M. tuberculosis, remains one of the top 10 causes of death worldwide. Macrophages, the first cells to encounter M. tuberculosis and critical for defense against infection, are hijacked by M. tuberculosis as a protected growth niche. M. tuberculosis-infected macrophages undergo metabolic reprogramming where key mitochondrial pathways are modulated, but the mechanisms driving this metabolic shift is unknown.Tuberculosis, the disease caused by the bacterium  Mycobacterium tuberculosis induces metabolic reprogramming in macrophages like the Warburg effect. This enhances antimicrobial performance at the expense of increased inflammation, which may promote a pathogen-permissive host environment. Since the NAD+-dependent protein deacetylase Sirtuin 3 (SIRT3) is an important regulator of mitochondrial metabolism and cellular redox homeostasis, we hypothesized that SIRT3 modulation mediates M. tuberculosis-induced metabolic reprogramming. Infection of immortalized and primary murine macrophages resulted in reduced levels of SIRT3 mRNA and protein and perturbation of SIRT3-regulated enzymes in the tricarboxylic acid cycle, electron transport chain, and glycolytic pathway. These changes were associated with increased reactive oxygen species and reduced antioxidant scavenging, thereby triggering mitochondrial stress and macrophage cell death. Relevance to tuberculosis disease in vivo was indicated by greater bacterial burden and immune pathology in M. tuberculosis-infected Sirt3−/− mice. CD11b+ lung leukocytes isolated from infected −/−Sirt3 mice showed decreased levels of enzymes involved in central mitochondrial metabolic pathways, along with increased reactive oxygen species. Bacterial burden was also greater in lungs of creSirt3L2/L2LysM mice, demonstrating the importance of macrophage-specific SIRT3 after infection. These results support the model of SIRT3 as a major upstream regulatory factor, leading to metabolic reprogramming in macrophages by M. tuberculosis. Mycobacterium tuberculosis. The bacillus blocks phagosome maturation, reprograms metabolism, and induces cell death in infected macrophages (M. tuberculosis skews macrophage metabolism from oxidative phosphorylation (OXPHOS) toward aerobic glycolysis, comparable to the Warburg effect. This altered metabolic state may represent a host defense to fuel a rapid inflammatory response  , 12, a fproteins . SIRT3, proteins –18 and iroteins –. Kim et mulation . HoweverM. tuberculosis infection of macrophages downregulated SIRT3 in a TLR2-dependent manner, resulting in perturbation of enzymes in the tricarboxylic acid (TCA) cycle, electron transport chain (ETC), and glycolytic pathways. This led to increased mitochondrial ROS (mtROS), and cell death. Using SIRT3-deficient primary bone-marrow derived macrophages (BMDM) and CD11b+ lung leukocytes isolated from M. tuberculosis-infected −/−Sirt3 mice, we show that the changes in TCA, ETC, and glycolytic enzymes were regulated in vitro and in vivo in a SIRT3-dependent manner. Finally, SIRT3 was shown to be required for optimal host protection against M. tuberculosis infection in vivo. We propose that SIRT3 modulation by M. tuberculosis is a key upstream event leading to metabolic reprogramming and macrophage dysfunction in tuberculosis (TB).Here, we show that M. tuberculosis Erdman infection on the expression of genes involved in central metabolic pathways using J2-immortalized murine macrophages and wild-type (WT) primary C57BL/6J mouse BMDM and resident peritoneal macrophages (RPM). The expression of hexokinase 2 (Hk2), the platelet isoform of phosphofructokinase (Pfkp), and glucose transporter 1 (Glut1), three rate-limiting enzymes in the glycolytic pathway  were decreased or unchanged (Hk2 at 24 h p.i. (see M. tuberculosis promotes glycolysis (M. tuberculosis-infected J2 macrophages (G6pd) and 6-phosphogluconate dehydrogenase (Pgd) were also increased  Gene expression in mouse resident peritoneal macrophages (n = 6 mice) infected with M. tuberculosis . The data are depicted as mean relative expression levels ± the SD. Sirt3, sirtuin 3; Idh2, isocitrate dehydrogenase 2; Ndufv3, NADH:ubiquinone oxidoreductase subunit V3; Ndufs7, NADH:ubiquinone oxidoreductase core subunit S7; Hk2, hexokinase 2. (B) Gene expression of Idh2 in mouse BMDM infected with M. tuberculosis for 24 or 48 h. The data are expressed relative to uninfected conditions at 24 h and show the means ± the SD of three independent experiments. (C and D) Gene expression of the ETC complex I subunits, NADH:ubiquinone oxidoreductase core subunit S7 (Ndufs7) and NADH:ubiquinone oxidoreductase subunit V3 (Ndufv3), in mouse BMDM infected with M. tuberculosis at 24 h (C) and 48 h (D) p.i. The data are expressed relative to uninfected conditions and error bars show the means ± the SD of three independent experiments. (E) Gene expression of Ndufs2 and Ndufs7, NADH:ubiquinone oxidoreductase core subunit S2 and S7, and Ndufv3, NADH:ubiquinone oxidoreductase subunit V3, in J2 macrophages infected with M. tuberculosis for 48 h. The data are expressed relative to uninfected conditions and analyzed as in panel A. (F and G) complex II, III, and IV protein subunits of the ETC in J2 macrophages infected with M. tuberculosis for 24 h (F) and 48 h (G) p.i. Gene expression was analyzed as described for panel A. Sdha and Sdhd, succinate dehydrogenase, subunits A and D; Uqcrc2, ubiqinol-cytochrome c reductase core protein II; Cox5a, cytochrome c oxidase subunit 5A. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Download FIG S1, PDF file, 0.3 MB.Copyright © 2021 Smulan et al.2021Smulan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Idh2), a TCA cycle enzyme that catalyzes the decarboxylation of isocitrate to 2-oxoglutarate  cycle and ETC components were analyzed. Expression of isocitrate dehydrogenase 2 , and NADH:ubiquinone oxidoreductase subunit V3 (Ndufv3) were transcriptionally downregulated in J2 macrophages at 24 h p.i. (Ndufs7 and Ndufv3 (see Ndufv3 (see Sdha) and subunit D (Sdhd), as well as the complex III subunit ubiquinol-cytochrome c reductase core protein 2 (Uqcrc2), were decreased by 48 h p.i.  in macrophages. We found that the GSH/GSSG ratio declined more than 2-fold in J2 macrophages at 24 h p.i. (M. tuberculosis-infected cells despite a 4-fold increase in Sod2 gene that detoxifies superoxide (Atf5) and heat shock protein 10 (Hsp10), mediators of the UPRmt associated with mtROS accumulation . Fi. FiM. tumulation –33, incrulation –. These r10.1128/mBio.03140-20.2FIG S2M. tuberculosis alters cellular redox in macrophages. (A and B) Analysis of reduced (GSH) (A) and oxidized (GSSG) (B) glutathione in mouse J2 macrophage total cell lysate infected with M. tuberculosis (MOI of 10) for 24 h. Error bars show the means ± the SD of four independent experiments. *, P < 0.05. Download FIG S2, PDF file, 0.04 MB.Copyright © 2021 Smulan et al.2021Smulan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. tuberculosis infection, we investigated the expression of SIRT3, an enzyme that is known to regulate mitochondrial metabolism and cellular redox homeostasis  Gene expression of mammalian sirtuins  was analyzed in wild-type (WT) and −/−Sirt3 BMDM infected with M. tuberculosis . Genes are expressed relative to uninfected WT BMDM and show the means ± the SD of three independent experiments. (B) Sirtuin 3 gene (Sirt3) expression in mouse BMDM infected with M. tuberculosis 24 or 48 h. The data are shown relative to uninfected macrophages at 24 h p.i., and error bars show the means ± the SD from three independent experiments. (C) Analysis of Sirt3 gene expression in J2 macrophages infected with M. tuberculosis for 3, 6, 18, and 24 h. Genes are expressed as relative to uninfected macrophages and show the means ± the SD of three independent experiments.  Representative immunoblot showing protein expression of SIRT3 and β-ACTIN in primary mouse BMDM total cell lysate infected with M. tuberculosis as in panel A. (Right) Densitometry analysis of immunoblots. The data are normalized to β-actin and expressed relative to uninfected conditions. Plots show the means ± the SD of three independent experiments. (E to H) Gene expression of sirtuin 3 (Sirt3) (E), the ETC complex I subunits, NADH:ubiquinone oxidoreductase core subunit S7 (Ndufs7) and NADH:ubiquinone oxidoreductase subunit V3 (Ndufv3) (F), isocitrate dehydrogenase 2 (Idh2) (G), and hexokinase-2 (Hk2) (H) in wild-type (WT) and −/−/Trif−/−Myd88 BMDM infected as described for panel A. Genes are expressed relative to uninfected WT BMDM and expressed as the means ± the SD of four independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Download FIG S3, PDF file, 0.5 MB.Copyright © 2021 Smulan et al.2021Smulan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. tuberculosis. To test that hypothesis, we challenged −/−/Trif−/−Myd88 double knockout BMDM lacking signal adapters essential for all TLR signaling  ligand lipopolysaccharide , suggestignaling . As predp.i. see . Next, wrophages . Like Sigulation  to H. AlM. tuberculosis  or the scrambled control sequence 24 h before infection with M. tuberculosis . The gene expression of Sirt3 (A), Idh2 (B), Ndufs7 (C), and Ndufv3 (D) was analyzed, and genes are shown relative to uninfected macrophages expressing the scrambled control. The data are depicted as the means ± the SD of three independent experiments. Sirt3, sirtuin 3; Idh2, isocitrate dehydrogenase 2; Ndufs7, NADH:ubiquinone oxidoreductase core subunit S7; Ndufv3, NADH:ubiquinone oxidoreductase subunit V3. (E) Gene expression of phosphofructokinase, platelet (Pfkp), muscle (Pfkm), and liver (Pfkl) types was analyzed in WT and −/−Sirt3 BMDM with infection conditions as described for panel A. Genes are shown relative to uninfected WT BMDM and expressed as the means ± the SD of three independent experiments. (F) Analysis of reduced (GSH) to oxidized (GSSG) glutathione in WT and −/−Sirt3 BMDM total cell lysate with infection conditions as described for panel A. The data are expressed relative to uninfected WT BMDM and show the means ± the SD of four independent experiments. (G) Sirt3 gene expression in J2 macrophages expressing single guide RNA for Sirt3 (sgSirt3) or the nontemplate control (NTC). The data are expressed relative to NTC and show the means ± the SD of three independent experiments. (H) Gene expression of activating transcription factor 5 (Atf5) was analyzed in WT and −/−Sirt3 BMDM with infection conditions as described for panel A. The data are expressed relative to uninfected WT BMDM and shown as the means ± the SD of three independent experiments. (I) Cell viability of WT, −/−Sirt3 and Phox–/–gp91 BMDM treated with the ROS scavenger MitoTEMPO (500 μM) or vehicle and infected as described for panel A. The data are expressed relative to uninfected, untreated WT BMDM and show the means ± the SD of five independent experiments. (J) Mitochondrial ROS (mtROS) analysis in WT, −/−Sirt3, and phox–/–gp91 BMDM treated with the ROS scavenger MitoTEMPO  or vehicle and infected as in panel A. The data are expressed relative to uninfected, untreated WT BMDM and show the means ± the SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Download FIG S4, PDF file, 0.5 MB.SIRT3 regulates genes involved in central metabolism. (A to D) J2 macrophages were transfected with silencing RNA for Copyright © 2021 Smulan et al.2021Smulan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the −/−Sirt3 BMDM. Expression of Hk2 was 1.6-fold higher in uninfected −/−Sirt3 BMDM compared to WT  prevented M. tuberculosis-induced mtROS accumulation in WT BMDM and partially prevented accumulation in −/−Sirt3 BMDM  Wild-type (WT) and −/−Sirt3 mice (n = 5 per group) were infected with M. tuberculosis Erdman by the aerosol route, and the CFU in lung (A) and spleen (B) homogenates were analyzed at 14 and 28 days p.i. The data are expressed as the means ± the SD. (C and D) CFU in lung (C) and spleen (D) homogenates from M. tuberculosis-infected female L2/L2Sirt3 and creSirtL2/L2LysM mice (n = 4 to 6 per group) were analyzed at 16 weeks p.i. The data are expressed as the means ± the SD. *, P < 0.05. Download FIG S5, PDF file, 0.3 MB.Bacterial burden in whole body Copyright © 2021 Smulan et al.2021Smulan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. tuberculosis shifts mitochondrial bioenergetic metabolism toward aerobic glycolysis in human and murine macrophages  provokes rapid mortality  and increased bacterial burden in −/−Sirt3 mice -Sirt3tm1.1Fwa/J, JAX 027975] mice (Lyz2 (B6.129P2-Lyz2tm1(cre)lfo/J, JAX 004781) and L2/L2Sirt3 mice , obtained from The Jackson Laboratory  . Mice were housed in the Animal Medicine facility at UMMS, where experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee and Institutional Biosafety Committee.Age-matched male C57BL/6J WT (JAX 000664) and 75] mice  were obtbor, Me) , were creviously . Myd88−/−/−Tlr2 and −/−Tlr4 mice, developed as described previously  or nontargeting control siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific). For BMDM, bone marrow cells from 8- to 10-week-old age-matched male C57BL/6J, −/−Sirt3, phox–/–gp91, or −/−/Trif−/−Myd88 mice were differentiated for 7 days in complete DMEM supplemented with 20% L929 supernatant. For all experiments, macrophages were split using 0.05% trypsin-EDTA, seeded at 6 × 105 cells/ml, and incubated at 37°C with 5% CO2 for 24 h before infection. RPM were obtained by washing the peritoneum with complete DMEM and isolating CD11b+ cells using magnetic bead separation (Miltenyi Biotec). CD11b+ cells were seeded at 1 × 106 cells/ml and maintained at 37°C with 5% CO2 for 24 h before infection.Murine J2-immortalized macrophages from (WT) C57BL/6J mice, eviously , were cut test was used to analyze significance. One-way analysis of variance (ANOVA) was used when comparing three or more groups, and a log-rank (Mantel-Cox) test was used for the survival study. A P value of <0.05 was considered statistically significant.GraphPad Prism 7.0 was used for statistical analysis. The results of experiments show combined data from three to four individual experiments and are expressed as the means ± the standard deviations (SD). For comparison to uninfected control cells, Student 10.1128/mBio.03140-20.6TABLE S1Sirt1 to Sirt7, Idh1, Idh2, Nduf2, and Nduf7 are indicated. Download Table S1, PDF file, 0.1 MB.Primer sequences for real-time PCR. Primer sequences for Copyright © 2021 Smulan et al.2021Smulan et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Peganum harmala L., Zygophyllum album, Anacyclus valentinus L., Ammodaucus leucotrichus, Lupinus albus, and Marrubium vulgare in Algerian empirical medicine prompted our interest in evaluating their antidiabetic activity by screening their free radical scavenging (DPPH), α-glucosidase, and nitric oxide (NO) inhibitory activities as well as the total phenolic content (TPC). Extracts of the selected plants were prepared using different ratios of ethanol . In this study, 100%, and 80% ethanol extracts of L. albus were found to be the most potent, in inhibiting α-glucosidase activity with IC50 values of 6.45 and 8.66 μg/mL, respectively. The 100% ethanol extract of A. leucotrichus exhibited the highest free radical scavenging activity with an IC50 value of 26.26 μg/mL. Moreover, the highest TPC of 612.84 μg GAE/mg extract was observed in M. vulgare, extracted with 80% ethanol. Metabolite profiling of the active extract was conducted using 1H-NMR metabolomics. Partial least square analysis (PLS) was used to assess the relationship between the α-glucosidase inhibitory activity of L. albus and the metabolites identified in the extract. Based on the PLS model, isoflavonoids , amino acids (asparagine and thiamine), and several fatty acids (stearic acid and oleic acid) were identified as metabolites that contributed to the inhibition of α-glucosidase activity. The results of this study have clearly strengthened the traditional claim of the antihyperglycemic effects of L. albus.Claims of effective therapy against diabetes using plants including  Peganum harmala L., Zygophyllum album, Anacyclus valentinus L., Ammodaucus leucotrichus, Lupinus albus, and Marrubium vulgare were selected on the basis of their ethnomedical records as antidiabetic agents and also due to the fact that they might contain several bioactive compounds against DM [Diabetes mellitus (DM) is the most widely recognized endocrine disorder in almost all countries. According to the World Health Organization (WHO) 2016), an estimated 1.6 million deaths were directly caused by diabetes. Due to the adverse effects of modern treatment such as hypoglycemic coma, renal complications, stomach upset, and weight gain, the use of medicinal plants as antidiabetic agents has been gaining increasing attention due to their effectiveness. The latter is strongly supported by several empirical therapies and clinical experiences accumulated over hundreds of years [6, an estP. harmala L. (family Nitrariaceae) is also known as Harmal or Suryin Rue [P. harmala possesses analgesic and anti-inflammatory properties [P. harmala are alkaloids that are found especially in the seeds and roots. These include β-carbolines such as harmine and harmaline , harmalol, harman, and the quinazoline derivatives; and vasicine and vasicinone [Z. album (family Zygophyllaceae) has been used as a diuretic, local anesthetic, antihistaminic, and antidiabetic agent [Z. album have demonstrated the presence of alkaloids, flavonoids, and saponins as the major compounds. Furthermore, it has been reported that Z. album contains glycosides, coumarins, sterols and/or triterpenes, tannins, and cardiac glycosides [A. valentinus L. (family Asteraceae), also known as “Gartoufa”, is a herb used by the Saharan population of Algeria for treating several diseases including diabetes. Phytochemical analysis of the aqueous extract of this species demonstrated the presence of a variety of compounds such as tannins, saponins, flavonoids, cardiac glycosides, coumarins, alkaloids, mucilage, and amino acids [A. valentinus possesses significant antidiabetic activity and the crude aqueous extract also contains certain mineral elements such as K and Ca, which have been claimed to participate in insulin secretion [A. leucotrichus (family Apiaceae), commonly known as “Hairy Cumin’’, is widely used in North African countries as a condiment or spice and in traditional medicine as a cure for cold, fever, and digestive disorders, particularly in children [A. leuchotrichus, but with the exception of a guaianolide and amolactone, there is not much information available concerning the chemical constituents of A. leuchotrichus [ryin Rue . Currentoperties , wound hoperties , and hypoperties  activitisicinone . In folkic agent . Phytochycosides . A. valeno acids . A. valeecretion . A. leucchildren . A few sotrichus .M. vulgare (family Lamiaceae) is known by the name Marriouth in Algeria; it has been frequently used in folk medicine to treat a variety of maladies. The plant has been reported to possess hypoglycemic [l-malic acid, acteoside, phenylethanoid glycoside, marruboside [L. albus (family Fabaceae) are also known to possess diuretic, emmenagogue, hypoglycemic, and vermifuge activities [L. albus is also a rich source of proteins (33–47%), oils (6–13%), and contain a high concentration of polyunsaturated fatty acids and fibers [L. albus an important crop for human nutrition.glycemic , anti-inglycemic . Phytochruboside , and vulruboside . The seetivities , and acctivities . L. albud fibers . These pP. harmala L., Z. album, A. valentinus L., A. leucotrichus, L. albus, and M. vulgare). Therefore, this comparative study was conducted to fill in the current research gap existing for these plants. The total phenolic content (TPC), 2,2-diphenyl-2-picrylhydrazyl (DPPH), α-glucosidase, and nitric oxide (NO) inhibitory activities were evaluated in this study. In addition, the nuclear magnetic resonance (NMR) metabolomics approach was applied to identify the potential bioactive compounds present in the most active extract. To the best of our knowledge, this study provides the first detailed metabolite composition and correlation with the biological activities of L. albus by using a NMR-based metabolomics approach.Nevertheless, no sufficient data are currently available regarding the effect of binary solvents on the biological activity of the six Algerian medicinal plants that are traditionally used for the treatment of hyperglycemia , whereas a significant difference (p < 0.05) was observed between the same solvent ratio of the six species as shown by the results in P. harmala L., Z. album, and A. valentinus L., the effect of the solvent on the phenolic content was noticeable in A. leucotrichus, L. albus, and M. vulgare. The high ethanol ratio mixtures could be associated with the solubility factor of the phenolic compounds rather than the water mixtures, as observed by the highest value shown by M. vulgare extracted using 80% ethanol (612.84 μg GAE/mg extract). This finding is consistent with those reported by Amri et al. [M. vulgare was used for the same assay. On the other hand, A. leucotrichus showed the least amount of phenolic compounds among the samples tested, with a value of 124.98 μg GAE/mg extract, which may indicate the type of phenolic compounds represented in A. leucotrichus that are insoluble in water [There was no statistically significant difference between the four solvent ratios in every species  was noticed between the DPPH free radical scavenging activities of the four solvent ratio extracts in the same species and the six species extracted using the same solvent ratio. The highest free radical scavenging inhibition activity was achieved with the 100% ethanolic extract of A. leucotrichus (81.60% ± 0.56) with an IC50 value of 26.26 μg/mL. This value was almost similar to that reported by Sebaa et al. [leucotrichus that provided an inhibitory activity of 93.31% ± 4.87, in contrast to the results reported by Dahmane et al. [A. leucotrichus possessed weak antioxidant activity. It should be emphasized that since all previous studies have only investigated the activity of the essential oil and due to the lack of studies on the free radical scavenging activity of the crude extract of A. leucotrichus, an appropriate comparison could not be made between the results.According to the findings shown in a et al. , who useA. leucotrichus, the 100% and 80% ethanolic extracts of M. vulgare exhibited high activities of 75.84% ± 2.95 and 78.57% 1.88, respectively, with IC50 values of 24.08 and 19.67 μg/mL respectively. These values were found to be higher than the results reported by Pukalskas et al. [M. vulgare showed a free radical scavenging activity of 58.20% ± 0.7. On the other hand, A. valentinus was screened for the first time, which resulted in a moderate activity of 65.57% ± 1.32 with an IC50 value of 25.17 μg/mL by the 80% ethanolic extract. This result was higher than that observed with Anacyclus clavatus, a species of the same genus that exhibited its activity with an IC50 value of 54.77 μg/mL [Anacyclus pyrethrum, another species of the same genus, which exhibited high activity with an IC50 value of 5.60 μg/mL [50 of some extracts was not determined due to the samples’ low activities, which were less than 50% inhibition at 5000 μg/mL.In addition to s et al.  where th77 μg/mL . From th60 μg/mL . Its notM. vulgare had the highest total phenolic content, whereas its antioxidant activity was lower than that reported of A. leucotrichus; which exhibited a very low value of total phenolic content compared to that with M. vulgare. This can be due to the type of phenolics and the amount of the individual phenolic compounds present in the sample, or to the high flavonoid content of the sample. Ismail et al. [Various studies have outlined the strong relationship between the phenolic content and the antioxidant activity ; howeverl et al.  reportedl et al. .p < 0.05) was observed between the α-glucosidase inhibitory activities of all of the samples. The 100% ethanolic extract of L. albus showed the maximum percentage inhibition of 96.78% ± 1.75 with an IC50 value of 6.45 μg/mL, which was higher than the inhibition of the 80% ethanolic extract of the same species of 64.57% ± 1.96 with an IC50 of 8.66 μg/mL; the α-glucosidase inhibitory activity of L. albus in this study was directly proportional to the increase in ethanol ratio. Piślewska et al. [L. albus; the capability of absolute ethanol in liquefying the chemical composites may be related to the degradation of the plant cell wall, thereby allowing the extraction of more endocellular compounds [L. albus. Moreover, the 80% ethanol extract of M. vulgare exhibited an acceptable activity of 68.06% ± 2.15 with an IC50 value of 12.6 μg/mL. Despite the strong hypoglycemic potential that was highlighted by the wide use of these plants in Algerian folk medicine and the fact that previous studies have reported these species were a natural and infinite source of the phenolic compounds as well as the flavonoids. In addition, no significant activity was exhibited by any of the remaining samples including P. harmala, Z. album, A. valentinus, and A. leucotrichus. This finding was consistent with the results reported by [50 of some extracts was not determined due to their low inhibition activity at the highest concentration (5000 μg/mL).Based on the results shown in a et al.  highlighompounds . This maorted by . As mentA. leucotrichus with 50.53% and M. vulgare with 49.87%, which have been reported for their weak inhibitory activity for NO by Hammami et al. [Nitric oxide performs several functions including the modulation of inflammatory responses. This assay determines the nitric oxide concentrations based on the enzymatic conversion of nitrate into nitrite by nitrate reductase. This assay is useful in monitoring individual condition and provides a measure of the innate immune response. All of the plant species in this study did not exhibit any significant nitric oxide inhibitory activity, as indicated by their low values, which were measured via the Griess assay. The highest nitric oxide inhibitory activity values were observed with 100% ethanol extracts of i et al. . The othL. albus was the best candidate among the selected plants for further study using 1H-NMR metabolomics. The identification of the metabolites in the L. albus extract was primarily based on the 2D-J-resolved spectra, the Chenomx database, and comparison with the NMR signals of standard references. The 1H-NMR spectrum of L. albus contains a multitude of overlapping signals, with multiple signals from each detected metabolite. L. albus , which clearly shows that the intensity of peaks in the aliphatic region (δ 0.5–3.5) of the 100% ethanol extract was higher than those obtained with 80%, 50%, and 0% ethanolic extracts. In contrast to the aliphatic region, the intensity of peaks in the carbohydrate region (δ 3.5–5.5) was the lowest among the four spectra.According to the results and on the basis of the important α-glucosidase inhibitory activity value, L. albus. Flavonoids were also identified such as wighteon at (δ 6.49 (s), δ 5.28 (br t), δ 3.37 (br d), δ 1.78 (s), 1.65 (s)); kaempferol at , δ 6.92 , δ 6.35 , δ 6.20 ); and (epi)catechin at (δ 7.05 (br s), δ 6.96 (m), δ 6.12 , δ 6.08 , δ 5.01 (br s), δ 2.94 (m), δ 2.78 (m)). The characteristic signals of some isoflavonoids that were practically found in L. albus were successfully detected at the level of the aromatic region including, lupisoflavone , δ 6.88 , and δ 3.88 (m)); lupinoisolone A (δ 13.40 (s), δ 8.18 (s), δ 6.33 (s), δ 5.33 , δ 3.89 (m), δ 1.70 (brs), δ 1.25 (brs)); lupinisolone C (δ 13.41 (s), δ 7.94 (s), δ 7.03 , δ 6.44 brs, δ 6.41 , δ 3.75 , δ 1.62 (s), δ 1.28 (s)); lupinisol A (δ 13.58 (s), δ 8.12 (s), δ 6.45 (s), δ 7.33 (s), δ 7.29 , δ 6.89 , δ 5.35 , δ 4.72 (brs), δ 4.4 (m), δ 3.35 ); lupinisol B (δ 13.00 (s), δ 8.18 (s), δ 6.95 , δ 6.52 (s), δ 5.32 , δ 4.94 (br s), δ 4.77 (br s), δ 4.48 , δ 3.48 ); lupinisol C (δ 13.01 (s), δ 8.14 (s), δ 7.05 , δ 6.56 (s), δ 5.28 , δ 5.04 (br s), δ 4.85 (br s), δ 4.37 , δ 3.38 ); chandalone (δ 13.46 (s), δ 8.14 (s), δ 7.35 , δ 7.29 , δ 6.88 , δ 6.69 ); isoderrone (δ 13.00 (s), δ 8.25 (s), δ 7.29 , δ 6.48 , δ 6.44 , δ 6.31 , δ 5.77 , δ 1.41 (s)); lupinalbin F (δ 13.58 (s), δ 8.12 (s), δ 7.33 (s), δ 7.29 , δ 4.40 (m), δ 3.35 ); and lupinoisoflavone G (δ 8.14 (s), δ 7.35 , δ 7.29 , δ 6.37 (s), δ 5.33 , δ 3.18 , δ 1.73 (br s), δ 1.29 (s), δ 1.25 (s)) [As presented in J = 7.5, 4 Hz), δ 2.96 (m), δ 2.88 (m)); thiamine (δ 5.47 (s), δ 3.87 , δ 3.20 , δ 2.55 , and proline (δ 2.33 (m), δ 4.11 (m), δ 3.35 (m), δ 3.30 (m)) [The primary and secondary metabolites were assigned and confirmed by a comparison of the data and literature review including amino acids such as asparagine ) ,40. In a.30 (m)) .L. albus; these samples were used for this analysis based on their high α-glucosidase inhibitory activity. As shown in L. albus when compared to those in the 50% and 0% ethanol extracts. However, compounds such as hydroxyiso-lupalbigenin, lupinisol B, palmitic acid, lupeol, and epicatechin had a higher content in the 50% and 0% ethanolic extracts.Principal component analysis (PCA) and partial least square analysis (PLS) plots were obtained from the different ethanolic extracts of L. albus. The PLS biplot for the 1H-NMR data of the L. albus ethanolic extracts  biplot analysis was conducted to correlate the α-glucosidase, DPPH, and NO inhibitory activities with the phytochemical contents of the four ethanolic extracts of extracts  revealedL. albus.However, the clusters of the 50% ethanolic and water extracts, which did not exhibit any significant activity, were grouped separately in the opposite and negative side, presenting a negative correlation with the bioactivities. This dissimilarity may be attributed to the effect of the ethanol ratio in the extraction of the chemical compounds that contributed to this differentiation between the extracts of L. albus [L. albus seeds act as antioxidants in oils and fats [L. albus .The Fabaceae family has been shown to possess a wide range of bioactive metabolites including saponins and phenolic compounds . Within L. albus . Furtherand fats . In addiand fats . All theY-axis intercepts of R2 and Q2 for the three assays were in the range of 0.28–0.56 and −0.4 to −0.42, respectively. A permutation test with 200 random permutations was performed to confirm the validity of the PLS biplot model with the goodness of fit of several models as well as the predictive ability of the model (R2/Q2).The variable importance to projections (VIP) was acquired to measure the contribution that a variable makes to the model . A regioL. albus. Overall, a high significant difference (P ≤ 0.001) was observed in the metabolites of the 50% ethanolic and aqueous extracts, which were found to contain a higher amount of thiamine (δ 5.46), proline (δ 3.3), lupeol (δ 3.22), stearic acid (δ 2.42), betulinic acid (δ 3.54), luteone (δ 3.44), and lupinoisoflavone G (δ 6.38). These extracts also had a high polar solvent ratio , which was characterized by a significantly higher concentration of alpha-tocopherol that was detected at (δ 1.26), unknown 35 (δ 1.24), oleanolic acid (δ 1.3), linoleic acid (δ 2.3), caprate (δ 0.86), and lipinisol A (δ 5.38). These findings were consistent with the PLS results  in the projection (VIP) was performed to further validate whether these metabolites affected the discrimination among samples.  results , which s2CO3), gallic acid (purity of 97%), quercetin (purity of >95%), curcumin (purity of >98%), phosphate buffer, α-glucosidase enzyme, glycine, 2,2-diphenyl-2-picrylhydrazyl (DPPH), and p-nitrophenyl- α-D-glucopyranoside (PNPG) were supplied by Sigma-Aldrich . Absolute ethanol was purchased from Merck Millipore International . The reagents used for cell culture including media Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and L-glutamine (with and without phenol red), penicillin-streptomycin antibiotic solution, fetal bovine serum (FBS), 3--2,5-diphenylte-trazolium bromide (MTT), and TrypLE Express enzyme were obtained from Gibco/BRL Life Technologies Inc. . Recombinant murine IFN-γ, lipopolysaccharide (LPS), and phosphate-buffered saline (PBS) were supplied by Sigma-Aldrich Co. . Dimethyl sulfoxide-d6 (DMSO) and trimethylsilyl propionic acid-d4 sodium salt (TSP) were purchased from Merck Millipore International . Water was purified by a MiliQ system .Folin-Ciocalteu reagent, sodium carbonate . However, the aerial parts of Z. album, A. valentinus L., A. leucotrichus, and the seeds of L. albus were collected from the Saharian region (Southwestern Algeria). The plants used in this study were identified by an in-house botanist (Dr. Idder Boubaker) at the Department of Microbiology and Plant Biology, University of Abdelhamid Ibn Badis, Mostaganem, Algeria. The vouchers specimens P. harmala (LMPB-0015-2019), M. vulgare (LMPB-0014-2019), Z. album (LMPB-0013-2019), A. valentinus (LMPB-0018-2019), A. leucotrichus (LMPB-0016-2019), and L. albus (LMPB-0017-2019) were deposited in the herbarium at the University. All samples were cleaned from any impurities with clean tissue before air-dried at ambient temperature until a constant weight was achieved. The dried samples were ground using a laboratory blender .All samples were harvested during the summer of 2015 from various areas of Algeria. The aerial parts of To produce the crude extract, 4 g of powdered samples were soaked in 100 mL of different ratios of ethanol . The mixtures were subjected to sonication for 1 h in an ultrasonic bath sonicator . The ultrasonic was operated at a power voltage of 220 V and frequency of 53 kHz. Each mixture was filtered thrice using filter paper; this extraction procedure was repeated six times for each solvent ratio, with a total of 24 extracts for each species. The filtered extracts were collected and concentrated using a rotary evaporator under vacuum at 40 °C to yield the crude extract, which was subjected to freeze drying to ensure that no water remained. The extracts obtained were stored in amber bottle at 4 °C until further analysis.Total phenolic content of the six species were evaluated using the Folin–Ciocalteu method according to Lee et al. . The sam50 values in µg/mL, which denote the concentration of sample needed to scavenge 50% of the DPPH free radicals. The IC50 for the active samples was calculated from the graph of different concentrations of the extract against percentage inhibition. The IC50 values were calculated using the fitted line: Y = a × X + b, IC50 = (0.5 – b)/a [Radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was conducted according to the protocol described by Wan et al. . Samples5 – b)/a .50 values in μg/mL.The α-glucosidase inhibitory activity was conducted using the procedure reported previously by Lee et al. with minor modifications . Briefly2 at 37 °C. When the cells were at 80–90% confluence, cell detachment followed by centrifugation for 10 min in 1000 rpm at 4 °C were performed. A viable cell counting was performed on the Hemocytometer using the Trypan blue cell exclusion of live cells staining technique. A total of 50 μL of cells (1 × 106 cells/mL) was then seeded into a 96-well culture plate with 50 μL of DMEM and incubated for 24 h. The cells were then stimulated by 10 µg/mL of LPS  and 1 µg/mL of IFN-γ in the well with the presence or absence of the extract for 17 h. The sterility of the extract was achieved by filtering the extracts using a 0.2 um filter prior to the assay. The Griess reagent was prepared by adding 1% sulfanilamide and 0.1% N-1-naphthylethylenediamine dihydrochloride in 2.5% H3PO4, and used to measure the nitrite (NO-2) concentration, according to the stable nitric oxide conversion product. A 50 μL of Griess reagent was mixed with an equal volume of the supernatant of the cultured cells for 15 min at room temperature and in the dark. The absorbance was measured at 550 nm using a Spectramax Plus (Molecular Devices) UV/V microplate reader, the percentage of inhibition was calculated according to Abas et al. [2 incubator. The medium was then aspirated, and the formed formazan crystals were solubilized by adding 100 μL of DMSO per well and kept for 30 min in the incubator. The intensity of the dissolved formazan crystals (purple color) was measured at 570 nm [The nitric oxide inhibition assay was performed according to the method reported by Maulidiani et al. . RAW 264s et al. . The asst 570 nm . The res1H-NMR analysis. A 500 MHz Varian INOVA NMR spectrometer  was used at 26 °C. The NMR analysis was performed for all the crude extracts with their six replicates. Each 1H-NMR spectrum was collected with a preset acquisition time of 3.53 min, comprising of 64 scans with a spectral width of 20 ppm. Two-dimensional J-resolved (2D JRES) was used as an additional support for the assignment and a confirmation of some compounds. The J-resolved spectrum was acquired in 50 min 18 s using eight scans per 128 increments for the axis of the spi-spin coupling constant, and spectral widths of 66 Hz and 8 K for the chemical shift axis with spectral widths of 5000 Hz.The sample preparation for the NMR measurements was performed according to Kim et al.  by addinChenomx software  was used to correct the phasing and baseline of all the NMR spectra. The spectral region (δ 0.5–10.0) was binned with a width of δ 0.04, resulting in 237 integral regions per NMR spectrum. The residual signal of DMSO at δ 2.50 was removed from the spectra. The multivariate data analysis (MVDA) was performed using SIMCA-P software . Principal component (PCA) and partial least-squares analyses (PLS) were conducted to evaluate the correlation between the activities tested and the metabolites identified. The Pareto scaling was applied in all analysis.p < 0.05.The statistical analysis of TPC, DPPH radical scavenging, α-glucosidase, and nitric oxide inhibitory activities were determined using MS Excel (Version 2010), Minitab software , and the InStat V2.02 statistical package . All experiments were performed in six replicates and the results are expressed as mean ± SD. Analysis of variance (ANOVA) was performed to identify significant differences with a probability of P. harmala L., Z. album, A. valentinus L., A. leucotrichus, M. vulgare, and the seeds of L. albus were assessed for their α-glucosidase, free radical scavenging (DPPH), and nitric oxide (NO) inhibitory activities as well as the total phenolic contents (TPC). Among the species used in this study, L. albus exhibited the highest α-glucosidase inhibitory activity. On the basis of these findings, it can be concluded that the extract obtained from L. albus using 100% EtOH was preferable to yield metabolites that might contribute to its α-glucosidase inhibitory activity. 1H-NMR metabolomics successfully identified metabolites that were responsible for the activity. In total, 34 metabolites were identified in the ethanolic extract of L. albus comprising flavonoids , amino acids , vitamin , fatty acid (linoleic acid), triterpenoids , and isoflavonoids . The metabolites identified could be used as chemical markers that are responsible for its strong α-glucosidase inhibitory activity exhibited by the ethanolic extract of L. albus.In this study, the ethanolic extracts at different ratios  were prepared from the aerial parts of


Human noroviruses (HuNoVs) are a major cause of nonbacterial gastroenteritis in all age groups worldwide. HuNoVs can be detected in vitro using molecular assays such as RT-PCR and RT-qPCR. However, these molecular-based techniques require special equipment, unique reagents, experienced personnel, and extended time to obtain results. Besides, the diversity of viral genotypes is high. Therefore, methods that are rapid, broad-range and effective in the detection of HuNoVs are desiderated for screening the feces or vomit of infected people during outbreaks.6 genomic copies per gram of stool sample (gc/g) and 4.4 × 105 gc/g for genogroup I and II (GI and GII) HuNoVs, respectively. A total of 122 clinical samples were tested for HuNoVs by ICA and compared against RT-qPCR. The relative sensitivity, specificity and agreement of ICA was 84.2% (95% CI: 83.6–84.8%), 100.0% (95% CI: 98.5–100.0%) and 87.7% (95% CI: 85.6–89.8%), respectively. No cross-reaction with other common enteric viruses or bacteria was observed. The ICA detected a broad range of genotypes, including GI.1, GI.3, GI.4, GI.6, GI.14, GII.2, GII.3, GII.4, GII.6, GII.13, and GII.17 HuNoVs.In this study, a colloidal-gold-based immunochromatographic assay (ICA) was developed for effective detection of HuNoVs in clinical samples. Monoclonal antibodies (MAbs) against the shell (S) domain in the major capsid protein of HuNoVs were used in the ICA. The limitations of detection for HuNoVs in clinical samples were 1.2 × 10This study demonstrates that ICA targeting the S domain of VP1 is a promising candidate for effectively identifying the different genotypes of HuNoVs in clinical samples with high sensitivity and specificity.The online version contains supplementary material available at 10.1186/s12866-020-02084-z. Caliciviridae family. The genome has three open reading frames , where ORF2 encodes a major capsid protein referred to as VP1. VP1 consists of a shell (S) domain and a protruding (P) domain. The S domain is the most highly conserved region in VP1, and forms a shell surrounding the RNA genome, while the P domain of VP1 contains the most variable sequence  were provided by Dr. Ningbo Liao ). In total 117 clinical diarrheal samples were provided by the Affiliated Hospital of Guangzhou Medical University (3), the Affiliated Hospital of Sun Yat-sen University (7), Chinese CDC (39), and Anhui Provincial CDC (68). The samples were tested by both RT-qPCR and the developed ICA. Clinical samples provided by Zhejiang Provincial and Chinese CDC were obtained from patients with acute gastroenteritis in 2018. The samples from Anhui provincial CDC and Affiliated Hospital of Guangzhou Medical University were collected in 2017. The samples from the Affiliated Hospital of Sun Yat-sen University were obtained between 2015 and 2017.2O were added to make a total volume of 25.0 μl. The amplification reactions were as follows: reverse transcription at 50 °C for 30 min; heat-denaturation at 95 °C for 3 min; 40 cycles with denaturation at 95 °C for 10 s, annealing and extension at 60 °C for 30 s. Fluorescence signals were collected at the end of each extension step. The highest dilution from real-time RT-PCR was used to generate a positive cycle threshold (Ct) signal, which was one real-time RT-PCR unit (RT-qPCRU) [t values versus log10 viral genomic copies were generated from a continuous 10-fold dilution .Real-time RT-PCR was performed using a commercial one-step RT-qPCR kit , consisting of 12.5 μl 2 × one-step RT-qPCR Master Mix (with SYBR Green), 0.65 μl RT enzyme Mix, and 0.4 μl of each primer (0.16 μmol/l)  . Linear Pretreatment included physical or chemical methods. Heat-denaturation was used for physical treatment. Briefly, 10.0% (w/v) fecal sample was diluted with PBS, placed in a water bath at a specific temperature  for 1 to 5 min at an interval of 1 min. A reducing agent (DTT)  and alkaline conditions were used for chemical treatment. The pH in PBS was adjusted from 6.0 to 10.0. The fecal samples were dissolved to 10.0% with PBS buffers  at room temperature (25 °C) for 5, 10, 15, 20, 25 and 30 min. DTT was added to the homogenized solution of the clinical sample at a final concentration of 1.0%. Sandwich ELISA was used to detect HuNoVs, as previously described .The procedure used to prepare ICA strips is described in Additional file 5 to 5 × 106 gc/g (GI) and 1.1 × 105 to 3.5 × 106 gc/g (GII). All experiments were performed in triplicate to confirm the reproducibility of the results.The purified S domain of the VP1 was used at concentrations of 22.4 ng/ml, 11.2 ng/ml, 5.6 ng/ml, 2.8 ng/ml, 1.4 ng/ml and 0.7 ng/ml. PBS was used as blank control. Two HuNoVs positive samples  were two-fold diluted with 1.6 × 10Salmonella  were tested. The copies of Rotavirus, Sapovirus, Astrovirus, and Adenovirus RNA were more than 107 gc/g feces.To determine the specificity of antibodies in the colloidal gold test, 4 Rotavirus, 3 Sapovirus, 2 Astrovirus and 4 Adenovirus  and 3 Thermal acceleration tests were used to determine the stability of the ICA strips, at 60 °C . The actwww.spssau.com).One-way ANOVA was used for data analysis. SPSSAU, an online data analysis tool, was used to perform all the statistical analyses . Figure S2. A sandwich ELISA analysis of HuNoVs after treatment of pH at different time. .Additional file 2: Table S1. The Ct data of five stool samples used in the LOD test. Table S2. The Ct data and viral genomic copies of 15 stool samples not detected by ICA.Additional file 3: Table S3. Commercial kits for sensitivity, specificity and agreement comparison.Additional file 4: Table S4. Optimal BSA concentration for blocking. Table S5. Selection of conjugate pad and dilution multiple of gold-labeled antibody. Table S6. (A) Selection of the Nitrocellulose Membrane. (B) Optimization of the capture antibody and control antibody concentration. (C) The optimal concentration of sucrose (w/v) for the immobilized antibody on the NC membrane.Additional file 5: Figure S3. Selection of optimal conditions for gold-labeled antibody complex.Additional file 6: Table S7. Primers used in RT-qPCR.Additional file 7: Figure S4. Standard curves for calculation of genomic copies of GI and GII HuNoVs.Additional file 8. Preparation of the sample pad and others and assembling of the ICA.


Candida species. We analyzed the molecular SL signatures of drug-resistant clinical isolates of Candida auris, which have emerged as a global threat over the last decade. This included Indian hospital isolates of C. auris, which were either resistant to fluconazole (FLCR) or amphotericin B (AmBR) or both drugs. Relative to Candida glabrata and Candida albicans strains, these C. auris isolates were susceptible to SL pathway inhibitors such as myriocin and aureobasidin A, suggesting that SL content may influence azole and AmB susceptibilities. Our analysis of SLs confirmed the presence of 140 SL species within nine major SL classes, namely the sphingoid bases, Cer, αOH-Cer, dhCer, PCer, αOH-PCer, αOH-GlcCer, GlcCer, and IPC. Other than for αOH-GlcCer, most of the SLs were found at higher concentrations in FLCR isolates as compared to the AmBR isolates. SLs were at intermediate levels in FLCR + AmBR isolates. The observed diversity of molecular species of SL classes based on fatty acyl composition was further reflected in their distinct specific imprint, suggesting their influence in drug resistance. Together, the presented data improves our understanding of the dynamics of SL structures, their synthesis, and link to the drug resistance in C. auris.Independent studies from our group and others have provided evidence that sphingolipids (SLs) influence the antimycotic susceptibility of  •Candida auris isolates are susceptible to sphingolipid inhibitors myriocin and aureobasidin A.•C. auris isolates resistant to different antifungals.The distribution of sphingolipid species is distinct among •Phytoceramides are the most abundant class of sphingolipid.•C. auris.Cer(d18:1/18:1) is the major of ceramide species in •C. auris isolates.d19:2 glucosylceramide backbone is typically in abundance in AmB resistant  Most of systems . In contathogens . The rec concern . Each cl concern ,6.C. auris isolates were resistant to fluconazole (FLC), ~35% to amphotericin B (AmB), ~41% resistant to two antifungals (FLC and AmB) and ~4% resistant to all three major classes of antifungals  (4). Azoles are front-line drugs in systemic infections against most Candida species that target the ergosterol biosynthesis pathway by inhibiting 14α-demethylase encoded by ERG11 [C. auris isolates identified well-characterized examples of azole resistant in C. auris [C. auris isolates confirmed the presence of mutations in azole target ERG11 [CDR1/CDR2 and MDR1 [C. auris isolates displayed resistance to polyenes that bind fungal membrane ergosterol [C. auris with the treatment of AmB also revealed altered expression of sterol biosynthesis genes [C. auris is incomplete. It remains unclear why C. auris isolates can easily acquire resistance to multiple antifungals [A multicenter study on three continents reported that ~93% of by ERG11 . NotablyC. auris . Whole-gand MDR1 ,10,11. Igosterol ,13. The C. albicans transposon-mediated mutant library revealed that the null mutants of CaFEN1 or CaFEN12 (encoding enzymes for the synthesis of very-long-chain fatty acids) became resistant to FLC [CaFEN1 and CaFEN12 are also crucial for AmB susceptibility, cell wall integrity, hyphae, and biofilms formation in C. albicans [2C) depletion in ipt1Δ mutant in S. cerevisiae demonstrated increased susceptibility to plant defensins and decreased susceptibility to oxidative stress [C. neoformans [C. albicans [ORM gene that codes for SL regulatory proteins knockout mutants imposed ER stress responses and influenced azoles susceptibility in A. fumigatus [LAC1, SUR2, LCB2, and IPT1 genes have Pdr1/Pdr3 response elements (PDREs) suggesting regulation by the Pdr1 transcription factor, which is a known regulator of azole resistance in several fungi [Several antifungal drugs target membrane ergosterol and enzymes involved in lipid biosynthesis, including sphingolipid (SLs), biosynthesis which are critical for many cell homeostatic processes ,16. Thest to FLC . Mass spt to FLC . CaFEN1 albicans . The mane stress . The gluoformans  and dispalbicans . Furtherumigatus . Interesal fungi .Candida by our group and others demonstrated that lipids are tightly linked to MDR (Multiple Drug Resistance), and that membrane composition can influence the drug susceptibilities [C. albicans [C. auris, has reported a semi-quantitative assessment of the lipidome [C. auris and prime drug target to treat fungal infections and therefore demand attention. In this study, we focused on the characterization of the dynamics of SLs specific to recovered clinical drug resistance in C. auris. To assess the relevance of different sphingolipidomic signatures, we used three categories of clinical isolates of C. auris: (i) FLC-resistant (FLCR), (ii) AmB-resistant (AmBR) and (iii) both FLC and AmB resistant (FLCR + AmBR). Our data revealed that all the C. auris strains in this study were susceptible to SL pathway inhibitor MYR, compared to C. albicans and C. glabrata. High-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) based sphingolipidomics identified 140 SLs species in C. auris and defined the SL molecular species imprints specific to drug resistance. Our data provide insights into comprehensive SL landscape with some correlating with antifungal resistance in C. auris.Previous independent studies on bilities ,26. Largbilities ,28. For lipidome . This dalipidome . The rol22.1Lipid standards were purchased from Sigma Aldrich, USA, and Avanti polar lipids Inc., USA. All solvents and chemicals, unless specifically mentioned, were LCMS (liquid chromatography-mass spectrometry) grade purchased from Sigma Aldrich, USA, and Fisher Scientific, UK.2.2C. auris strains were provided by National Culture Collection of Pathogenic Fungi (NCCPF), Indian Council of Medical Research (ICMR), New Delhi, sponsored National facility at the Mycology Division, Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research (PIGMER), Chandigarh, India. C. albicans and C. glabrata strains used in this study were susceptible to FLC and AmB  was originally procured from ATTC  and C. glabrata (BG2) was obtained from Centre for DNA Fingerprinting and Diagnostics, India. Cultures were maintained on YEPD agar plates at 30 °C. For lipid extraction, cells grown to an OD600 of 0.1 were inoculated in YEPD medium, then allowed to grow to OD600 0.8 to 1. Approximately 5 × 108 cells were harvested, washed with dH2O thrice before lipid extraction. and AmB . The lab2.3C. auris strains, were determined by broth microdilution with two-fold serial dilutions in RPMI-1640 medium, as described in Clinical and Laboratory Standards Institute  [600 = 1.0, and 10 μL of each culture was mixed with 190 μL YEPD broth in 96 well plates, and OD600 was measured at every 30 min of an interval to up to 24 h. FLC (3 mg/mL) and AmB (2 mg/mL) and MYR (2.4 mg/mL) were dissolved in DMSO, AbA (1 mg/mL) was prepared in ethanol. C. krusei (ATCC 6258) and C. parapsilosis (ATCC 22019) were used as quality control reference strains.Minimum inhibitory concentrations (MIC) for FLC, AmB, against the LS, USA) . The gro2.48 cells were suspended in 1.5 mL Mandala buffer ). To each sample 50 mg of glass beads (0.4–0.6 mm), C17 ceramide  and C17 sphingosine   were added and cells were then broken using Fast prep . The samples were then kept at 60 °C for 1 h, with periodic vortexing and sonication at intervals of 15 min. Cell debris was removed by centrifugation  and the supernatant was dried using N2 gas. Next, Bligh and Dyer's extraction was performed by dissolving the pellet into chloroform: methanol in ratio 1:2 (v/v) and incubated at 37 °C for 1 h [2O with vortexing. The hydrophobic layer was taken, dried using N2 gas. Further, mild alkaline hydrolysis was performed using 0.5 mL chloroform, and 0.5 mL 0.6 M methanolic KOH was added into the dry lipid samples, then vortexed and incubated at room temperature for 1 h. Then 0.325 mL 1 M HCl and 0.125 mL dH2O was added and vortexed. The hydrophobic layer was dried with N2 gas and stored at −20 °C until further use.Lipid extraction was performed as described earlier . Briefly for 1 h . Phase s2.52O (Buffer A) and Buffer B. Quantification of SL species achieved by internal standard normalization method. The lipid data were further normalized to per O.D. cells, and after that, mol% calculated.SLs were detected in base hydrolyzed lipid samples by multiple reaction monitoring (MRM) approaches described previously , using t2.6t-test was used to determine the statistical significance, and a p-value of ≤0.05 was considered significant. The SYSTAT software (Version 13.0) was used to perform the principal component analysis (PCA).Each analysis was performed in triplicates. Student's 33.1C. auris have not been formally established. Our findings are based on the breakpoint suggested by the Centers for Disease Control and Prevention  by analyzing the data on related Candida spp. The MICs of all the isolates used in this study were measured against two antifungals, namely AmB and FLC. MIC values ≥ 32 μg/ml and ≥2 μg/ml for FLC and AmB respectively were considered as resistant. Our selection included four FLCR isolates NCCPF_470147, NCCPF_470026 and NCCPF_470157, with MIC50 ≥ 64 μg/ml and NCCPF_470055 with MIC50 32 μg/mL. Three AmB resistant (AmBR) clinical isolates were selected in which NCCPF_470140 and NCCPF_470161 have MIC50 at 4 μg/mL, and NCCPF_470097 have MIC50 at 2 μg/mL. These seven isolates did not display cross-resistance to other antifungals classes (data not shown). Three clinical isolates NCCPF_470154, NCCPF_470114, and NCCPF_470151 were also included in this study, with resistance to both FLC and AmB (FLCR + AmBR)   . Growth ctively) a.3.250 ranged between 0.01 and 0.5 μg/mL in comparison with C. glabrata (BG2) and C. albicans (SC5314) strains (MIC50 was ≥4 μg/ml) (R + AmBR) showed the maximum MIC50 0.5 μg/mL for MYR, which was relatively high from other isolates. AbA MIC50 ranged from 0.06 to 0.25 μg/mL, which was also less than C. albicans (SC5314) and C. glabrata (BG2)  and aureobasidin A (AbA). Irrespective of resistance types, all the drug-resistant isolates were susceptible to MYR with MIC4 μg/ml) . Isolateta (BG2) . IndepenMIC data b.3.3C. auris. The identified SLs of C. auris belong to nine classes namely, sphingoid bases (or LCB's), dihydroceramide (dhCer), ceramides (Cer), αhydroxy ceramides (αOH-Cer), phytoceramide (PCer), αhydroxy phytoceramide (αOH-PCer), GlcCer, αhydroxy glucosylceramide (αOH-GlcCer) and inositol phosphoryl ceramides (IPCs). Further sphingoid bases (LCBs) were divided into seven subclasses namely, dihydrosphingosine (DHS), sphingosine (SPH), sphingosine-1-phosphate (S1P), dihydrosphingosine-1-phosphate (DHS1P), phytosphingosine (PHS), phytosphingosine-1-phosphate (PHS1P) and glucosyl sphingosine (Glucosyl-SPH). The total sphingoid bases were low in abundance, except for DHS, which exhibited relatively higher abundance (0.144 mol%). Moreover, the total sphingoid base amount did not show significant variation between FLCR, AmBR, and FLCR + AmBR isolates , which significantly varies between the FLCR and AmBR isolates. For instance, the amount of dhCer was higher in FLCR isolates with an average of 25.5 mol%, and, in comparison, a much lesser amount was present in AmBR isolates (15.5 mol%) (The utilization of sphingoid bases in the SL pathway results in the dhCer structures formed by an amide linkage with fatty acid . In C. a.5 mol%) , Fig. 1.C. auris isolates, both the Cer and αOH-Cer classes followed a similar distribution pattern, as was the case with dhCer. Thus, Cer and αOH-Cer classes exhibited a higher amount in FLCR than in the AmBR isolates. The next intermediate of the SL biosynthetic pathway is PCer, which was found to be the most abundant biosynthetic intermediate in C. auris, representing 46 mol% of total SLs. However, its levels showed no significant variations in contents and remained unchanged between our tested drug-resistant isolates. Our analysis also detected α-hydroxylated PCer (αOH-PCer) with 5.6 mol% . In comparison with AmBR isolates, αOH-PCer was significantly higher in FLCR isolates. The major complex SLs in C. auris were represented by αOH-GlcCer, followed by GlcCer and IPCs. Among these, glycosylated SL, GlcCer was higher in FLCR (2.7 mol%), while αOH-GlcCer was significantly higher in AmBR isolates (25.2 mol%). IPCs are the third complex SL class found in our analysis, which did not exhibit any significant differences among these isolates. In general, except for αOH-GlcCer, most of the class of SLs were significantly higher in FLCR isolates than in the AmBR. It is important to note here that the higher classes of IPCs, i.e., MIPC and M(IP)2C of complex SL, were below detection in our analysis in positive ion MS.Among 3.4C. auris. However, the other sphingoid backbones with d19:2 and d18:2 have also been detected, albeit much lower in number.In fungi, the SLs consist of the LCB backbone, which is linked to a fatty acid at the C2 position by an amide-linkage and has esterified polar head at the C1 position . In our 3.4.1R and FLCR isolates and was higher in FLCR as compared to AMBR isolates. FLCR NCCPF_470157 isolate had the highest SPH (0.038 mol%) level, while the least SPH level (0.006 mol%) was found in AmBR NCCPF_470140 isolate. No significant differences in SPH contents were observed in the isolates resistant to both FLCR and AMBR antifungals. Phosphorylated sphingosine (S1P) had also been detected in our analysis but displayed no significant variation among the tested clinical isolates.Sphingoid bases are long-chain aliphatic amino alcohols (LCB), which serve as precursors to a variety of SLs. SPH specifically refers to -2-amino-4-octadecen-1,3-diol, C18 aliphatic chain with an amine group in C2, C1, and C3 hydroxyl group, and C4 double bond, which forms the backbone structure of complex SLs. SPH content showed significant variation between AmBR isolates (0.28 mol%) and lowest in all AmBR isolates (0.083 mol%)  a; howeveF_470161 . In contC. auris a.3.4.2C. auris isolates varied in the carbon chain length from 12C to 26C, while the sphingoid backbone (d18:0) was the common denominator of all the species of dhCer. Among these, the dhCer with C18:0 fatty acid (stearic acid) was the most abundant species showing a relatively higher level in FLCR isolates (9.7 mol%) as compared to AmBR (7.7 mol%). Interestingly, dhCer with 24C was particularly higher in FLCR isolates (9.53 mol%) as compared to AmBR isolates (5.02 mol%). Among the three AmBR isolates, NCCPF_470140 showed the highest amount (10.9 mol%) of Cer(d18:0/18:0) (C. auris isolates. Other dhCer species Cer(d18:0/12:0) and Cer(d18:0/20:1) also displayed a significant difference between the FLCR vs. AmBR and AmBR vs. FLCR + AmBR isolates. The mol% distinction was also evident for Cer(d18:0/14:0), Cer(d18:0/18:1) and Cer(d18:0/26:0) species between FLCR and AmBR isolates  and very-long-chain fatty acid (VLCFA) synthesis arm of the SL pathway. The fatty acid chain length of dhCer in :0/18:0) . The Cerisolates b.3.4.3R and AmBR, but there was a difference between FLCR vs. FLCR + AmBR and between AmBR vs. FLCR + AmBR isolates. Cer with C14:0, C16:0, C20:1, C20:0, C22:0 and C22:1, fatty acids were low in abundance in C. auris, and Cer(d18:1/12:0) was either not detected or not present in these isolates. Cer with VLCFA  exhibited a significant variance within FLCR, and AmBR isolates where all these three Cer structures were abundant in FLCR and scarce in AmBR (R) isolate was particularly rich in Cer content as compared to all the isolates while the NCCPF_470151 and NCCPF_470161 isolates showed lowest Cer content ), was the most abundant species and showed significant variance between FLCR and AmBR isolates (R isolates. NCCPF_470154 (FLCR + AmBR) had the highest αOH-Cer content (3.5 mol%), and NCCPF_470097 (AmBR) had the lowest αOH-Cer content (1.25 mol%). αOH-Cer molecular species contents with hydroxy fatty acid C12:0(2OH), C14:0(2OH), C16:0(2OH), C16:1(2OH), C18:0(2OH), and C24:0(2OH) varied significantly , was most abundant, followed by C26:0 (Cerotic acid). The distribution of Cer(t18:0/24:0) among resistant isolates showed its higher content in AmBR isolate (26.7 mol%) and significantly lower in FLCR (21.3 mol%) isolates. The second most abundant species Cer(t18:0/26:0), displayed different distribution among these isolates (R (1.2 mol%) and AmBR (1.8% mol%) isolates. Other species with C12:0, C18:1, and C24:1 fatty acyls showed a significant disparity between at least two selected sets of isolates, while others showed strain-specific variations that could not be grouped. C. auris isolate, which was most abundant in PCer, was NCCPF_470097 with 53.8 mol%, while NCCPF_470026 was with the least amount of PCer with an average of 42 mol%  as compared to AmBR (4.48 mol%). Cer(t18:0/26:0(2OH)) was the other abundant species but showed no significant variation among the isolates ), and this species displayed the significant difference between FLCisolates . Unlike isolates a.3.4.5C. auris, implying that the GlcCer branch of the SL pathway is active in C. auris. GlcCer formation requires the transfer of glucosyl-group of UDP-glucose to the αOH-Cer C1 hydroxyl group [R and AmBR isolates; however, it displayed a significant change in concentrations between AmBR (0.75 mol%) and FLCR + AmBR (1.29 mol%) isolates. GlcCer species with VLCFA  were most variable in terms of mol% between the FLCR and AmBR isolates ), with a hydroxylated fatty acid of 18C, was the most abundant species. This species showed a higher level in AmBR (24.2 mol%) than in the FLCR (13.3 mol%) isolates and also exhibited a significant difference between FLCR and FLCR + AmBR isolates  isolates had a higher abundance of IPCs among tested C. auris isolates, while NCCPF_47014 isolate recorded the least amount of IPCs with 0.27 mol%  and dhCer  are abundant in FLCR, and AmBR + FLCR isolates and significantly lower in AmBR isolates.PCA was performed using the mol%  of the SL molecular species datasets between FLCicted in . We can R + FLCR and AmBR isolates. SL species IPC46:0;4B, Cer(d18:1/16:1(2OH)), Cer(t18:0/28:1) and GlcCer(d18:1/16:0) were particularly lower in content in AmBR isolates, while IPC44:0;4B and Cer(d18:0/24:1) were lower in content only in AmBR + FLCR isolates. Likewise, the loading values of PCA 3 showed that αOH-Cer  and αOH-PCer  were significantly lower in AmBR isolates while abundant in AmBR + FLCR isolates. Several GlcCer species  were significantly lower in AmBR + FLCR, and AmBR isolates, while abundant in FLCR isolates. Together, the PCA analysis identified specific SL species characteristics to each of the isolate groups and also validated the statistical significance of these datasets , IPC structures , were lower in abundance in AmBdatasets .3.6To identify the specific SL species variations that could be associated with drug resistance, we compared the SL profile of a drug-susceptible isolate (CBS10913T) with the various drug-resistant isolates. Our analysis revealed that drug-susceptible isolate (CBS10913T) has a distinct sphingolipid profile as compared to resistant isolates. For instance, the sphingoid bases (like S1P and PHS1P), dhCer and Cer (18C and 20C FA containing), αOH-Cer (16C and 30C FA containing) and PCer (28C FA containing), αOH-PCer (28C FA containing), IPC species, among others, were in abundance in CBS10913T strain . On the C. auris isolates than those depicted in It is important to point out that while some of the SL species were present in higher amounts in 4C. auris as a new NAC spp. Notably, C. auris clinical isolates display raised resistance to FLC, but many also show collateral resistance to AmB or echinocandins [The available knowledge of the drug resistance mechanisms is unable to explain, global emergence of highly drug resistance ocandins ,37. Withocandins . With thocandins . SLs serocandins .C. auris remain concealed. Apart from a single multi-omics study in C. auris that led to the identification of a few SLs species [C. auris is demanded to unravel the relevance of these lipids in the impressively higher observed resistance in this pathogen. In the present study, we focused on high throughput ESI-MS/MS analysis of SLs extracted from the ten drug-resistant clinical isolates C. auris. These isolates based on their single or collateral drug susceptibility to antifungals were clustered into three groups. In our analysis, we could identify a total of 154 SL species belonging to nine SL classes based on their structures. Detailed analysis of these SL classes unraveled the characteristics of SL species present in C. auris, starting from simple sphingoid base to complex SLs such as GlcCer and IPC.Notwithstanding, several independent studies on SL composition and their diverse roles in other yeasts and fungi, SL biosynthetic pathways in  species , this asR, FLCR, and AmBR + FLCR isolates. Many pathways of lipid biosynthesis and metabolism are conserved across the species ranging from yeast to human. Nevertheless, a notable difference exists in the SLs pathway of yeasts and mammals [C. auris  mutants in A. nidulans and C. albicans [RTA2 was shown to be linked with FLC resistance in C. albicans [Among the identified nine classes, sphingoid bases are the simplest form of SLs intermediates. DHS (18:0) was the most common, and this exhibited significant variation in mol% distribution in different strains. For example, FLCalbicans . DHS traalbicans . Togethealbicans .FEN1 and SUR1 mutants of C. albicans [Cryptococcus, where Cer synthase preferably used 18:0 fatty acyl [C. auris primarily uses 18:1 fatty acyl as substrate. However, fatty acyls of 16:0, or 24:0 or 26:0 to form dhCer structures, were also detected. dhCer notably displayed enrichment in FLCR isolates than the AmBR. PHS gets converted into PCer by the Cer synthase gene. For the synthesis of PCer, Cer synthases predominantly used C24:0 fatty acyl carbon followed by C26:0, C28:0, and C18:0. PCer was the most abundant SL species found in our analysis. Hydroxylation at the C2 position of PCer by B9J08_001759/SCS7 results in conversion of PCer into αOH-PCer. The final production of SL biosynthesis is M(IP)2C, which is generated in three irreversible steps, mediated by enzymes B9J08_005313/IPC1, B9J08_003885/SUR1, B9J08_000315/CSG2, B9J08_004082/IPT1, and B9J08_002262/SKN1. Interestingly, the IPT1 gene displays 57.42% sequence identity with the S. cerevisiae counterpart and 45.4% with C. albicans (IPT1 (M(IP)2C). More focused MS analysis using the negative ion approach is needed to analyze IPCs higher derivatives.DHS has two fates. It can be hydroxylated at the C4 position into PHS (sphingoid base) by B9J08_002536/SUR2 or can be converted to Cer by Cer synthases encoded by B9J08_004239/LAG1, B9J08_002101/LAC1, and B9J08_004173/LIP1 . Among talbicans . Our dattty acyl , C. aurialbicans . Our anaSLD1, B9J08_000787/DES1, B9J08_002918/MTS1, and B9J08_000009/HSX11. First, LCB undergoes Δ4-desaturation by a desaturase (B9J08_000787/DES1) and form d18:1 Cer backbone ) is the main GlcCer species in C auris, also reported in Cryptococcus [The analysis of GlcCer biosynthesis structures revealed the enzymes involved in this process; however, they remain uncharacterized. The second branch (Cer branch) of the SL pathway culminates at the formation of GlcCer by enzymes encoded by B9J08_005054/backbone . A desatalbicans ,43. Cer tococcus .C. auris. We suggest that resistance to a specific antifungal could be linked to the amount and molecular species of SL. For example, in αOH-Cer class, a different sphingoid backbone (Cer(d19:2/18:0(2OH))) is rich in AmBR strains as compared to other isolates. αOH-GlcCer was the major complex SLs present in all isolates, and its levels were raised in AmBR isolates. SL pathway inhibitors such as N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0) highly synergistic when combined with FLC and AmB, suggesting a strong link between SL contents and drug resistance mechanism [C. auris, and confirmed the PCA based differences among different isolates  the IPC biosynthetic pathways slow down leading to an accumulation of precursors PCer and αOH-PCer; or (ii) upregulation of IPC degradation pathway (via ISC1) leading to an accumulation of PCer and αOH-PCer structures. In either scenario, the altered levels of PCer in membranes can alter the overall homeostasis affecting cellular functions in pathogenic fungi. Specific structural features like hydroxylated backbones and fatty acyls, are important for proper surface localization and organization of membrane proteins. For example, in C. neoformans, it was earlier reported that oligomerization of Pma1 (a plasma membrane ATPase) is severely affected in conditions where PCer levels are low [C. auris [We extended our analysis to include a drug susceptible bilities . A compabilities . Specifibilities . The com are low . LikewisC. auris ,10,18. HC. auris. Additionally, the study shows that some of the most abundant and distinguished sphingolipid structures present in C. auris include Cer(d18:1/18:1) and Cer(d18:0/18:0). While Cer(t18:0/26:0) is the major PCer structure, which represents the most abundant class of sphingolipids in C. auris, high levels of GlcCer with d19:2 glucosylceramide backbone were detected in AmB resistant C. auris isolates ; F: FLCR, FA: FLCR + AmBR, A: AmBR, CBS: CBS10913T; * represents species and classes that differ in at least 2 groups, ** represents species and classes that differ in at least 3 groups.Supplementary file 1Loading values of principal components 1, 2 and 3. The 12 highest and 12 lowest values are indicated.Supplementary file 2C. albicans and S. cerevisiae SLs protein sequences by protein BLASTp search.% Identity of C. auris SLs protein sequences with Supplementary file 3C. auris. Average ± SEM of all classes is shown. *Represents the SL species which show statistically significant variation (p ≤ 0.05) between CBS10913T and all the other three groups of isolates. The complete dataset can be found in Supplementary file 1.The abundant SLs species of nine SLs classes that are found in the drug susceptible and all three sets of resistant isolates of Supplementary Table 1The following are the supplementary data related to this article.All the datasets can be accessed either in the main manuscript or in supplementary files and access to all other primary data sets is available upon request.Mohit Kumar: Conceptualization, Methodology, Investigation, Software, Data curation, Writing - original draft, Formal analysis, Validation, Writing - review & editing. Ashutosh Singh: Conceptualization, Methodology, Data curation, Writing - original draft, Formal analysis, Writing - review & editing, Funding acquisition. Sonam Kumari: Data curation, Investigation, Methodology, Writing - review & editing. Praveen Kumar: Data curation, Formal analysis. Mohd. Wasi: Data curation, Formal analysis. Alok K. Mondal: Supervision, Resources. Shivaprakash M. Rudramurthy: Data curation, Writing - review & editing. Arunaloke Chakrabarti: Data curation, Writing - review & editing. Naseem A. Gaur: Conceptualization, Resources, Writing - original draft, Writing - review & editing, Project administration, Funding acquisition, Supervision. Neil A.R. Gow: Supervision, Funding acquisition, Writing - review & editing, Project administration. Rajendra Prasad: Conceptualization, Resources, Writing - original draft, Writing - review & editing, Project administration, Funding acquisition, Supervision.The authors declare that there is no conflict of interest.


Two phase II studies in Japan examined the efficacy and safety of nivolumab, a programmed cell death 1 receptor inhibitor, in patients with advanced squamous and non-squamous non-small cell lung cancer (ONO-4538-05 and ONO-4538-06). We examined the long-term efficacy and safety of nivolumab in these patients treated for up to 5 years.N = 35) or non-squamous (N = 76) non-small cell lung cancer received nivolumab (3 mg/kg every 2 weeks) until disease progression/death. Overall survival and progression-free survival were assessed at 5 years after starting treatment in separate and pooled analyses. Safety was evaluated in terms of treatment-related adverse events.Patients with squamous (P = 0.0703). Overall survival prolonged with increasing programmed death ligand-1 expression, with 5-year survival rates of 11.8, 21.8 and 41.7% in patients with programmed death ligand-1 expression of <1, ≥1–<50 and ≥50%, respectively. Treatment-related adverse events in ≥10% of patients  included rash (15.3%), malaise (14.4%), decreased appetite (14.4%), pyrexia (14.4%) and nausea (10.8%).A total of 17 patients were alive at the database lock (26 July 2019). The median overall survival  and 5-year survival rate were 16.3 (12.4–25.2) months and 14.3% in squamous patients, 17.1 (13.3–23.0) months and 19.4% in non-squamous patients and 17.1 (14.2–20.6) months and 17.8% in the pooled analysis, respectively. Programmed death ligand-1 expression tended to be greater among 5-year survivors than in non-survivors (Long-term survival with nivolumab was observed in patients with squamous or non-squamous non-small cell lung cancer. No new safety signals were reported after ≥5 years of follow-up. This long-term follow-up demonstrated ongoing efficacy and safety of nivolumab over ≥5 years in patients with squamous or non-squamous non-small cell lung cancer in Japan. EGFR) gene-tyrosine kinase inhibitors, may be used as second-line therapy, these drugs generally failed to show superior or non-inferior effects on overall survival (OS) relative to docetaxel in this setting  tumour biopsy specimens using a validated, automated immunohistochemical assay (Dako North America) with a rabbit anti-human PD-L1 antibody . Tumour PD-L1 expression was confirmed when the tumour cell membranes were stained (at any intensity) in a section with ≥100 evaluable tumour cells.The primary endpoint in both studies was the ORR, which was assessed by an Independent Review Committee and was calculated as a best overall response of complete response (CR) or partial response (PR) defined by Response Evaluation Criteria in Solid Tumors guidelines version 1.1. Secondary endpoints included the disease control rate , duration of response (DOR), OS and progression-free survival (PFS). Safety was evaluated in terms of adverse events (AEs) graded according to the National Cancer Institute Common Terminology Criteria for AEs version 4.0. Tumour PD-L1 expression was assessed in pre-treatment tumour-biopsy specimens by immunohistochemistry, as described in the previous study .2 or Wilcoxon signed-rank tests were used to compare non-parametric variables at baseline. OS was calculated as the time (days) as follows: ‘date of death from any cause’ − ‘date of the first dose of nivolumab’ + 1. PFS was calculated as the time (days) as follows: ‘date of disease progression or death, whichever comes first’ − ‘date of the first dose of nivolumab’ + 1. OS and PFS curves were plotted using the Kaplan–Meier method, and the OS and PFS rates were calculated at 36, 48 and 60 months. We also compared OS and PFS according to tumour PD-L1 expression  in the pooled population and in non-SQ patients separately. AEs are reported as the number (percent) of patients with treatment-related AEs (TRAEs) according to SQ/non-SQ NSCLC type and in the pooled analysis.The sample sizes of both studies were calculated based on the threshold response rate for nivolumab, and the efficacy and safety outcomes were assessed in all patients who received at least one dose of nivolumab in each study ,12. For A total of 111 patients were enrolled and treated, 35 with SQ and 76 with non-SQ NSCLC. Among SQ NSCLC patients, the median duration of exposure to nivolumab and median number of doses of nivolumab were 110 days (range 15–2216 days) and 8 (range 2–152), respectively. The corresponding values in non-SQ NSCLC patients were 74.5 days (1–2290 days) and 6 (range 1–152). One SQ NSCLC patient and five non-SQ NSCLC patients were still receiving nivolumab at the data cut-off, with ongoing responses and administration for >60 months. A total of 17 patients were alive at the database lock (26 July 2019).In SQ NSCLC patients, ORR , DCR and median DOR were 25.7% (95% CI 14.2–42.1%), 54.3% (95% CI 38.2–69.5%) and not reached (range 3.0–68.9 months), respectively. In non-SQ NSCLC patients, the ORR , DCR and median DOR were 22.4% (95% CI 14.5–32.9%), 47.4% (95% CI 36.5–58.4%) and 41.5 months (range 1.6–72.1 months), respectively. In SQ and non-SQ NSCLC patients combined, ORR, DCR and median DOR were 23.4% (95% CI 16.5–32.1%), 49.5% (95% CI 40.4–58.7%) and 41.5 months (1.6–72.1 months), respectively.P = 0.070).The median OS in SQ NSCLC patients was 16.3 months (95% CI 12.4–25.2 months). The estimated 4- and 5-year survival rates were 14.3 (95% CI 5.2–27.7%) and 14.3% (95% CI 5.2–27.7%), respectively . The medCentrally assessed PFS was determined in SQ NSCLC patients, non-SQ NSCLC patients and in the pooled population . In SQ NPD-L1 expression was measurable in 59/111 patients and was classified as <1, ≥1–<50 and ≥50% in 17 (28.8%), 30 (50.8%) and 12 (20.3%) patients, respectively. OS increased in parallel with PD-L1 expression, with 5-year survival rates of 11.8, 21.8 and 41.7% in patients with PD-L1 expression of <1, ≥1–<50 and ≥50%, respectively . PFS proTRAEs of any grade occurred in 68.6% of SQ NSCLC patients and 86.8% of non-SQ NSCLC patients, and included grade 3–4 TRAEs in 8.6 and 25.0% of patients, respectively . SeriousIn a pooled analysis, TRAEs that occurred in ≥10% of patients included rash (15.3%), malaise (14.4%), decreased appetite (14.4%), pyrexia (14.4%) and nausea (10.8%). Other TRAEs in ≥5% of patients overall are listed in The ONO-4538-05 and ONO-4538-06 phase II studies were performed to examine the efficacy and safety of nivolumab in Japanese SQ NSCLC and non-SQ NSCLC patients. Here, we report the results of the long-term OS and PFS outcomes after ≥5 years of follow-up. Responses to the established conventional therapies are generally poor, as illustrated in CheckMate 017 and 057, in which the 5-year OS and PFS rates for docetaxel-treated patients were just 3 and 0%, respectively . Of noteEGFR gene status were significantly associated with 3-year OS (EGFR status were not significantly associated with 5-year OS in the present analyses, although the 5-year survival rate was numerically high in patients with wild-type EGFR. OS and PFS tended to be better in patients with higher PD-L1 expression, with median OS of 18.6 and 21.1 months in patients with PD-L1 expression of ≥1–<50 and ≥50%, respectively. It was reported that upregulation of PD-L1 might be detrimental to prognosis (We found no marked differences in characteristics between patients who survived for ≥5 years and non-survivors . In the -year OS . Howeverrognosis . The poorognosis , consistrognosis .Among the 5-year survivors in the present study, 89.5% showed a response to nivolumab. This is similar to the value of 75% reported for 5-year survivors in the previous phase Ib study . These rIn terms of the long-term safety of nivolumab, several TRAEs were reported after the 3-year follow-up. The frequency of TRAEs resulting in discontinuation of nivolumab increased slightly after the 3-year follow-up. However, there were no new safety signals during this period of time and, as in the previous report , the safThe main limitation of these phase II studies is the lack of a comparator group. In addition, the number of SQ patients was relatively small compared with the number of non-SQ patients. Nevertheless, their outcomes did not differ materially from those of non-SQ patients.In this long-term follow-up of patients with SQ or non-SQ NSCLC enrolled in two phase II studies in Japan, 17.8% survived for ≥5 years after starting nivolumab, with six patients still receiving nivolumab and showing an ongoing response at the last follow-up. Of note, long-term survival was observed even in patients with low PD-L1 expression. In terms of safety, no new safety signals were reported after ≥5 years of follow-up, including in patients treated with nivolumab for ≥5 years.At this time, individual participant data have not been made available. However, reasonable requests made to the corresponding author for de-identified patient-level data that underlie the results reported in this article will be considered.ONO-4538-0506_5-year_OS_Supplementary_Figures_hyaa157Click here for additional data file.


CXCL9 and CXCL10) in recipient. Moreover, triptolide decreased the numbers of B lymphocytes and plasma cells, as well as the levels of donor specific antibodies (DSAs) in recipient. Furthermore, triptolide not only inhibited vascular smooth muscle cell (VSMC) viability and promoted VSMC apoptosis but also significantly inhibited VSMC migration in vitro. These results emphasize the efficacy of triptolide in inhibiting TV development and provide a basis for developing new treatments to prevent TV-related complications and improve the long-term survival of transplant recipients.Transplant vasculopathy (TV), a hallmark of chronic allograft rejection, is the primary cause of allograft loss after organ transplantation. Because multiple mechanisms are involved in TV pathogenesis, effective therapy for it remains elusive. Here, we identify the role of triptolide, which has a wide spectrum of immuno-suppressive activities, in inhibiting TV development. Murine aortic transplants models were constructed and divided into triptolide-treated and untreated groups. We found that triptolide significantly alleviated intima thickening of allografts by inhibiting multiple pathways. Triptolide significantly reduced infiltration of T lymphocytes and macrophages and inhibited the levels of pro-inflammatory  and pro-fibrotic factors  in the graft. Additionally, triptolide significantly decreased the numbers of IFN-γ-producing T lymphocytes, as well as the expression of IFN-γ and IFN-γ-inducing factor ( Organ transplantation is an ideal and final solution for patients suffering end-stage organ diseases . HoweverIt is well known that multifactorial events participate in the development of TV. Previous studies have reported inflammation as a primary mechanism of TV development , moreoveTripterygium wilfordii Hook F, which is broadly used in clinic due to its strong immunosuppressive and anti-proliferative properties  weighing between 20 and 25 g, were used as donors or recipients, respectively. Animals were housed in a specific pathogen-free facility at Sun Yat-sen University , and all animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals  and according to the Sun Yat-sen University Institutional Ethical Guidelines for animal experiments. Abdominal aortic transplantation was performed using a previously described technique with modifications . Brieflyn = 5/group): the triptolide group, which was subcutaneously administered triptolide  every other day, initiating at day 0 after aortic transplant and continuing through the end of the experiment (day 28 after transplantation); the untreated group, which was subcutaneously administered an equal volume of normal saline. No other immunosuppressive medication was used.All mice were weighed before and during treatment. Recipients were randomly assigned to two groups  were cut, deparaffinized, and rehydrated, followed by staining with hematoxylin and eosin. The sections were examined for severity of luminal stenosis using a DMR Leica microscope  and Image-Pro Plus (IPP) 6.0 imaging software  by an experienced pathologist who was blinded to the groups. The cross-sectional area of luminal stenosis was calculated using the following formula: luminal occlusion (%) = / × 100. Thickness of intimal and intimal medial layers were measured from 10 sites per graft section and intima/intima + media ratios were calculated as described . FurtherFor IHC analysis, the cross-sections (4-μm thick) were deparaffinized and rehydrated, followed by incubation with antibodies against CD3 , CD4 , CD8 , and CD68  at 4°C overnight. The samples were then stained with Goat Anti-rabbit IgG/HRP  for 1 h at 37°C. Adventitial CD3+, CD4+, CD8+, and CD68+ cells were scored by cell counting using IPP 6.0 imaging software (Media Cybernetics) and expressed as cell number per vessel section (400× magnification).glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as an internal control. Primers used for qRT-PCR are provided in Levels of proinflammatory cytokine mRNA in allografts were determined by qRT-PCR. Total RNA was extracted from frozen graft tissue and mononuclear cells from recipient spleens using a homogenizer and TRIzol reagent , and cDNA was reverse transcribed using PrimeScript RT master mix . qRT-PCR was performed in triplicate using SYBR Green I master mix  in a LightCycler480 system (Roche), with Levels of circulating donor-specific antibodies  in recipient sera at the indicated time points were assessed by flow cytometry, as previously described . BrieflyTM  according to the manufacturer’s instructions for intracellular staining.Fresh recipient spleens were milled gently in phosphate-buffered saline (PBS) supplemented with 1% heat-inactivated fetal bovine serum using a needle on a 5-mL syringe, followed by pressing through a 200-um mesh nylon screen. Mononuclear cells from whole blood or spleen of recipient were collected by Ficoll density gradient centrifugation . Mononuclear cells were then stained with fluorochrome-conjugated antibodies against CD3, CD4, CD8a, IFN-γ, CD45, CD19, and CD38. For intracellular IFN-γ staining, cells were first stimulated at 37°C for 5 h with a leukocyte-activation cocktail , followed by staining with a surface marker, and further fixed and permeabilized using Cytofix/Cytopermin vitro, mononuclear cells from recipient spleens were seeded at a density of 1 × 106 cells/well and plated into a 24-well plate . The cells were then stimulated with anti-CD3  and anti-CD28  with or without tiptolide (4 ng/mL) at 37°C for 24 h. Then, cells were cultured in the presence of 1 μg/mL brefeldin A  for 12 h, harvested, followed by staining with a surface marker, and further fixed and permeabilized for intracellular staining. Data were analyzed using the FlowJo software . Antibodies used for flow cytometric analysis were purchased from BioLegend , including: FITC-CD4 , PerCP/Cy5.5-IFN-γ , BV570-CD45 , PerCP/Cy5.5-CD19 , and FITC-CD38 . Other antibodies were obtained from eBioscience , including: eFluor 450-CD8a  and Alexa Fluor 700-CD3 .To evaluate the effect of triptolide on IFN-γ-producing T cells 2.Aortic VSMCs (MOVAS-1) was a gift from Southern Medical University . The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin  at 37°C under 5% CO2.Mononuclear cells from recipient spleens were maintained in RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C with 5% CO4 cells/well in a 96-well plate (Corning Inc.), followed by exposure to PBS or various concentrations of triptolide  for 24 h and 48 h, and the subsequent addition of CCK-8 solution (10 μl). Cells were then incubated for 1 h at 37°C and 5% CO2, and the absorbance of each well was recorded at 450 nm using a microplate reader . Cell viability (%) was calculated as follows: (average absorbance of triptolide-treated cells/average absorbance of PBS-treated cells) × 100.Cell counting kit-8  assays were performed to evaluate the viability of VSMC following treatment with different concentrations of triptolide. Briefly, VSMCs were seeded at a density of 1 × 10in vitro effects of triptolide on VSMC apoptosis were determined using the Annexin V-FITC/propidium iodide assay. VSMCs were seeded at a density of 1 × 106 cells/well and plated onto a 6-well plate (Corning Inc.), followed by exposure to various concentrations of triptolide  for 24 h and 48 h, after which Annexin V-FITC/PI  was added to the cells and incubated for 10 min at 25°C in the dark according to manufacturer instructions. Cell apoptosis was analyzed by flow cytometry using a FACScan system .The 6 cells/well and plated in a 24-well plate (Corning Inc). Following exposure to various concentrations of triptolide  for 72 h, apoptosis assays were performed as described (Mononuclear cells from recipient spleens were seeded at a density of 1 × 10escribed .5 cells/well) in serum-free DMEM were placed in the upper chamber, followed by incubation with PBS or various concentrations of triptolide  for 24 h. Infiltrated cells were fixed in 4% paraformaldehyde  and stained with 0.1% crystal violet . Migrated cells were photographed under an inverted microscope , and five random high-power fields (200× magnification) were selected for quantification of cell number using the IPP 6.0 imaging software (Media Cybernetics). VSMC-migration ability was expressed as the ratio of the number of migrated cells to that of control cells.Vascular smooth muscle cell migration was determined using Transwell chambers, with a Transwell membrane containing 8-μm pores  and coated with 10 μg/mL fibronectin, inserted into a 24-well plate. The lower chamber was filled with 600 μL of DMEM with 10% FBS, and VSMCs . Probability values of P < 0.05 were considered significant.Data are expressed as mean ± standard deviation (SD). Normal distribution was first used to test the distribution of data using KS normality test. All data was normal distribution. Comparisons between two groups were performed using Student’s p < 0.05, p < 0.05).We established an allogeneic aortic graft model, and the recipients were treated with or without triptolide for 4 weeks; subsequently, allografts were harvested and tissue sections were stained with hematoxylin and eosin to visualize TV lesions. As shown in the representative photomicrographs, the arterial intima of the triptolide-treated group was observably thinner than those of untreated group . Additionally, qRT-PCR analysis of the mRNA levels of IFN-γ and IFN-γ-inducing factors (C-X-C-motif chemokine ligand (CXCL)9 and CXCL10) (TNF)-α, interleukin (IL)-2, and IL-6)] (TGF)-β, α-smooth muscle actin (SMA), and matrix metalloproteinase (MMP)-9 (p < 0.05).As shown in the representative images, the infiltration of CD38+ cells  were sig CXCL10) , pro-infd IL-6)] , pro-fib (MMP)-9  revealed+ (34.6% ± 2.7% vs. 28.5% ± 1.5%), CD3+ CD4+ (60.2% ± 1.9% vs. 52.2% ± 2.2%), CD3+ CD8+ (20.8% ± 2.0% vs. 17.8% ± 0.5%), CD4+ IFN-γ+ (5.7% ± 1.0% vs. 3.1% ± 0.6%), and CD8+ IFN-γ+ (8.4% ± 1.4% vs. 4.3% ± 1.5%) T cells in recipient blood , CD3+ CD4+ (55.7% ± 2.3% vs. 49.8% ± 2.4%), CD3+ CD8+ (21.7% ± 1.4% vs. 18.6% ± 0.6%), CD4+ IFN-γ+ (6.6% ± 0.7% vs. 2.7% ± 0.7%), and CD8+ IFN-γ+ (8.3% ± 1.3% vs. 4.2% ± 0.7%) T cells  were significantly inhibited  and plasma cells (CD45+ CD38+) in recipient blood, which revealed significant reductions of both B cells (9.00% ± 0.33% vs. 7.52 ± 0.26%) and plasma cells (12.91% ± 0.70% vs. 7.92 ± 0.25%) in the triptolide-treated group  of triptolide for 24 h and 48 h. Then, the viability was assessed by the CCK8 assay  associated with TV  to attenuate the prognosis of TV.Triptolide inhibited intimal thickening by suppressing the IFN-γ axis. Numerous studies had reported that triptolide inhibited the proliferation and activity of T lymphocytes , 29, and with TV . In our TNF-α, IL-2, and IL-6) and pro-fibrotic factors .Macrophages, pro-inflammatory and pro-fibrotic cytokines also play important roles in TV development –35. AlthPrevious studies confirmed that DSA levels were directly related to TV development . The effin vitro. Secondly, the mechanisms by which triptolide inhibits the migration of VSMC to improve the prognosis of TV remains to be further clarified. Finally, the concentration of triptolide used in vivo experiment is based on references and previous experience, so the toxic and side effects of triptolide are not described in the content.Several limitations of this study should be noted. Firstly, triptolide has a wide range of effects and this study does not make further in-depth study on the mechanisms, especially, direct allospecific T cell response has not been demonstrated In summary, our results reinforce the view that triptolide can significantly attenuate TV through inhibiting multiple pathways. These findings highlight the efficacy of triptolide in inhibiting TV and suggest triptolide as a potential ideal therapeutic strategy of great clinical value for preventing TV-related complications and improving the long-term survival of transplant recipients.The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the Sun Yat-sen University Institutional Ethical Guidelines.ZL: study design and drafting of the manuscript. TL and ZL: performed the transplantation model. YZ and FH: performed the experiments. QS and HZ: pathological scoring. ZY and ZL: statistical analysis; QS: conceived the study and critical revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.