Candida parapsilosis is an emergent opportunistic yeast among hospital settings that affects mainly neonates and immunocompromised patients. Its most remarkable virulence traits are the ability to adhere to prosthetic materials, as well as the formation of biofilm on abiotic surfaces. The Ndt80 transcription factor was identified as one of the regulators of biofilm formation by C. parapsilosis; however, its function in this process was not yet clarified. By knocking out NDT80 (CPAR2-213640) gene, or even just one single copy of the gene, we observed substantial alterations of virulence attributes, including morphogenetic changes, adhesion and biofilm growth profiles. Both ndt80Δ and ndt80ΔΔ mutants changed colony and cell morphologies from smooth, yeast-shaped to crepe and pseudohyphal elongated forms, exhibiting promoted adherence to polystyrene microspheres and notably, forming a higher amount of biofilm compared to wild-type strain. Interestingly, we identified transcription factors Ume6, Cph2, Cwh41, Ace2, Bcr1, protein kinase Mkc1 and adhesin Als7 to be under Ndt80 negative regulation, partially explaining the phenotypes displayed by the ndt80ΔΔ mutant. Furthermore, ndt80ΔΔ pseudohyphae adhered more rapidly and were more resistant to murine macrophage attack, becoming deleterious to such cells after phagocytosis. Unexpectedly, our findings provide the first evidence for a direct role of Ndt80 as a repressor of C. parapsilosis virulence attributes. This finding shows that C. parapsilosis Ndt80 functionally diverges from its homolog in the close related fungal pathogen C. albicans. Candida parapsilosis is a ubiquitous yeast, often recovered from domestic animals, soil, and marine environments, but is also a commensal of the human skin. Among hospital settings, this species is considered a major opportunistic pathogen involved in invasive fungal infections [Candida species isolated from blood cultures in Latin America, Asia, and Southern Europe countries [C. parapsilosis is of particular concern among susceptible populations, comprising low birth weight neonates, immunocompromised individuals, and patients requiring prolonged use of indwelling devices such as central venous catheters [C. parapsilosis stands out for its capacity to adhere to the abiotic surface of implanted devices, later involving biofilm formation [C. parapsilosis virulence and are critical for its involvement in hospital outbreaks [fections ,2. Its iountries . C. paraatheters ,9. Besidormation ,10,11. IC. albicans, C. parapsilosis does not form true hyphae and, therefore, its biofilm only involves yeast and pseudohyphal forms [C. parapsilosis biofilm regulators, more than 100 transcription factors were knocked-out and mutants were assessed for biofilm formation ability [C. albicans, Bcr1, Efg1 and Ace2 were also directly implicated in biofilm development in C. parapsilosis [C. parapsilosis [C. parapsilosis biofilm defective mutants, NDT80 was firstly pointed as a putative biofilm regulator, in analogy with C. albicans biofilm regulation network. However, in the case of C. parapsilosis, NDT80 role was undisclosed due to marked growth defects exhibited by ndt80 mutant [C. albicans, Ndt80 was first described as a key modulator of azole drug sensitivity, being involved in the control of ergosterol biosynthesis [C. parapsilosis Ndt80 ortholog to be a transcription factor upregulated following azole resistance acquisition [ndt80 mutant exhibits increased susceptibility to azoles and that, together with Upc2 transcription factor, also regulates the expression of various genes of ergosterol biosynthetic pathway, namely ERG25, ERG6, ERG2, ERG3 and ERG4 [Biofilm is an organized community comprised of a dense network of microbial cells embedded in an extracellular matrix of polymers, which clinically restricts drug access and the immune response ,13. The al forms ,18. To i ability . Previoupsilosis ,19–22, tynthesis  and actiynthesis . We firsuisition . Later, and ERG4 .C. parapsilosis as a repressor of virulence attribute expression, namely morphogenesis, adhesion, and biofilm formation. Additionally, we explore the morphological phenotypes, its constitutive filamentous growth, and the adhesion profile resulting from NDT80 knockout, as well as its interaction with host immune system by assessing macrophage-mediated response.In this study, we address the role of Ndt80 in SAT1 flipper cassette, transformants were incubated in YPM medium  overnight, with agitation (180 rpm); afterward, approximately 100 cells were plated on YPD plates supplemented with nourseothricin at final concentration of 20 μg ml−1. All C. parapsilosis strains were stored in YPD broth with 40% glycerol, at – 80°C.Yeast strains used in this study were routinely grown in YPD broth medium  at 30°C with agitation (180 rpm) or on YPD agar plates, following addition of 2% of agar. To recycle the −1 sodium pyruvate at 37°C in a humidified atmosphere with 5% CO2. The culture medium was changed every 2 days, until ~70% of cell confluence was reached. RAW 264.7 cells were resuspended in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% inactivated FCS, 23.8 mM sodium bicarbonate and 50 mM glucose for the experimental assays (initiated until the cells 15th generation).RAW 264.7 murine macrophages were obtained from the European Collection of Cell Cultures and maintained in DMEM (Sigma-Aldrich) with 10% non-inactivated Fetal Calf Serum (FCS), 10 mM HEPES, 12 mM sodium bicarbonate and 11 mg mlNDT80 gene in C. parapsilosis BC014S (wild-type strain) [et al. [NDT80 gene were amplified using CpNDT80up_F and CpNDT80up_R primers (containing recognition sites for KpnI and ApaI) and CpNDT80down_F and CpNDT80down_R primers (containing recognition sites for SacII and SacI), respectively, and cloned into the flanking sites of pCD8 plasmid [KpnI and SacI, pNG4 disruption cassette was introduced into the native locus of NDT80 gene of C. parapsilosis BC014S. All primer sequences are listed in To knockout  strain) , the pNG [et al.  was used plasmid . After ret al. [600 of 0.2 and incubated at 30°C until reaching approximately OD600 of 2.0. After being pelleted, yeast cells were resuspended in 10 ml of Tris-EDTA buffer  containing 10 mM dithiothreitol and incubated at 30°C for 1 h with agitation (100 rpm). Yeast cells were washed twice with 40 ml of cold water plus once with 10 ml 1 M Sorbitol and, subsequently resuspended in 125 μl of this solution. Approximately 1 µg of purified KpnI-SacI fragment of pNG4 was added to 50 µl of competent cells. The cell mixture was then transferred to a 1 mm electroporation cuvette. Electroporation shock was performed at 1.25 kV, using a Gene Pulser X-cell Electroporator (Bio-Rad). Afterward, 950 μl of YPD containing 1 M sorbitol was immediately added; the mixture was incubated at 30°C for 4 h with agitation; afterward 100 μl were plated on YPD agar supplemented with nourseothricin at final concentration of 200 μg ml−1. Transformants were obtained after 24 h of incubation at 30°C.Transformation of wild-type strain was performed by electroporation as described by Ding et al. . Brieflyet al. [g for 5 min and washed twice with phosphate buffer saline (PBS) (Sigma-Aldrich). A yeast suspension was standardized to 2 × 106 cells ml−1 in the same buffer and mixed with 2 × 107 microspheres ml−1 of 1 μm uncoated carboxylated highly green fluorescent polystyrene microspheres (Molecular Probes). This mixture was incubated at room temperature for 30 min at 150 rpm. The suspensions were vortexed, and 50,000 events were analyzed using a FACS Calibur flow cytometer (BD Biosciences). Cell adhesion results are expressed as the percentage of cells with microspheres attached, representative of at least three independent experiments, performed in triplicate.Yeast adhesion was quantified by flow cytometry, as described by Silva-Dias et al. . Brieflyg for 5 min, washed once with PBS and standardized to obtain a suspension of 1 × 106 yeast cells ml−1 in RPMI-1640 medium supplemented with L-glutamine and buffered with MOPS acid (Sigma-Aldrich). One ml of such cell suspension was placed in each of a 12-well polystyrene microplate and incubated for 24 and 48 h at 37°C. Following incubation, total biomass was quantified by Crystal Violet (CV) assay, as previously described by Silva-Dias et al. [After overnight growth at 37°C with agitation (180 rpm) in Sabouraud broth medium, yeast cells were collected by centrifugation at 10,000 s et al. . BiofilmC. parapsilosis strains were set up as previously described, except the standardization of the cell suspension, which was diluted to an OD600 of 1; afterward, 5 ml were distributed in each well of a 6-well polystyrene plate. After 24 and 48 h of incubation at 37°C, adherent biofilms were washed with PBS, scrapped from the bottom of the wells, and vacuum filtered, as described by Holland, et al. [For dry mass assessment, , et al. . The aveColony phenotypes were observed and photographed under 20× magnification using a Stereo zoom S9i (Leica Microsystems) dissection microscope, after growth on YPD agar at 30°C, for 72 h. Images of yeast cell morphology were taken with a Zeiss Axioplan microscope, coupled with an AxioVision image acquisition system (Zeiss), after staining with Calcofluor White (Sigma-Aldrich) and mounting on glass slides. Yeast cells were photographed under 1000× magnification, oil immersion.280/A260 ratios ranging from 1.6 to 2.2 and showing no signs of degradation, after electrophoresis, were used in subsequent analyses.RNA was extracted as described by Kohrer and Domdey . ConcentNDT80 (CPAR2_213640), OCH1 (CPAR2_404930), ALS3 (CPAR2_404770), ALS7 (CPAR2_404800), GZF3 (CPAR2_800210), ALS7 (CPAR2_404800), BCR1 (CPAR2_205990), EFG1 (CPAR2_701620), and the orthologues of Candida albicans STP3 (CPAR2_200390), CWH41 (CPAR2_501400), STP3 (CPAR2_200390), MKC1 (CPAR2_800090), CPH2 (CPAR2_603440), RHR2 (CPAR2_503990), ACE2 (CPAR2_204370), CPH2 (CPAR2_603440), UME6 (CPAR2_803820) and CZF1 (CPAR2_501290).From 100 ng of total RNA, the first-strand cDNA was synthesized using the SensiFAST cDNA Synthesis Kit (Bioline) according to the manufacturer´s instructions. The resulting cDNA was stored at −20°C prior to use for real-time quantitative polymerase chain reaction (RT-qPCR). The genes analyzed were the followed: ACT1 gene expression was used to normalize the signal obtained for each gene. Data obtained were analyzed with REST software.For each real-time quantitative PCR, five replicates per strain were analyzed. All primers used are detailed in C. parapsilosis CDC317 open reading frames (ORFs) plus 1000 bp upstream and downstream  were downloaded from the Candida Genome Database . To identify putative Ndt80-regulated genes, a search for the MSE consensus motif (gNCRCAAAY) was performed in the promoter regions (1000 bp upstream the start codon). The resulting ORFs containing MSE sequences were grouped according to Gene Ontology (GO) terms using the CGD Gene Ontology Slim Mapper with the default parameters.Sequences from Macrophage-yeast interaction assays were carried out as previously described . BrieflyC. parapsilosis as described below. At the end of each incubation period , coverslips were washed twice with ice-cold PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. After 3 washing steps with PBS, cell membranes were stained with WGA, for 10 min, protected from light. Macrophages were treated with a blocking solution of 10% bovine serum albumin in PBS for 30 min at 37°C. Cells were then incubated overnight, at room temperature, with the primary rabbit polyclonal antibody against Candida , diluted (1:200) in blocking solution. Coverslips were washed and incubated for 2 h at room temperature with the AlexaFluor 488 donkey anti-rabbit IgG secondary antibody . Finally, after a washing step, macrophage cells were incubated with DAPI 0.02% for 10 min at room temperature. Cells were subsequently washed and the coverslips were mounted in glass slides with DAKO mounting medium and kept at −20°C until observation under confocal or fluorescence microscopy. Digital images were captured using a Carl Zeiss LSM 710 Confocal Microscope, using Plan-ApoChromat 40x/63x/1.4 oil objectives; Zen Blue and FiJi software’s were used to analyze the images.Macrophages grown in coverslips were incubated with The yeast cell viability following interaction with RAW 264.7 macrophage cells was assessed by a colony-forming unit (CFU) assay. After 30 min and 3 h of co-incubation, supernatants were collected and plated on YPD agar, to count non-internalized or non-adhered yeast cells. The remaining adhered RAW 264.7 macrophages were scraped and lysed with 0.5% Triton X-100. This cell suspension, representing the amount of yeast cells internalized was plated on YPD agar, using serial dilutions. Following 3 days of incubation, at 30°C, the number of yeast colonies per ml was calculated.For macrophage viability assay, after 30 min and 3 h of co-incubation, viable, and death macrophage cells were calculated using a hemocytometer, after staining with Trypan Blue .For live cell imaging assays, culture media without phenol-red was used and macrophage cell membranes were stained with Wheat Germ Agglutinin, Tetramethylrhodamine conjugate . Image acquisitions were conducted during at least 45 min, using a confocal Cell Observer Spinning Disk microscope (Zeiss), equipped with an LCI PlanNeofluar 63x/1.3 glycerol objective; Zen Blue software was used to analyze the time-lapse videos obtained.Statistical analysis of results of adhesion, biofilm and infection assays was performed using one-way ANOVA followed by a Dunnett post hoc test. Differences were considered statistically significant for a p-value <0.05. Significant differences were marked with an asterisk character (*), in which *p < 0.05, **p < 0.01, ***p < 0.001. All results are presented as mean ± standard deviation, of at least three independent experiments.C. parapsilosis virulence attribute expression, two independent lineages lacking one (ndt80Δ – NG2 strain) or both (ndt80ΔΔ – EF16 strain) copies of NDT80 were generated from C. parapsilosis strain BC014S (wild-type strain) [NDT80 locus of the wild-type strain, generated NG1 clone, which after cassette recycling, resulted in the NG2 strain. To delete the second copy of NDT80 gene, a second round of integration/recycling were performed, generating EF15 and EF16 clones, respectively. Gene knockout was confirmed by PCR (To gain insight into the role of Ndt80 in  strain) . Deletio strain) . The intd by PCR  and (b))NDT80 had a major effect upon colony and yeast cell morphology )ndt80Δ and ndt80ΔΔ mutants flocculate in liquid medium, suggesting that Ndt80 negatively affects the cell-cell adhesion process . The abeviously . Comparendt80ΔΔ mutant are two known enhancers of biofilm formation. We assessed wild-type and mutants strains regarding the ability to form biofilm, using two independent methods, Cristal Violet (CV) staining [C. parapsilosis lacking one or both copies of NDT80 gene exhibits enhanced capacity to form biofilm compared to wild-type strain )C. parapsilosis. In an attempt to identified Ndt80 targets involved in triggering virulence factors, we quantified the expression of the above-mentioned genes by RT-qPCR  and also adhesins Als-like , were identified by several authors ,31,32 as RT-qPCR .Figure 4ALS7 in ndt80Δ and ndt80ΔΔ mutants was upregulated 210- and 180-fold, respectively, compared to wild-type. In contrast, ALS3 gene expression was not changed significantly among the studied mutant strains. The expression of UME6 was upregulated, approximately, 5-fold in the ndt80Δ haploid mutant and a 13-fold in the ndt80ΔΔ diploid mutant, compared to the wild-type. MKC1 expression was also upregulated 2.8-fold and 36-fold in haploid and diploid mutants, respectively, comparatively to the wild-type. CPH2 gene exhibited a 1.2-fold upregulation in ndt80Δ mutant and of approximately 4-fold increased expression in ndt80ΔΔ mutant, in comparison to the wild-type.Relatively to adhesin-like genes, the expression of ACE2, CWH41 and OCH1 genes displayed similar expression values of approximately 3-fold, 2-fold, and 1.2-fold, respectively, in the haploid and diploid mutants. BCR1 gene was 1.5 and 1.7-fold upregulated in ndt80Δ and ndt80ΔΔ mutants in comparison to wild-type. The expression of STP3 was increased approximately 1.8-fold in ndt80Δ mutant but remained unchanged in ndt80ΔΔ mutant. In contrast, EFG1, GZF3 and RHR2 were downregulated in ndt80ΔΔ mutant comparatively to the wild-type; ndt80Δ mutant exhibited a slight upregulation of expression of such genes . CZF1 gene was progressively downregulated following sequential NDT80 gene copy deletion, by approximately 30% and 70%, respectively.NDT80 transcript was observed in the null strain. Interestingly, the expression of NDT80 in ndt80Δ mutant was 1.6-fold up-regulated. Since NDT80 gene has in its promoter region the MSE binding sequence, we could hypothesize that to cope with one copy gene deletion, Ndt80 up-regulates itself expression, as described in S. cerevisiae and A. nidulans [As expected, no nidulans ,34.C. albicans and S. cerevisiae ORFeomes [Ndt80 was found to bind to the middle sporulation element (MSE) (5ʹ-CACAAA-3ʹ) in the target gene promoter region  of C. alORFeomes ,23. The ORFeomes ,35, the C. parapsilosis ORFeome. This analysis allowed the retrieval of 417 ORFs containing MSE motifs in their promoters. These were mapped to GO terms and grouped according to Biological Process, Molecular Function or Cellular Component , while this process is more lagging for the wild-type macrophage interaction; after 30 min of interaction, most of the C. parapsilosis cells were outside of the macrophages, adherent or not . Howeveg fading ); the ndt or not , i). Folll types , ii).Figndt80ΔΔ mutant cells, caused a significant reduction of the number of viable macrophages soon after 30 min . FolΔ strain , ii).C. albicans, several studies addressing the regulatory networks of non-albicans species, like C. glabrata and C. parapsilosis, demonstrate a significant difference in the evolutionary adaptation of such yeasts to the human host [C. parapsilosis virulence attributes is still somewhat limited, this species displays many biological features that are presumed to be directly related to its environmental colonization and pathogenicity, such as enhanced adherence and biofilm development on abiotic surfaces.While molecular mechanisms are well characterized in man host ,39. AlthC. albicans [ALS3 and HWP1), being required for their normal expression [NDT80 reduces C. albicans virulence in vivo, by blocking yeast to hyphal transition, as well as the expression of genes involved in the filamentous transcriptional program [Adhesion, morphogenetic variations and biofilm formation are virulence attributes clearly depicted for albicans ,41 and aalbicans ,15. Ndt8pression . Thus, d program .C. albicans, the disruption of C. parapsilosis NDT80 gene triggers two noticeable phenotypic changes: morphogenesis in a spontaneous and constitutive manner , whose expression is extraordinary increased. This adhesin was previously identified as a mediator of C. parapsilosis adhesion to human buccal epithelial cells [ALS7 and ALS3 are included in this group.Surprisingly, and opposing to what was described for e manner , and proal cells . AlthougUME6 and CPH2, described as inducers of yeast to pseudohyphae transition [C. albicans. Other genes like OCH1, the orthologs of C. albicans CWH41 and STP3 are also involved in C. parapsilosis phenotypic switching, as positive and negative regulators, respectively [OCH1 and the ortholog of C. albicans STP3; interestingly, the ortholog C. albicans CWH41 expression doubles in ndt80ΔΔ mutant, suggesting that this gene could be a target for Ndt80, which putatively represses the expression of this pseudohyphae formation factor.According to our findings Ndt80 can have a dual role in yeast to pseudohyphae transition: on one hand, by impairing the expression of ansition ; on the ansition ,35, two ectively ,36. We fC. albicans biofilm development [NDT80 deletion significantly compromises biofilm formation either in vitro or in vivo models [C. parapsilosis NDT80 gene promotes biofilm growth in vitro, suggesting that this transcription factor is acting as a repressor of genes involved in such process. Other biofilm regulators, acting as repressors and activators in a circuit system were already previously identified in C. albicans and C. parapsilosis [C. parapsilosis [C. parapsilosis, deletion of CZF1, GZF3, UME6, and CPH2 was associated with a reduced biofilm formation ability. Although Ndt80 was not identified as a component of C. parapsilosis regulatory network due to the inherent growth defects [et al. [ndt80ΔΔ mutant revealed an approximately 36-fold, 13-fold, 4-fold and 3-fold upregulation of MKC1, UME6, CPH2 and ACE2, respectively, while other genes also described to be required for biofilm formation, such as GZF3 and CZF1, were demonstrated to be downregulated. These findings strongly suggest the role of Ndt80 as a negative regulator of MKC1, UME6, CPH2 and ACE2 expression and as an activator of GZF3 and CZF1 expression. Thus, in Ndt80 absence, and despite GZF3 and CZF1 genes exhibiting a reduced expression, the upregulation of MKC1, UME6, CPH2 and ACE2 genes occurs and biofilm development is promoted  responsible for the regulation of elopment . In thiso models . Converspsilosis . Efg1, Bpsilosis . In C. p defects , we anal [et al.  for the promoted . RHR2 waormation . Neverthndt80ΔΔ mutant led us to explore its interaction with immune system cells. The ability to switch from yeast to a filamentous form is a key factor that allows successful phagocytosis evasion of C. albicans [C. parapsilosis, several studies have elucidated distinct virulence traits of this species that could modulate the mechanism by which phagocytosis and the immune response proceed [in vitro infection assays a prompter interaction of both mutants with the macrophage cells in comparison to the wild-type strain. This finding is also in accordance with results obtained with the adhesion assays to abiotic surfaces and to other yeast cells.The virulence-related phenotypes exhibited by albicans . In the  proceed . We founet al. [C. parapsilosis pseudohyphae did not correlate with the engulfment time. In our assays, after 3 h of co-culturing, only the ndt80ΔΔ mutant induced a significantly increase of macrophage killing with concomitant higher yeast viability, while neither the wild-type nor the ndt80Δ mutant promoted significant damage of the macrophage cells. These results show that the phenotype prompted by NDT80 knockout results in a more virulent C. parapsilosis strains, more resistant to macrophage attack, associated with a decrease of macrophage cytoplasmic membrane integrity and a concomitant increase of macrophage cell death. Virulence features are not exclusively related to the constitutive pseudohyphal form; notably, the promoted expression of ALS7 and MKC1 transcripts  [ndt80ΔΔ mutant, with impact upon adhesion and recognition by immune system cells [Toth et al.  using otodeling) ,48 proviem cells .NDT80-like genes recognize the conserved DNA-binding domain motif, MSE, through an Ig fold. As other members of the Ig-fold family of transcription factors, such as p53 or NFAR from mammals, NDT80-like genes share a similar regulation mechanism [NDT80-like genes are divergent among fungal species and even within species [NDT80 absence, as seen in Schizosaccharomyces pombe, to a family of six members, as seen in Fusarium oxysporum. While in Saccharomyces cerevisiae, NDT80 single gene functions as a master regulator of meiosis process and sporulation [NDT80-like genes the unraveling of its function and regulation mechanism is laborious and far from being obtained. NdtA and XprG are two of the Ndt80-like proteins in the filamentous fungal species Aspergillus nidulans. The former has a high homology with Ndt80 and like in S. cerevisiae, it is crucial for sexual reproduction. The later, under carbon starvation, regulates positively fungal response by controlling its extracellular proteases, mycotoxin, and penicillin expression, which could result in autolysis, hyphal fragmentation and ultimately in cell death [Neurospora crassa possesses three Ndt80-like proteins, Vib-1, Ncu04729 and Fsd-1. Vib1, closely related to XprG, is an activator of extracellular protease production and is also associated with apoptosis [C. albicans has three NDT80-like DNA-binding domain genes, NDT80, RON1 and REP1 [MDR1 and is required for yeast growth on presence of N-acetylglucosamine (GlcNAc) and galactose. Ron1 is associated with GlcNAc regulation signaling.In fungi, echanism . However species . These drulation , in othell death . Neurospand REP1 . These NC. albicans and C. parapsilosis, although it diverged to opposite functional roles. Our study highlights the importance of Ndt80 on the complex regulation of C. parapsilosis virulence attributes, as a major repressor.Notably, Ndt80 was identified as a morphogenesis and biofilm regulator, in Click here for additional data file.


This is the first demonstration of C15:0’s direct role in attenuating multiple comorbidities using relevant physiological mechanisms at established circulating concentrations. Pairing our findings with evidence that (1) C15:0 is not readily made endogenously, (2) lower C15:0 dietary intake and blood concentrations are associated with higher mortality and a poorer physiological state, and (3) C15:0 has demonstrated activities and efficacy that parallel associated health benefits in humans, we propose C15:0 as a potential essential fatty acid. Further studies are needed to evaluate the potential impact of decades of reduced intake of OCFA-containing foods as contributors to C15:0 deficiencies and susceptibilities to chronic disease.Dietary odd-chain saturated fatty acids (OCFAs) are present in trace levels in dairy fat and some fish and plants. Higher circulating concentrations of OCFAs, pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0), are associated with lower risks of cardiometabolic diseases, and higher dietary intake of OCFAs is associated with lower mortality. Population-wide circulating OCFA levels, however, have been declining over recent years. Here, we show C15:0 as an active dietary fatty acid that attenuates inflammation, anemia, dyslipidemia, and fibrosis  In the U.S., during the 20-year period following this initial recommendation, the average individual’s daily intake of whole fat milk was reduced more than 4-fold, from 283 to 65 grams per day, in an effort to lower dietary saturated fats3. Despite this drop in whole fat milk intake, the global prevalence of obesity, type 2 diabetes, metabolic syndrome, and nonalcoholic fatty liver disease has increased6. Further, an 18-year longitudinal study including over 25,000 individuals demonstrated that children fed whole fat milk had a lower risk of having obesity compared to children who were provided fat-free or 1% fat milk, and multiple studies have demonstrated associations between higher dietary intake of full-fat dairy and reduced risk of type 2 diabetes and cardiovascular disease9. As such, there is a need re-evaluate potential health risks versus benefits of dietary dairy fats10.Over the past 40 years, diets with lower saturated fats have been recommended to decrease cholesterol levels and subsequent risks of heart disease11. In contrast, odd-chain saturated fatty acids (OCFAs), pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0), represent only 1% and 0.5%, respectively, of fatty acids in whole fat milk11. While dietary ECFAs have been associated with increased risk of inflammation, heart disease, and type 2 diabetes in humans15, higher dietary intake and circulating levels of OCFAs have been associated with lower risks of adiposity, chronic inflammation, cardiovascular disease, metabolic syndrome, type 2 diabetes, nonalcoholic steatohepatitis (NASH), chronic obstructive pulmonary disease, pancreatic cancer and other conditions27. In a prospective cohort involving over 14,000 people followed for an average of 14 years, increased dietary intake of OCFAs was associated with lower mortality in both men and women, while higher ECFA intake was associated with higher mortality in women28. Current dietary saturated fat recommendations do not discriminate between ECFAs and OCFAs, and the growing body of literature demonstrating opposing associations of ECFAs and OCFAs with disease and health have led to calls to refine global saturated fatty acid dietary guidelines30. This may be particularly important since population-wide concentrations of circulating OCFAs are decreasing31. While OCFAs have been routinely measured as biomarkers of dairy fat intake, and epidemiological studies provide evidence of associations between OCFAs and better health32, there has been a paucity of controlled studies to evaluate OCFAs as active dietary fatty acids that directly confer the health benefits to which they are associated.Among fatty acids present in whole fat milk, approximately 68% are even-chain saturated fatty acids (ECFAs), including myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0)Tursiops truncatus) are long-lived, large-brained mammals that can naturally develop metabolic syndrome and NASH34. We previously reported that, similar to humans, dolphins with higher serum OCFAs have a lower risk of metabolic syndrome and liver disease35. In an effort to continually improve dolphin health and to understand how changes in the ocean may impact wild dolphins’ dietary needs, a group of dolphins was provided a modified fish diet higher in OCFAs. The modified diet effectively increased serum C15:0 and C17:0 concentrations, and components of metabolic syndrome normalized within three months35. This pilot study supported that increased dietary OCFAs may improve metabolic health. Further, our study showed that beyond dairy products, dietary OCFA intake can be increased using specific types of fish. This is consistent with previous studies documenting measurable OCFAs outside of dairy products, including some types of fish, plants, and bacteria39. Because our study used modified fish diets and not pure OCFAs, however, we were not able to conclude that increased OCFAs directly caused the observed health benefits.Bottlenose dolphins (in vitro and in vivo studies with pure (99%) OCFAs to assess their potential causative roles in safely attenuating targeted chronic comorbidities. Herein, given the known role of omega-3 and other dietary fatty acids as natural ligands for the body’s orchestrators of metabolism  that may protect against insulin resistance, type 2 diabetes, inflammation and NASH, we tested OCFAs for PPAR agonist activities43. Due to potential roles of fatty acids on mitochondrial function and reactive oxygen species (ROS) that can, in turn, increase the risk of cardiometabolic diseases, we evaluated impacts of OCFAs on mitochondrial function and ROS production44. OCFAs were tested across a variety of human cell systems mimicking chronic inflammatory and fibrotic disease states. The effects of OCFA oral supplementation were then studied in in vivo models of cardiometabolic, inflammatory, liver, hematologic, and fibrotic diseases. Based on our findings, we reviewed current scientific literature related to OCFAs, with a lens on potential support for OCFAs as essential dietary fatty acids.Due to the need for robust, controlled studies to evaluate potential direct activities of OCFAs, we conducted a series of 40. Here, OCFAs C15:0 and C17:0, as well as ECFAs C14:0 and C16:0, were evaluated at 10 concentrations for cell-based PPAR agonist activities, including all three PPAR isoforms, α, δ, and γ. C15:0 was a dual, partial PPARα and PPARδ agonist, with maximum activities of 65.8% and 52.8%, respectively, compared to that of positive controls   are present throughout the body and help to regulate metabolism and inflammation by detecting and responding to dietary fatsDue to detected dual, partial PPARα/δ agonist activities, C15:0 was further tested at 10 concentrations for other agonist and antagonist activities across 76 assays and 47 genes and targets that are relevant to compound safety and potent pharmacological mechanisms of action, including G-protein coupled receptors, kinases, transporters, ion channels, nuclear receptors, and non-kinase enzymes. No off-target activities were detected with C15:0 when tested up to 20 µM , 20 µM  and 50 µM  compared to non-supplemented control cell systems (23.4 ± 4.3%) , is a key component of the degradative process of aging and numerous chronic diseases, including cardiometabolic diseasesSafety of C15:0 was further assessed by exposing 12 primary human cell systems with venular endothelial cells, peripheral blood mononuclear cells, B cells, bronchial epithelial cells, dermal fibroblasts, coronary artery smooth muscle cells, keratinocytes, lung fibroblasts or macrophages, at multiple concentrations . Based on a definition of cytotoxicity in which more than 50% of total protein in the cell system was reduced, C15:0 did not induce cytotoxicity in any of the 12 cell systems  and ECFAS (C14:0 and C16:0) were tested for anti-inflammatory and antifibrotic activities, including measuring 148 biomarker readouts across 12 primary human cell-based systems mimicking inflammation and fibrosis. C15:0 was tested at four concentrations , and the remaining saturated fatty acids were tested at 20 µM. Here, C15:0 had dose-dependent, annotated anti-inflammatory activities, including reduced monocyte chemoattractant protein 1 (MCP-1) and secreted immunoglobulin G (IgG) , we first sought to understand oral doses of C15:0 needed to achieve these plasma concentrations in appropriate models. Here, 8- to 10-week old Sprague Dawley rats  dosed orally once with C15:0 at 35 mg/kg body weight had increased C15:0 plasma levels within 30 minutes Fig. . A maximTo further evaluate the safety of C15:0 at increasing doses, Sprague Dawley rats  were dosed orally once daily for 14 days with C15:0 at 35, 175 and 350 mg/kg body weight. A non-dosed vehicle control group was included. Safety assessments included clinical observations, body weight, food intake, clinical chemistries, and histology . Additionally, total plasma C15:0 and C17:0 concentrations were measured at Day 14. There were no mortalities or observed abnormal behaviors in animals throughout the 14-day study across all study groups, and there were no significant differences when comparing body weights and organ weight-to-body weight ratios or the prevalence of abnormal clinical chemistry values or histologic observations between C15:0-supplemented and non-supplemented control animals  and daily oral C17:0  on inflammation and cardiometabolic conditions in obese C57BL/6 J mice  fed a high-fat diet . Mice supplemented with oral C15:0 for 90 days at low doses (5 mg/kg) had lower circulating levels of the proinflammatory chemokine, monocyte chemoattractant protein 1 (MCP-1), and the proinflammatory cytokine, interleukin 6 (IL-6) compared to non-supplemented controls Fig. . In cont46. After 2 weeks on this diet, animals were supplemented with daily oral C15:0 (35 mg/kg body weight) for 11 weeks and compared to both non-supplemented diseased controls and standard diet healthy controls . Daily, oral C15:0 supplementation attenuated a regenerative hemolytic anemia induced by the high fat, high cholesterol diet  developed hypercholesterolemia, anemia, and NASHols Fig. . Additio47. Given our demonstration of C15:0 and C17:0 as active dietary fatty acids, we reviewed the literature for evidence supporting or negating C15:0 and C17:0 as potential essential fatty acids  are required to maintain a healthy physiological state, (2) are not made at adequate levels endogenously, and (3) require dietary intake in order to maintain healthy concentrations in the body are requin vivo efficacy that align with health benefits, including lower risk of inflammation, cardiometabolic diseases, and NASH, to which they have been associated in numerous large epidemiological studies in humans29.Our series of studies herein support that dietary odd-chain saturated fatty acids (OCFAs), especially C15:0, are active fatty acids having cell-based activities and 50. Here, daily oral supplementation with C15:0 and C17:0 lowered proinflammatory states in obese mice with metabolic syndrome, as well as lowered proinflammatory biomarkers in primary human cell systems mimicking chronic inflammation. C15:0 and C17:0 lowered monocyte chemoattractant protein 1 (MCP-1), a proinflammatory chemokine; C15:0 also lowered proinflammatory chemokines , cytokines , and adipokines . MCP-1, which can be induced by the even-chain saturated fatty acid (ECFA), C16:0, contributes to chronic inflammation present in chronic cardiometabolic diseases, and elevated MCP-1 concentrations have also been associated with pancreatic cancer53. Further, circulating MCP-1 has been proposed as a measurement of biological age in mice and frailty in humans54. Our studies suggest that active lowering of proinflammatory states, including lowered MCP-1, by OCFAs may contribute to their associations with lower adipokines and lower risks of type 2 diabetes, cardiovascular disease, pancreatic cancer, and mortality28.Chronic low-grade inflammation, driven by proinflammatory chemokines and cytokines, contributes to cardiometabolic comorbidities and the aging process55. Metabolic syndrome increases the risk of type 2 diabetes, heart disease, and all-cause mortality. In our studies, daily oral C15:0 supplementation over 12 weeks lowered total cholesterol and glucose in an in vivo model with metabolic syndrome. Similarly, a case-cohort study involving 15,919 humans across eight European countries demonstrated associations between higher plasma phospholipid C15:0 and C17:0 concentrations and lower total cholesterol and triglycerides; conversely, higher ECFA concentrations were associated with higher triglycerides56. While higher circulating concentrations of both C15:0 and C17:0 have been associated with lower risks of type 2 diabetes, a controlled study in an animal model demonstrated that plasma increases of C17:0, and not C15:0, had linear correlations with decreased insulin and glucose57.Dyslipidemia and hyperglycemia are components of metabolic syndrome, a cluster of conditions impacting approximately 1 in 3 people globallyin vivo, suggesting that ingestion of C15:0 may offer benefits of both raised circulating C15:0 and C17:0, including improved glucose control. Elongation of C15:0 to C17:0 has been demonstrated in MCF7 human cells transfected with human elongases encoded by elongation of very long-chain fatty acid 6 (ELOVL6), as well as ELOVL7, providing a feasible mechanism for increased C17:0 concentrations following C15:0 ingestion58.Our study data, paired with observational associations in human population studies, support that increased dietary and circulating C15:0 may have direct cholesterol- and triglyceride-lowering effects in humans. Further, our studies demonstrate that ingestion of C15:0 alone increases both C15:0 and C17:0 plasma concentrations 59. There is currently has no cure for NASH, which is soon become the leading cause of hepatocellular carcinoma and end-stage liver failure globally59. Here, in addition to lowering cholesterol and triglycerides, C15:0 supplementation attenuated anemia, liver iron deposition, and severity of liver fibrosis in our in vivo NASH model. The NASH phenotype in our model specifically mimics anemia of inflammation  involving fragile red blood cells and associated dysmetabolic iron overload syndrome with severe liver fibrosis, which are risk factors for more severe comorbidities, including cardiovascular diseases and NASH in humans63. Consistent with our in vivo study, we also demonstrated that C15:0 causes a dose-dependent reduction in fibroblast proliferation, collagen I and III, and proinflammatory PAI-1 in primary human cell systems mimicking fibrotic diseases. Aligned with our findings, higher circulating C15:0 concentrations have been previously associated with lower liver enzymes and lower risks of NAFLD and severe NASH in humans64. Our study provides evidence that orally administered C15:0 can improve red blood cell stability and attenuate anemia, liver iron deposition and liver fibrosis that could prevent or decrease the severity of NASH.Nonalcoholic steatohepatitis (NASH) is an advanced stage of nonalcoholic fatty liver disease, which is present in approximately 25% of the global population and involves fat deposition in the liver paired with inflammation and fibrosis65. Here, in addition to reducing a proinflammatory state and improving red blood cell stability to attenuate anemia in vivo, our studies demonstrate the ability for C15:0 to reduce reactive oxygen species and repair mitochondrial function in human cells. Similar effects were observed with C17:0. These results are consistent with previously proposed mitochondrial-protective effects of OCFAs due to increased downstream production of succinate, which, in turn, can rescue mitochondrial function via mitochondrial complex II66. Due to the demonstrated ability for OCFAs to attenuate multiple components of aging, there is a need to better understand how OCFAs may have a causative role in observed reduced mortalities among people who ingest higher dietary OCFAs compared to those who have lower OCFAs in their diets28.Aging is a degradative process involving decreased cellular stability (i.e. cellular senescence), increased reactive oxygen species, mitochondrial dysfunction and chronic inflammation that contribute to the onset and severity of comorbidities of aging, including cardiovascular disease, type 2 diabetes and chronic anemiade novo to maintain a healthy physiological state67. As components of the cell membrane lipid bilayer, essential fatty acids, and fatty acids in general, play important roles in cellular stability, cell signaling, and immune function68. Alpha-linolenic acid and linoleic acid have long been considered essential fatty acids, and recent studies have demonstrated that their downstream polyunsaturated fatty acid derivatives, arachidonic acid and docosahexaenoic acid, can alone support healthy development in mice70. Our studies demonstrate C15:0 and C17:0 as active dietary fatty acids that cause cell-based activities and in vivo efficacy paralleling previously established associations with lower risks of numerous chronic inflammatory, cardiometabolic and liver conditions in humans. Further, C15:0 and C17:0 attenuated key hallmarks of aging, which may help explain why higher dietary OCFA intake has been associated with lower mortality28. These outcomes led to our review of the literature regarding the potential for OCFAs to be essential fatty acids.Essential fatty acids are defined as active dietary lipids not produced in high enough quantities de novo. Multiple studies have demonstrated that dietary C15:0 intake and associated circulating concentrations are linearly correlated71. This supports that, although C15:0 can be elongated from propionate or C13:0, there does not appear to be strong endogenous, de novo production of C15:072. In addition to linear correlations between dietary and circulating C15:0 concentrations, evidence of C15:0 as a primarily exogenous fatty acid includes lack of peroxisomal 2-hydroxyacyl-CoA lyase and liver-based α-oxidation involvement in endogenous production of C15:0, lack of strong correlations between orally administered propionate with plasma C15:0 concentrations, and no evidence of effects of changing gut microbiota on circulating C15:073. In contrast, C17:0 appears to be readily produced de novo via propionate, liver-based α-oxidation, and elongation of C15:0 via ELOVL673. Combining these publications with our studies, we propose dietary C15:0 as an important diet-derived building block for C17:0, in part by elongation of C15:0 to C17:0 via ELOVL6; this hypothesis is further supported by in vivo dose response studies demonstrating a linear correlation between dietary and serum C15:0 concentrations and near-doubling of serum C17:0 concentrations over what would be expected given actual C17:0 consumed57.First, there is strong evidence supporting that circulating C15:0 concentrations are primarily driven by exogenous, diet-based sources, while C17:0 is readily produced 74. These levels are consistent with our studies, which demonstrated cell-based PPARα/δ agonist, anti-inflammatory, antifibrotic, and mitochondrial protective C15:0 activities between 10 and 50 µM, with most of our studies demonstrating optimal activities at 20 µM. Human pharmacokinetic studies support that a single dose of 200 mg of C15:0 results in 20 µM circulating C15:0 concentrations (approximately 5 µg/ml)75. Further, the average reported dietary intake of C15:0, based on a population of women from the 1990s, was 220 ± 100 mg per day, which is consistent with the amount needed to achieve approximately 20 µM circulating C15:0 concentrations76. Combined, these studies support potential targeted circulating C15:0 concentrations from 20 to 30 µM. Further studies are needed to determine whether these concentrations may be defined as ‘adequate’ to help sustain physiological health.Second, there is moderate to strong evidence that ‘adequate’ circulating C15:0 concentrations can be defined. A human population pharmacokinetic model, based on a series of studies involving oral administration of pure C15:0 to healthy adults and children, estimated that the average baseline circulating C15:0 concentration in people is 24.9 µM28. These studies, however, are associative and do not establish causality. Our studies demonstrate OCFAs as active dietary fatty acids that directly attenuate multiple hallmarks of aging, as well as cardiometabolic, liver and inflammatory conditions, at concentrations consistent with circulating concentrations in humans, supporting a causal role of OCFAs in maintaining cardiometabolic, liver, and immune health. Our reliance on dietary C15:0 and maintained healthy circulating C15:0 concentrations is further supported by observed decreasing serum C15:0 concentrations in human populations over a recent 13-year period that are concurrent to the increasing prevalence of obesity, type 2 diabetes and NASH31. These studies support that targeted OCFA concentrations may be needed to maintain metabolic health, but more studies are needed to thoroughly evaluate the potential for OCFA deficiencies and associated deficiency disorders.Third, there is moderate supporting evidence that adequate circulating OCFA concentrations are needed to maintain physiological health. Large-scale population studies conducted globally repeatedly demonstrate that higher circulating concentrations of OCFAs (C15:0 and C17:0) are associated with lower risks of mortality, as well as having or developing obesity and numerous cardiometabolic and liver diseasesin vivo supplementation studies measured total plasma C15:0 and C17:0, they did not measure OCFA deposition in other sources, including red blood cells, lymph, and adipose tissue, where OCFAs can accumulate77. Further, while our mouse efficacy study included measurements of various serum lipid species containing C15:0 and C17:0, our efficacy studies lacked total circulating C15:0 and C17:0 concentrations, limiting our ability to confirm actual total circulating OCFA concentrations. Our pharmacokinetic studies, however, paired with previously published studies conducted with orally administered OCFAs using a variety of mammalian models with deuterated and C14-labeled C15:0, repeatedly confirm that orally administered OCFAs are bioavailable77. Because our efficacy studies were limited to males, we were unable to evaluate potential differences in OCFA efficacy based on sex. Thus, future studies with OCFAs would benefit from evaluating total OCFA concentrations in serum or plasma, red blood cells, lymph, and adipose tissues, and should include both males and females. It is worthy to note that our efficacy studies used diets containing relatively higher percentages of fat, especially saturated fats, with or without higher cholesterol compared to baseline diets to induce illness; given that our study design used dietary odd-chain saturated fatty acids to effectively attenuate illness in these models, more studies are needed to differentiate among various types of saturated fats and ‘high fat diets’ commonly used in in vivo cardiometabolic studies. While we propose C15:0 as a potential essential fatty acid, controlled studies feeding C15:0-depleted diets in appropriate models and evaluating them for impaired health could help confirm C15:0 as essential and define potential C15:0 deficiency syndromes.Our studies had several limitations. While the 24-hour and 14-day in vivo, 2) may provide optimal health benefits against numerous cardiometabolic, liver and aging-associated conditions at circulating concentrations around 20 to 30 µM following ingestion of 200 to 300 mg C15:0 per day, and 3) may be needed in the diet to preserve a healthy metabolic state.In conclusion, our series of studies and review of the literature demonstrate that C15:0 (pentadecanoic acid), a trace dietary odd-chain saturated fatty acid present in dairy fat and some fish and plants: 1) is an active, exogenous fatty acid with anti-inflammatory, antifibrotic, red blood cell-stabilizing, and mitochondrial-reparative activities resulting in lowered cholesterol, triglycerides and glucose, and attenuated inflammation, anemia, and liver fibrosis Based on these studies, we propose C15:0 as a potential essential fatty acid that may be needed to stem the global pandemics of obesity, cardiometabolic diseases and NASH. Further studies are needed to confirm this hypothesis, including assessing potential public health implications of population-wide declines in dietary whole dairy fat and other food-based sources of OCFAs over the past 40 years.In vivo study protocols described herein were reviewed and approved by Institutional Animal Care and Use Committees at the facilities and were in accordance with the Final Rules of the Animal Welfare Act regulations , the Public Health Service Policy on Humane Care and Use of Laboratory Animals from the Office of Laboratory Animal Welfare, and the Guide for the Care and Use of Laboratory Animals from the National Research Council.All studies herein utilized synthetic free saturated fatty acids, including pentadecanoic acid, purchased from Millipore Sigma .Based on known PPAR ligand and agonist activities of polyunsaturated fatty acids, it was hypothesized that C15:0, as a fatty acid, may also be a PPAR ligand. Chinese hamster ovary  cell-based functional protein interaction assays for PPARα, δ, and γ that are part of the Eurofins/DiscoverX PathHunter NHRscan panel  were used to evaluate C15:0 PPARα, δ and γ agonist activities at 10 concentrations (up to 100 µM). Effective concentrations (EC50) and maximum activities were determined in relation to positive controls . Detailed methods used for these assays and assay readouts are available from Eurofins/DiscoverX.It was hypothesized that C15:0 would not have off-target pharmacology. To test this hypothesis, the Eurofins/DiscoverX SafetyScan47 panel was used to test C15:0 at 10 concentrations for agonist and antagonist activities across 76 assays and 47 genes and targets that are relevant to compound safety and mechanisms of action, including G-protein coupled receptors, kinases, transporters, ion channels, nuclear receptors, and non-kinase enzymes  on stressed HepG2 cell systems with dysfunctional mitochondria producing ROS. The basis of our assay was triphenylphosphonium dihydroethidium, which allowed for electrophoretically driven uptake of the probe in actively respiring mitochondria (unmodified dihydroethidium is limited to the cytosol). The dihydroethidium moiety reacts with superoxide and generates a fluorescent signal by hydroxylating the 2 position, generating fluorescent hydroxyethidium. This allowed detection of ROS in the mitochondria of live cells. HepG2 human liver carcinoma cells were cultured in High Glucose Dulbecco’s modified eagles medium (DMEM Gibco 11965092) with 10% fetal bovine serum (FBS). Cells were passed every 3 days using TrypLE disassociation reagent and grown at 37 °C with 5% COIncubation of cells under various conditions were started 18–24 hours before the assay. T25 flasks were aspirated of high glu media, washed with 7 ml’s DPBS (Gibco 14040-133) and the media replaced with 7 ml’s of Substrate Limited media supplemented with 5% FBS and 20% of appropriate test condition. The typical final concentration was 33 μM BSA and 200 μM FFA as per Agilent protocol for a 6:1 FFA:BSA ratio. HepG2 cells in T25 flasks were aspirated of media, washed with 7 ml of Dulbecco’s phosphate buffered saline (DPBS) and aspirated to remove wash. 2 ml’s of TrypLE was added to the flask and placed back in a humidified incubator for 5–10 minutes until cells were fully detached. Cells were transferred to a 15 ml conical tube and the flask was washed with an additional 2 ml’s of DPBS which was also added to the 15 ml conical tube. Cells were spun at 140 RCF for 5 minutes and supernatant was decanted. 5 ml’s of DMEM (Substrate Limited media + 10% 170 μM BSA solution) was added to cell pellet and cells were re-suspended by pipette.6 cells/ml in serum starved DMEM (Substrate Limited Media + 20% test solution) and 300ul was plated into each well of a 96 well conical polypropylene plate (Nunc 339651). Cells were incubated for 1 hr at 37 °C with gentle agitation. After incubation, the cells were spun at 300 RCF for 5 minutes and decanted. 300ul of 2uM MitoSOX red in DPBS was added to each well except for blanks. Cells were incubated for 30 minutes at 37 °C with gentle agitation. Cells were spun at 300 RCF for 5 minutes and decanted. Cells were washed (3×) with 300ul of warmed (37 °C) DPBS, re-suspended by pipette and spun at 300 RCF for 5 minutes, then decanted and the procedure repeated. Cells were re-suspended by pipette during each wash. After final wash, cells were re-suspended in another 300ul of DPBS. Re-suspended cells were then transferred to 1.5 ml Eppendorf tubes for analysis on Attune NxT flow cytometer. Cells were analyzed by flow cytometry with the following parameters. FSC = 8, SSC = 300, BL2 (corresponding to ~510ex/580em) =400, 100 ul/min flow rate, 150 ul total volume. Raw FCS files were analyzed by FlowJo (v10.5.0) to determine the percent ROS positive cells within those cells determined to be live and singlets (FSCH vs FSCA ~ 1). Seven replicate measurements were made for every concentration. Wilcoxon tests and two-sided P values were used to compare percent mitochondrial ROS between each C15:0 supplemented group (by concentration) and the non-supplemented control group. To evaluate Multiple saturated fatty acids  at 20 µM were then assessed for reduction of mitochondrial ROS. Due to low sample sizes and inability to assume normal distributions, Kruskall Wallis tests were used to evaluate differences in percent mitochondrial ROS among the different groups. Significance for all studies was defined as a two-sided P value ≤ 0.05.Re-suspended cells were diluted to ~ 2.0 ×10th percentile significance envelope and had at least one concentration with an effect size> 20% (log10 ratio)> 0.1. Three cell systems  were the focus of our study due to detected annotated activities by C15:0. The SAg system mimics chronic inflammation and autoimmune disease. In this system, human primary venular endothelial cells and peripheral blood mononuclear cells were exposed to TCR ligands (1×) to model Th1 type chronic inflammation. The BT system mimics asthma, oncology, autoimmunity and allergy. In this system, human primary peripheral blood mononuclear cells and B cells were exposed to α-IgM and TCR ligands  to model T cell dependent B cell proliferation. The HDF3CGF system mimics fibrosis and chronic inflammation. In this system, human primary dermal fibroblasts were exposed to IFNγ, TNFα, IL-1β, EGF, bFGF, and PDGF-BB to model fibrosis and Th1-type inflammation. Effects of C13:0, C14:0, C16:0 and C17:0 (20 µM) on these three cell systems were assessed and compared to C15:0.It was hypothesized that, given its role as a natural PPARα/δ ligand, C15:0 would have anti-inflammatory and antifibrotic activities in human cell systems and that these activities would be better than other saturated fatty acids . To test this hypothesis, primary human cell systems from the BioMAP Diversity PLUS panel  mimicking inflammation and fibrosis were exposed to C15:0 at four concentrations . Quantitative measurements of 148 biomarker activities across this broad panel, along with comparative analysis of biological activities from known bioactive agents, were used to predict and compare the efficacy and function of C15:0 across these systems compared to non-supplemented control systems. Activated BioMAP systems were incubated with C15:0 for 24 to 72 hours. Biomarker activities were annotated when 2 or more consecutive concentrations changed in the same direction relative to at least six vehicle controls, were outside of the 9579.Cell-based activity studies demonstrated C15:0 anti-inflammatory and antifibrotic activities between 6.7 and 20 µM. As such, a 24-hour pharmacokinetic study was conducted with 8- to 10-week old male Sprague Dawley rats (body weights ranging from 260 to 288 g) to evaluate if a single oral dose of C15:0 at 35 mg/kg body weight via gastric gavage could achieve 20 µM concentrations in the serum . Briefly, six healthy rats were dosed once with deuterated C15:0 at 35 mg/kg orally in a methylcellulose suspension (10 mL/kg). The first 3 animals had blood collected pre-dose, and 15 min, and 1, 4, and 12 hrs post dosing. The final 3 animals had blood collected pre-dose, and 30 min, and 2, 8, and 24 hrs post dosing. Plasma deuterated C15:0 and C17:0 measurements were performed by the Genetics Laboratories at the Kennedy Krieger Institute. Fatty acids were analyzed by capillary gas chromatography/mass spectrometry of pentaflourobenzyl bromide fatty acid esters using an AT-Silar-100 column  as previously described79. At necropsy, liver, kidneys (combined), heart, and adrenal glands (combined) of animals were weighed and fixed in 10% neutral buffered formalin. Standard trimming, processing, slide preparation and histopathology of liver, kidney, heart, and adrenals from the vehicle (Group 4) and high dose (Group 3) groups were completed.The purpose of this study was to determine the safety of daily oral pentadecanoic acid at higher doses over 14 days in 7- to 8-week old Sprague Dawley rats. There were four main study groups, with 5 animals/sex/group. The first three groups received pentadecanoic acid daily via gastric gavage for 14 days in a 10 mL/kg suspension: Group 1 = 35 mg/kg, Group 2 = 175 mg/kg, Group 3 = 350 mg/kg doses. Group 4 was the vehicle control group. Animals received the daily oral doses on Days 0–13. Once daily clinical observations were made during acclimation, and twice daily clinical observations were made during the dosing period. Weekly cage food consumption was measured. Body weights were measured pre-study (2 times), on Day 0, then weekly . On Day 14, blood was collected and processed for clinical chemistries. Plasma total C15:0 and C17:0 measurements were performed by the Genetics Laboratories at the Kennedy Krieger Institute. Fatty acids were analyzed by capillary gas chromatography/mass spectrometry of pentaflourobenzyl bromide fatty acid esters using an AT-Silar-100 column  as previously describedForty 6-week old male C57BL/6J mice  were fed a high fat diet  for eight weeks. Animals were randomly assigned to one of the following four study groups (10 per group) on Day -1 based on body weight: Group 1 (HFD non-supplemented controls), Group 2 , Group 3 , and Group 4 . Group sizes for this pilot study were based on numerous published studies initially evaluating the effects of other natural small molecules on similar metabolic indices in the same model. Animals were provided the vehicle (1x PBS) or test articles daily via gastric gavage for 12 weeks. Animals were fed the HFD throughout the study. The study was conducted by Charles River Laboratories , where animals were housed singly in solid bottom polycarbonate cages with Beta-chip contact bedding and enviro-dri nesting material, with temperature and humidity ranges of 18° to 26 °C and 30 to 70%, respectively. A 12 hr light cycle was used, and water and food were provided ad libitum. The following blood-based variables were measured after 12 weeks of supplementation: glucose, insulin, triglycerides, cholesterol , IL-18, IL-6, monocyte chemoattractant protein 1 (MCP-1), plasminogen activation inhibitor 1 (PAI-1), tumor necrosis factor (TNF)α, free fatty acids, and ferritin. Body weights and body weight changes were recorded weekly throughout the study. Due to low sample sizes and inability to assume normal distributions, Wilcoxon tests were used to compare blood-based and body weight values between supplemented and non-supplemented controls; a two-sided P value ≤ 0.05 was defined as significant.Sixteen male New Zealand white rabbits, 12- to 13- weeks old and weighing approximately 3 kg upon ordering  were fed a high fat, high cholesterol diet  for two weeks. Animals were randomly assigned to one of the following two study groups (8 per group) on Day -1 based on body weight: Group 1 (HFHC diet supplemented controls) and Group 2 . Animal group numbers for this study were based on a power analysis on data from the prior mouse study; using a type-I error rate of 0.05  and 80% power for two independent study groups with a continuous endpoint, the minimum sample size per group was six. An additional negative control, low fat diet group  with 8 animals was included. Animals in Groups 1 and 2 were fed the HFHC diet throughout the supplement period (11 weeks). The study was conducted by PreClinical Research Services , where animals were housed singly in steel or plastic cages, with temperature ranges of 16° to 22 °C. A 12 hr light cycle was used, and water was provided ad libitum. Complete blood cell count, red blood cell indices, clinical chemistries, and iron panels were measured in blood samples collected from animals after 11 weeks of supplementation. Liver histology scoring, including fibrosis, hepatocyte ballooning, lobular inflammation, microgranuloma, steatosis, and iron deposition, was conducted by Charles River Pathology Services . Due to low sample sizes and inability to assume normal distributions, Wilcoxon tests were used to compare blood-based and liver histology values between supplemented and non-supplemented controls; a two-sided P value ≤ 0.05 was defined as significant.Supplementary information.


The immunomodulatory activity of flavonoids is increasingly appreciated. Macrophage phospholipids (PLs) play crucial roles in cell-mediated inflammatory responses. However, little is known on how these PLs are affected upon flavonoid treatment. In this work, we have used mass-spectrometry-based lipidomics to characterize the changes in the phospholipidome of proinflammatory human-macrophage-like cells (THP-1-derived and LPS+IFN-γ-stimulated) incubated with non-cytotoxic concentrations of three flavonoids: quercetin, naringin and naringenin. One hundred forty-seven PL species belonging to various classes were identified, and their relative abundances were determined. Each flavonoid displayed its own unique signature of induced effects. Quercetin produced the strongest impact, acting both on constitutive PLs  and on minor signaling lipids, such as phosphatidylinositol (PI) and phosphatidylserine (PS) species. Conversely, naringin hardly affected structural PLs, producing changes in signaling molecules that were opposite to those seen in quercetin-treated macrophages. In turn, albeit sharing some effects with quercetin, naringenin did not change PI and PS levels and interfered with a set of phosphatidylcholines distinct from those modulated by quercetin. These results demonstrate that flavonoids bioactivity involves profound and specific remodeling of macrophage phospholipidome, paving the way to future studies on the role of cellular phospholipids in flavonoid-mediated immunomodulatory effects. Inflammation is a complex physiological response to potentially harmful stimuli, with a fundamental role in protecting and restoring homeostasis . HoweverSeveral plant phenols display strong anti-inflammatory activity in vitro and in vivo , which mPhospholipids are not only the main building blocks of cell membranes, but they are also critically involved in cell signaling and membrane remodeling processes required for cells to perform multiple functions. Macrophage phospholipids play crucial roles in cell-mediated inflammatory responses, namely by providing precursors for the synthesis of potent lipid mediators, including proinflammatory eicosanoids, such as prostaglandins, and specialized pro-resolving mediators (SPMs) like resolvins . MoreoveThe phospholipid (PL) composition of THP-1-derived macrophage-like cells, activated by proinflammatory stimuli (LPS+IFN-γ) and subsequently treated with different flavonoids, was profiled using HILIC-LC-MS/MS, according to the experimental setup schematically represented in As a first approach to assess the impact of proinflammatory activation on the cells’ phospholipidome, we compared the relative abundances of the different PL classes in M0 and M1 macrophages (LPS+IFN-γ-stimulated) by considering the summed integrals of all peaks belonging to the same class. As shown in To identify the individual PL species affected by LPS+IFN-γ stimulation, multivariate analysis was applied. The two sample groups (M0 and M1) were clearly separated in the PCA and PLS-DA scores scatter plots . The varProinflammatory (M1) prepolarized macrophage-like cells were incubated for 24 h with non-cytotoxic concentrations of quercetin, naringin or naringenin. As reported in our preceding study , all flaMultivariate analysis of all the PL species identified further helped to reveal the specific effects produced by each flavonoid. In the PCA scores scatter plot obtained for the four groups compared , left, qA deeper understanding of the immunomodulatory activity of widely available flavonoids may contribute to expand their range of applications as nutraceuticals or anti-inflammatory drugs. Lipids are important players in immune-cell-mediated inflammatory responses, and hence, in this work, we have looked at how three different flavonoids modulate the phospholipidome of proinflammatory human macrophages. An initial comparison of LPS+IFN-γ-stimulated cells (M1 macrophages) with unstimulated (M0) macrophages corroborated the previously reported shift in the PL composition of THP-1-derived macrophages from short-chain saturated/monounsaturated FAs to long-chain polyunsaturated FAs upon M1 polarization . In lineThe relative abundances of several PC species were strongly and differentially affected by quercetin and naringenin, while showing milder changes upon naringin treatment. Quercetin increased the levels of eight diacyl PCs containing longer and more polyunsaturated FAs, while naringenin upregulated diacyl PCs with shorter and less unsaturated FAs. Additionally, while several of these changes were common to proinflammatory activation, naringenin caused diacyl PC (30:0) to increase, contrarily to the variation induced in M1 vs. M0 macrophages. Phosphatidylcholines are major components of mammalian cell membranes, with a key role in regulating membrane properties like the position of proteins, membrane fluidity and permeability . MoreoveThe levels of several alkyl-acyl and alkenyl-acyl  PCs were also markedly affected by flavonoids. Consistent increases were observed in eight of these species upon treatment with quercetin, while naringenin induced decreased levels of two other alkyl/alkenyl-acyl PCs. Additionally, three ether PEs were upregulated by the two flavonoids, especially by quercetin. Naringin modulated PC and PE ether lipids to a lesser extent and towards lower relative abundances, compared to M1-Ct. The functions of ether PLs in mammalian cells are still largely unknown, although there is increasing awareness of their importance in the regulation of membrane homeostasis and in cell signaling . For insThe lysoglycerophospholipids LPC 16:0, 18:0 and 18:1 and LPE 16:0, which showed depleted levels in M1 vs. M0 macrophages, increased their relative abundance in flavonoid-treated proinflammatory cells. Previous studies have proposed LPCs to be inflammatory mediators acting via multiple mechanisms, including the perpetuation of macrophage proinflammatory phenotype . On the Flavonoids additionally modulated PI and PS species, which are widely recognized as signaling molecules ,34,35. IWhile this study was designed to assess flavonoid-induced modulation of proinflammatory macrophages, the impact of the studied compounds towards unstimulated (M0) and anti-inflammatory, proresolution (M2-like) macrophages should be addressed in future work. Indeed, based on the close link between these cells’ phenotype and metabolism , we expeStock solutions of quercetin , naringenin  and naringin  were prepared with 99.5% DMSO at a concentration of 80 mM. Phorbol 12-myristate 13-acetate  was prepared in 99.5% DMSO at a concentration of 100 μg/mL. Powdered LPS  was dissolved in Gibco Water for Injection (WFI) at a concentration of 1 mg/mL. Interferon-γ  was kept in the original formulation at a concentration of 0.1 mg/mL. All solutions were kept at −20 °C and protected from light.2. THP-1 monocytes were differentiated into macrophages (M0) by 24-h incubation with 50 ng/mL PMA in RPMI medium, which was followed by a 24-h rest in new standard complete medium. Proinflammatory macrophage activation was carried out by incubation with 20 ng/mL IFN-γ and 100 ng/mL LPS for 24 h, and it was confirmed by increased production of proinflammatory cytokines, as reported in our previous work [Human monocytic THP-1 cells  were cultured in Roswell Park Memorial Institute (RPMI 1640) culture medium that contained 10% heat-inactivated fetal bovine serum  and 1% penicillin/streptomycin and was supplemented with 2.0 g/L sodium bicarbonate. Cells were maintained in culture in a humidified atmosphere at 37 °C and 5% COous work .6 cells/mL in cell-culture-treated 100-mm Petri dishes, differentiated and polarized to proinflammatory M1 cells as described above. Then, macrophages were treated with quercetin (60 µM), naringin (200 µM), naringenin (100 µM), or medium alone (M1-Ct) for a period of 24 h. The flavonoid concentrations used were the highest concentrations tested that did not cause significant reduction of cell viability, as determined by the Alamar Blue assay  were seeded at a density of 1 × 10s et al. ). v/v)  was added to the dish to quench metabolic activity, and cells were scrapped off. The cell suspension was transferred into a glass tube containing 0.5-mm glass beads (to aid cell breakage). After 2 min of vortexing, 400 µL of cold chloroform  were added to each tube, followed by vortexing and another addition of 400 µL of chloroform and 360 µL cold milli-Q water. Samples were vortexed again and left to rest on ice for 20 min. Then, after centrifugation at 3000 × g for 10 min, the bottom organic phase was transferred into an amber glass vial, and the remaining sample was re-extracted with 400 µL of chloroform to collect the organic phase again and add it to the previous vial. Organic extracts containing lipids, obtained from five cell-sample replicates for each condition, were then dried under a nitrogen gas stream and stored at −80 °C.Lipids were extracted according to a modified Bligh and Dyer extraction protocol ,44, usin2PO4.2H2O). Dried aliquots of samples and standards were resuspended in 125 µL of perchloric acid. Samples were then heated at 180 °C in a heating block  for 40–60 min, followed by cooling at room temperature. Thereafter, 825 μL of milli-Q water, 125 μL of 2.5% ammonium molybdate solution and 125 μL of 10% ascorbic acid were added to samples and standards, which were vortexed after each addition and incubated for 10 min at 100 °C in a water bath. After cooling in a cold-water bath, the absorbance of samples and standards was measured at 797 nm in a microplate reader . Four independent measurements were performed for each sample.In order to determine the total phospholipid (PL) content in each lipid extract, the phosphorous assay was performed according to Bartlett and Lewis . PhosphaDried samples were dissolved in dichloromethane to obtain a PL concentration of 1 µg/µL. A volume of 5 µL was taken from each sample and transferred to an appropriate vial, followed by addition of 91 µL of a solvent system consisting of two mobile phases in a proportion of 60% eluent B  and 40% eluent A  and 4 µL of an internal standard mixture containing PC (14:0/14:0), 0.02 µg; PE (14:0/14:0), 0.02 µg; PG (14:0/14:0), 0.012 µg; PI (16:0/16:0), 0.08 µg; PS (14:0/14:0), 0.04 µg; PA (14:0/14:0), 0.08 µg; LPC (19:0), 0.02 µg; Ceramide (d18:1/17:0), 0.04 µg; SM (d18:1/17:0), 0.02 µg; and CL (14:0/14:0/14:0/14:0), 0.08 µg. m/z values of 200–1600, with a resolution of 70.000, automatic gain control of 1 × 106 and 2 microscans. The 10 most abundant ions were selected for ion fragmentation at collision cell HCD. Collision energy varied between 25, 30 and 35 eV. The tandem mass spectra (MS/MS spectra) were obtained at a resolution of 17.500, automatic gain control of 1 × 105, 1 microscan and an isolating window of 1 m/z. Ion selection was limited to 2 × 104 counts. Maximum accumulated ions were established at 100 ms for MS spectra and 50 ms for MS/MS spectra. Dynamic exclusion was defined as 60 s.Phospholipids were separated through hydrophilic interaction liquid chromatography (HILIC-LC) using an Ascentis Si HPLC Pore  and a high-performance liquid chromatography (HPLC) system  with an autosampler coupled online to the Q-Exactive hybrid quadrupole Orbitrap mass spectrometer . An aliquot of 5 µL of each sample mixture was injected into the HPLC column at a flow rate of 40 µL/min and a temperature of 30 °C. Initially, 40% of mobile phase A was held isocratically for 8 min, followed by a linear increase to 60% of mobile phase A for 7 min, a 5 min maintenance period and the return to initial conditions within 5 min. A 10-min interval was given between injections to allow return to equilibrium. The mass spectrometer with a Q-Exactive orbitrap and a heated electrospray ionization (HESI) operated simultaneously in positive mode (voltage of 3 kV) and negative mode (voltage of −2.7 kV). The sheath gas flow rate was maintained at 15 units and the capillary temperature was 250 °C, while S-lenses RF was of 50 units and the probe’s temperature was 100 °C. Mass spectral acquisition method was at full scan in a scale of 5 were excluded. Relative quantification was performed by exporting peak area values into a computer spreadsheet . For normalizing the data, peak areas of the extracted-ion chromatograms (XICs) of each lipid molecular ion were divided by the sum of total XIC areas of the identified lipid molecular ions.Data acquisition was performed using the software Xcalibur v3.3 . Identification of the different lipid species was performed based on retention time, exact mass measurements of the ions assigned as lipid species (less than 5 ppm) and on manual interpretation of the MS/MS spectra. All the MS/MS fragmentation patterns characteristic of the lipid classes analyzed in the present study were acquired in positive and negative ion modes, as reported previously ,47, and Multivariate analysis of data matrices  was performed using Metaboanalyst . The datThis work demonstrates that the flavonoids quercetin, naringin and naringenin differentially modulate the phospholipidome of proinflammatory THP-1-derived macrophages. Quercetin produced the strongest impact, acting both on more abundant constitutive PLs  and minor signaling lipids, like a few PI and PS species. On the other hand, naringin hardly affected structural PLs, producing changes in PI and PS species that were opposite to those seen in quercetin-treated macrophages. In turn, albeit sharing some effects with quercetin, naringenin did not change PI and PS levels and interfered with a set of PCs distinct from those modulated by quercetin. These results highlight macrophage plasticity and stimuli-dependent responses, contributing to revealing the intricate bioactivity of flavonoids and exploring their pharmacological potential in modulating proinflammatory processes.


Owing to the strong inter or intra molecular hydrogen bonding, TATB shows poor solubility in most solvents. As the result, the particle shape and size of TATB products is hard to regulate, which closely related to the weapons stability. Herein, a new recrystallization method is provided to refine TATB using bicarbonate ionic liquids. Bicarbonate ionic liquids exhibited the record solubility (26.7 wt%) for dissolving TATB explosive. The recrystallized TATB were spherical particles with uniform size and showed extremely insensitivity to impact (>100 J) and friction (>360 N). Moreover, the experimental  These pollutions are considered as the most serious nuclear weapon accidents ever occurred7. For this reason, the related militaries and governments invested a great deal of effort to ensure the safety of strategic weapons. In 1970s, a new type of energetic materials called insensitive high explosives (IHE) were developed and applicated9. Among them, 1,3,5-triamino-2,4,6-trinitrobenzene (TATB) is the most attractive and famous IHE. TATB has many unique advantages including high density, low toxicity and low vapor evaporation, which are suitable for long-term storage of the strategic weapons11.It is well known that traditional energetic compounds like 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5-triazine (RDX) are cheap, easily prepared with good performance. However, most classical energetic materials are not qualified for strategic weapons because of their unsatisfactory mechanical stability. This apparent defect is an important reason leading to the Palomares H-bomb accident and Thule air base nuclear weapon accident, when 6 of the 8 nuclear bombs detonated by air crash, causing more than 2000 m12. This method is also of great significance for improving the stability of TATB and ensuring the safety of strategic weapons. Similar to many insoluble hydrogen-bond-rich materials14, TATB has strong intramolecular and intermolecular hydrogen bonding interactions, leading to a closely packed structure. Thus, TATB is insoluble in common solvents, causing a series refining problems during its production and application16. Dimethyl sulfoxide (DMSO) is currently used for recrystallizing TATB. Whereas, solubility of TATB in DMSO is too low (0.7 wt%) in room temperature and the solution need be heated at least 135 °C before carrying out17. Concentrated sulphuric acid (H2SO4) can dissolve TATB up to 20 wt%18, no significant improvement on quality of TATB can be assured after refinement19. For other nucleophilic polar solvents like 40% NaOH solutions or ammonia (NH3 H2O), new impurities such as 1,3,5-trihydroxy-2,4,6-trinitrobenzene (THTNB) lower the sensitivity of recrystallized TATB products20. Besides, these harsh recrystallization solvents are also a challenge to equipment, and harmful to environment and human body. So, mild solvents with high solubility for refining TATB is much looking forward to.Recrystallization is a favorable approach to increasing the quality of energetic compounds, for instance, improving the stability of 1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX)23. The task-specific ionic liquids have many advantages in dissolution and extraction25. Ionic liquids have been reported to dissolve a series of hydrogen-bond-rich natural products and 2D layered materials including cellulose, chitin, WS2, and graphene, showing their great potential in biomass, catalysis, solar cell and semiconductor27. For this reason, finding a kind of ionic liquid as TATB recrystallizing solvent will overcome the shortcomings of molecular solvents. Therefore, developing new kinds of ionic liquids to solve TATB’s refining problem is of great interesting in the field of energetic materials.Compared with molecular solvents, ionic liquids are green designable materials exhibiting unique physical and chemical behaviors3) and N,N,N,N-tetrapropylammonium bicarbonate (N3333HCO3) ionic liquids.  and DMF (0.0027 wt%) currently in use. Bicarbonate ionic liquids also have a higher solubility than the harsh solvents including concentrated H2SO4 (20 wt%) and NaOH (6.1 wt%)The solubility is measured by UV-Vis spectrophotometer from room temperature to 115 °C. Fig.  BmimHCO31H and 13C NMR spectra of TATB in solution were collected for the first time. 29. Besides, the IR spectra of N3333HCO3 as solvent did not have obvious change from room temperature to 110 °C. TATB maintains its own structure and spectral properties in bicarbonate ionic liquids, which are different from other nitrobenzene intermediates30.To better understand the solubility behaviour of TATB in bicarbonate ionic liquids, IR spectra of saturated TATB in Nes. Fig.  The spec3 and N3333HCO3 are decreased obviously to 187 mPa·s and 171 mPa·s, respectively. Compared with the common recrystallization solvents like DMSO (1.99 mPa·s) and H2SO4 (23.8 mPa·s), BmimHCO3 and N3333HCO3 are still in a higher viscosity but have no effect on TATB dissolution in bicarbonate ionic liquids, which means the solvation of TATB is dominated by other physicochemical parameters of the solvents.We also investigated the viscosity of bicarbonate ionic liquids under the standard atmospheric pressure Fig. . Owing t3 and N3333HCO3 ionic liquids were assessed by thermogravimetric analysis (TGA)  Fig. . TGA res3333HCO3 recrystallized TATB exhibits smaller particles size under the higher magnification. Angular and defective particles composed of tightly packed flaky crystals are observed in the raw TATB . In comparison, refined TATB  exhibited smooth sphere and uniform size after recrystallized by BmimHCO3, and the TATB product  has smaller particles with regular size distribution after recrystallization by N3333HCO3. Different orientation of crystalline growth is an important reason causing obvious defects of raw TATB. Furthermore, the size distribution histograms were performed in column (e) in Fig. 3333HCO3 recrystallized TATB 3 displays much more regular particle size within 2.0 ± 0.5 μm. Bicarbonate ionic liquids make TATB a smaller size and a more regular morphology.The particle shape and size of energetic compounds are closely related to the sensitivity. We analyzed the morphology and particle size of raw and recrystallized TATB by optical microscopy (OM) and scanning electron microscopy (SEM). Fig.  The appe31, the TATB recrystallized by BmimHCO3 and N3333HCO3 are much more insensitive against impact. This phenomenon is attributed to the improved morphology of recrystallized TATB, which can be explained by hot spot theory of Bowden et al.32. Hot spots can be reduced by modifying the particles shape, surface roughness and size of energetic compounds. The defective particles in raw TATB are modified by recrystallization of bicarbonate ionic liquids, which can reduce the quantity of hot spots generated by external stimulus. Besides, with uniform particle surface and particle size, recrystallized TATB particles can lower the probability of hot spots and disperse the localized heat in time.Common TATB explosive owns a low mechanical sensitivity with 50 J to impact and >360 N to friction. Table  Lowering3 and N3333HCO3 ionic liquids. The superior TATB has great potential for replacing existing TATB products, and it will make the weapons more secure. Furthermore, this method provides a new idea for dissolving the insoluble hydrogen-bond-rich materials. After obtaining high concentration solutions of hydrogen-bond-rich materials, NMR analysis could be introduced to study more details on their structures and properties.In conclusion, we provided a new strategy to greatly improve the solubility and impact stability of TATB explosive. The super impact intensive TATB was obtained by recrystallization using BmimHCO1H and 13C NMR spectra are recorded by Bruker AVANCE III HD with magnetic intensity 9.4 T and resonance frequency of 400 MHz and 100 MHz respectively. The samples are dissolved in locking solvent d6-DMSO and their chemical shifts are analyzed and recorded in ppm by compared with the internal standard tetramethylsilane (TMS). Differential scanning calorimetry (DSC) test were taken on TA Q20 calorimeter with nitrogen as shield gas and indium as standard. The temperatures were recorded from −80 °C to 400 °C in a heading speed of 10° min−1. Thermogravimetric analysis (TGA) were finished on NETZSCH TG 209F1 with nitrogen as shield gas of 70 mL min−1 and heating from 25 to 500 °C at 0.1 MPa in 10 °C min−1 Hydrogen, carbon and nitrogen elemental analyses is finished by Elementar Vario MICRO CUBE instrument. Schimadzu-xrd-6100 recorded PXRD data from 5° to 80 ° in a rate of 0.2°/min. Microscopic morphologies of the TATB were taken by HLP-85C polarizing microscope. The SEM test were obtained by Hitachi TM3000. Sensitivity were test by BCJ drop hammer testing machine and BMC friction testing machine.All chemical reagents were purchased in analytical grade. IR were recorded on Bruker ALPHA infrared spectrometer by using attenuated total reflection (ATR) mode. 34. Single-point energies of all the molecules were also calculated at the level of B3LYP/6-311 + G36. The optimized structures were get at the local energy minima on their potential energy surfaces and without any imaginary frequency. The NMR analysis results of TATB molecule was shown and analyzed by GaussView 5.0 program37.The computations were taken by the Gaussian09 (Revision A.02) programs to analysis the geometric optimization and frequencies of molecules by using functional group B3LYP and basis set 6-311 + G38, the sensitivity was performed by BAM method. The sample of raw TATB and recrystallized TATB is enclosed in a special impact device which receiving and transferring the energy by drop weight. The impact sensitivity of explosive can be defined as the lowest impact energy leading to the explosive decomposed from at least one out of at least six trials. Impact energy is calculated from the drop weight (kg), the acceleration of gravity (m/s2) and the fall height (m). If the sample at lower energy is no reaction, the test is continued with increased impact energies until the explosion is observed firstly. As for friction sensitivity test, the sample is test between the porcelain plate and peg at a load up to 360 N. The friction sensitivity of explosive can be defined as the highest friction load leading to the explosive decomposed from at least one out of at least six trials. If the sample at highest friction load (360 N) is no reaction, the result should be recorded as >360 N.According to the UN Recommendations on the Transport of Dangerous Goods-Tests and Criteria2 atmosphere at room temperature for 4 h. A colorless solution was gotten after filtered the precipitate. Excess carbon dioxide was bubbled into the solution in air at room temperature. After removing the ethanol solvent and dried in the vacuum for 24 hours, a pale-yellow liquid of BmimHCO3 was obtained in 83% yield. 1H NMR : δ = 9.30 , 7.85 , 7.77 , 4.20 , 3.88 , 1.78 , 1.26 , 0.90  ppm. 13C NMR : δ = 167.45, 136.49, 123.53, 122.22, 48.40, 39.52, 35.74, 31.33, 18.72, 13.24. IR (ATR): ν = 3420 (m), 3082 (s), 2959 (vs), 2868 (s), 2733 (w), 1701 (m), 1620 (s), 1566 (vs), 1459 (s), 1422 (m), 1373 (m), 1333 (m), 1307 (w), 1279 (w), 1248 (w), 1212 (w), 1159 (vs), 1134 (m), 1117 (m), 1083 (m), 1051 (m), 1013 (m), 956 (w), 891 (s), 835 (w), 808 (s), 753 (s), 699 (m), 655 (s), 631 (s), 412 (m).Solid NaOH  was added in batches into 20 mL ethanol solution of 1-butyl-3-methylimidazolium bromide  in Schlenk tube. The resulting mixture was stirred in N2 atmosphere at room temperature for 4 h. A colorless solution was gotten after filtered the precipitate. Excess carbon dioxide was bubbled into the solution in air at room temperature. After removing the solution and dried in the vacuum for 24 hours, a colorless liquid of N3333HCO3 was obtained in 87% yield. 1H NMR : δ = 3.15 , 1.63 , 0.90 . 13C NMR : δ = 158.10, 59.26, 14.80, 10.53. IR (ATR): ν = 3397 (m), 2962 (s), 2931 (s), 2879 (s), 2735 (m), 2674 (m), 1855 (m), 1641 (vs), 1490 (s), 1460 (s), 1381 (vs), 1368 (vs), 1334 (s), 1274 (m), 1189 (w), 1159 (w), 1105 (m), 1060 (s), 1031 (s), 1008 (s), 972 (vs), 908 (w), 835 (s), 768 (s), 683 (s), 645 (w).Solid NaOH  was added in batches into 20 mL ethanol solution of N,N,N,N-tetrapropylammonium chloride  in Schlenk tube. The resulting mixture was stirred in NSupplementary Information.