptors, such as TLRs and NLRs contribute to innate immune responses. Upon activation, some NLRs form multimeric protein complexes in the cytoplasm termed "inflammasomes" which induce gasdermin d-mediated pyroptotic cell death and the release of mature forms of IL-1β and IL-18. The NLRP6 inflammasome is documented to be both a positive and a negative regulator of host defense in distinct infectious diseases. However, the role of NLRP6 in polymicrobial sepsis remains elusive.
    Methods: We have used NLRP6 KO mice and human septic spleen samples to examine the role of NLRP6 in host defense in sepsis.
    Results: NLRP6 KO mice display enhanced survival, reduced bacterial burden in the organs, and reduced cytokine/chemokine production. Co-housed WT and KO mice following sepsis show decreased bacterial burden in the KO mice as observed in singly housed groups. NLRP6 is upregulated in CD3, CD4, and CD8 cells of septic patients and septic mice. The KO mice showed a higher number of CD3, CD4, and CD8 positive T cell subsets and reduced T cell death in the spleen following sepsis. Furthermore, administration of recombinant IL-18, but not IL-1β, elicited excessive inflammation and reversed the survival advantages observed in NLRP6 KO mice.
    Conclusion: These results unveil NLRP6 as a negative regulator of host defense during sepsis and offer novel insights for the development of new treatment strategies for sepsis.
    Keywords:  NLRP6; chemokine; cytokine; innate immunity; neutrophil
    DOI:  https://doi.org/10.3389/fimmu.2024.1248907
  13. Front Immunol. 2024 ;15 1287415
    The immune landscape of sepsis and using immune clusters for identifying sepsis endotypes.
    Guoxing Tang, Ying Luo, Huijuan Song, Wei Liu, Yi Huang, Xiaochen Wang, Siyu Zou, Ziyong Sun, Hongyan Hou, Feng Wang.
       Background: The dysregulated immune response to sepsis still remains unclear. Stratification of sepsis patients into endotypes based on immune indicators is important for the future development of personalized therapies. We aimed to evaluate the immune landscape of sepsis and the use of immune clusters for identifying sepsis endotypes.
    Methods: The indicators involved in innate, cellular, and humoral immune cells, inhibitory immune cells, and cytokines were simultaneously assessed in 90 sepsis patients and 40 healthy controls. Unsupervised k-means cluster analysis of immune indicator data were used to identify patient clusters, and a random forest approach was used to build a prediction model for classifying sepsis endotypes.
    Results: We depicted that the impairment of innate and adaptive immunity accompanying increased inflammation was the most prominent feature in patients with sepsis. However, using immune indicators for distinguishing sepsis from bacteremia was difficult, most likely due to the considerable heterogeneity in sepsis patients. Cluster analysis of sepsis patients identified three immune clusters with different survival rates. Cluster 1 (36.7%) could be distinguished from the other clusters as being an "effector-type" cluster, whereas cluster 2 (34.4%) was a "potential-type" cluster, and cluster 3 (28.9%) was a "dysregulation-type" cluster, which showed the lowest survival rate. In addition, we established a prediction model based on immune indicator data, which accurately classified sepsis patients into three immune endotypes.
    Conclusion: We depicted the immune landscape of patients with sepsis and identified three distinct immune endotypes with different survival rates. Cluster membership could be predicted with a model based on immune data.
    Keywords:  MDSCs; endotypes; immune indicators; prediction model; sepsis
    DOI:  https://doi.org/10.3389/fimmu.2024.1287415
  14. PLoS One. 2024 ;19(5): e0303516
    Itaconic acid inhibits nontuberculous mycobacterial growth in pH dependent manner while 4-octyl-itaconic acid enhances THP-1 clearance of nontuberculous mycobacteria in vitro.
    Paul Breen, Madsen Zimbric, Lindsay J Caverly.
      Increasingly prevalent, nontuberculous mycobacteria (NTM) infections affect approximately 20% of people with cystic fibrosis (CF). Previous studies of CF sputum identified lower levels of the host metabolite itaconate in those infected with NTM. Itaconate can inhibit the growth of M. tuberculosis (MTB) in vitro via the inhibition of the glyoxylate cycle enzyme (ICL), but its impact on NTM is unclear. To test itaconic acid's (IA) effect on NTM growth, laboratory and CF clinical strains of Mycobacterium abscessus and Mycobacterium avium were cultured in 7H9 minimal media supplemented with 1-10 mM of IA and short-chain fatty acids (SCFA). M. avium and M. abscessus grew when supplemented with SCFAs, whereas the addition of IA (≥ 10 mM) completely inhibited NTM growth. NTM supplemented with acetate or propionate and 5 mM IA displayed slower growth than NTM cultured with SCFA and ≤ 1 mM of IA. However, IA's inhibition of NTM was pH dependent; as similar and higher quantities (100 mM) of pH adjusted IA (pH 7) did not inhibit growth in vitro, while in an acidic minimal media (pH 6.1), 1 to 5 mM of non-pH adjusted IA inhibited growth. None of the examined isolates displayed the ability to utilize IA as a carbon source, and IA added to M. abscessus isocitrate lyase (ICL) decreased enzymatic activity. Lastly, the addition of cell-permeable 4-octyl itaconate (4-OI) to THP-1 cells enhanced NTM clearance, demonstrating a potential role for IA/itaconate in host defense against NTM infections.
    DOI:  https://doi.org/10.1371/journal.pone.0303516
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