Chaetomium produce compounds of this class, such as chetomin. To identify new antitumor agents, secondary metabolites of fungus Chaetomium sp 88194 were investigated and three new indole diketopiperazines, Chaetocochins G (1), Oidioperazines E (2) and Chetoseminudin E (3), along with two known compounds Chetoseminudins C (4) and N-acetyl-β-oxotryptamine (5), were obtained. Chaetocochins G and Chetoseminudin E were recrystallized in CHCl3 containing a small amount of MeOH, and their structures with absolute configuration were established by spectroscopic data interpretation and single-crystal X-ray diffraction analysis. The absolute configuration of Oidioperazines E was defined by comparing of experimental and calculated electronic circular dichroism spectra. These isolates were also evaluated the anticancer activity, and Chaetocochins G displayed more potent cytotoxicity in MCF-7 cells than the common chemotherapeutic agent (5-fluorouracil) associated with G2/M cell cycle arrest. More importantly, Chaetocochins G induced cell apoptotic death via caspase-3 induction and proteolytic cleavage of poly (ADP-ribose) polymerase, concomitantly with increased Bax and decreased Bcl-2 expression. Our findings suggested that indole diketopiperazines from endophytic Chaetomium sp 88194 may be potential resource for developing anti-cancer reagents.Diketopiperazines are important secondary metabolites of the fungi with variety bioactivities. Several species belonging to genus  Chaetomium produce many types of secondary metabolites, such as indole diketopiperazines which are widely found as mold secondary metabolites with bioactivities including antitumor, antimicrobial, antinematodal, and cytotoxicity effectsEndophytic fungi metabolites have gained increased attention as a new resource for the discovery of new therapeutic agents1Breast cancer is one of four oncology diseases that are most widespread in the world, it also one of leading causes of cancer-related deaths in female populationChaetomium cochliodes 88194 that obtained from China Forestry Culture Collection Center (cfcc 88194) was investigated, leading to the isolation of three new indole diketopiperazines (1–3) and two known compounds (4–5)61, 2, and 3), and Chaetocochins G (1) display antiproliferative effect, at least partially, due to the induction of apoptosis in MCF-7 cells.In order to find new antitumor agents, the extracts of the fermented rice substrate of the fungus 1–5) from Chaetomium cochliodes 88194 and investigating the antiproliferative effect of isolates against the MCF-7 human breast cancer cell line. The results showed that Chaetocochins G exhibited cytotoxicity with IC50 values of 8.3 μg/mL (48 h). Cell cycle arrest is regarded as one of effective strategies for eliminating the cancer cells811Our present research is focused on determining the structures of indole diketopiperazines . Its IR spectrum showed characteristic absorptions of hydroxyl group (3399 cm−1), and four carbonyl groups .Compound 1H NMR spectrum of 1 , three N-methyl , four methylene groups , two sets of aromatic protons , and an olefinic protons . Correspondingly, the 13C NMR and DEPT spectra showed the presence of the S-methyl, N-methyl, aromatic and olefinic carbons, together with five sp2 quaternary carbons , five sp3 quaternary carbons , one methines bearing heteroatoms (δC 81.7), and four carbonyl groups . In light of the above data, compound 1 was determined to be a chetomin-type indole diketopiperazine131 was deduced unambiguously from analysis of the 2D-NMR spectroscopic data, including 1H−1H COSY, HSQC, and HMBC experiments , colorless needle crystals; mp 209.6°C; c = 0.61, CDCl3); UV (CHCl3) λmax (log ε) 217 (4.79), 295 (4.06) nm; IR (KBr) νmax 3399, 3327, 2919, 1667, 1647, 1632, 1605, 1421, 1385, 1058, 751 cm−1; CD (CDCl2) 303 , 257  nm; 1H and 13C NMR data, see m/z 793.1934 [M + Na]+ .Chaetocochins G  observed in the HRESIMS corresponded to a molecular formula of C16H17N3O3S. The IR absorption bands at 1639, 1674 and 3429 cm−1 implied the presence of hydroxyl group and carbonyl groups. Comparison of 1H NMR data  and a N-methyl (δH 3.24), indicated the structure of 2 as shown (3 (δH 3.24) to C-1 (δC 161.7) and C-3 (δC 75.0) in the HMBC spectrum of 2 indicated that the N-methyl group was located at N-2. Similarly, the S-methyl group (δH 2.08) was located at C-3 (δC 75.0) (Compound NMR data  indicateas shown . The croδC 75.0) .2 was determined by comparing its experimental and calculated ECD spectra. The cotton effects in the experimental ECD spectrum were consistent with those in calculated ECD curve of (3R)-2 (2 was determined to be 3R configuration and named as Oidioperazines E.The absolute configuration of f (3R)-2 . So, the2): yellow amorphous powder; c = 2.07, CDCl3); UV (CHCl3) λmax (log ε) 227 (4.24), 339 (4.16) nm; IR (KBr) νmax 3429, 2928, 1674, 1639, 1530, 1432, 1103, 740 cm−1; CD (CDCl2) 236 , 335  nm; 1H NMR and 13C NMR data, see m/z 354.0883 [M + Na]+ .Oidioperazines E . The IR spectrum of 3 gave the absorption of hydroxyl group (3325 cm−1), and carbonyl groups (1685 and 1645 cm−1).Compound 1H NMR data of 3  and two carbonyl groups  could be observed.The ata of 3  exhibiteata of 3 . Accordi3/C-3. N-methyl assigned at the C-2 positions from the HMBC correlations between H-NCH3 from C-3 and C-1. Therefore, the planar structure of 3 is determined to be an analogue of Chetoseminudins BThe S-methyl moiety was located at the C-3 positions, as supported by the HMBC correlations  between 3. The absolute configuration of 3 was determined by the X-ray single crystal diffraction analysis with Cu Kα radiation. The results indicated that the crystal structure of compound 3 is shown in R, 6S and named Chetoseminudin E.As mentioned above, no NOESY correlations were observed to determine the relative configuration of 3): colorless cubic crystals; mp 193.3°C; c = 0.64, CH3OH); UV (CHCl3) λmax (log ε) 219 (4.66), 281 (3.89) nm; IR (KBr) νmax 3349, 3325, 2920, 1685, 1645, 1427, 1397, 1242, 1056, 752 cm−1; CD (CDCl2) 267 , 299  nm; 1H NMR and 13C NMR data, see m/z 372.0988 [M + Na]+ .Chetoseminudin E . The IC50 values of 48 h against MCF-7and Beas-2B cell were 8.3, and 9.7 μg/mL. Compounds 2, 3, 4 and 5 did not show obvious cytotoxicity (IC50 >40 μg/mL). So Chaetocochins G (1) was chosen for a further investigation.All compounds were evaluated for cytotoxic effect against MCF-7 cells, together with one noncancerous human pulmonary epithelial cell line BEAS-2B, using MTT methodμg/mL) for 48 h. The results  and ethidium bromide (EB) double stainingTo determine whether Chaetocochins G meditated inhibition of growth and proliferation was associated with apoptosis, the flow cytometry apoptosis assay was performed using Annexin V and PI staining202122μg/mL) for 48 h, the apoptosis of the cells were detected. As shown in After treatment at various concentrations MCF-7 cells were treated with Chaetocochins G as describe above. Changes of apoptosis-related protein, including Bax, Bcl-2, cleaved caspase-3 and PARP, were assessed by western blot analysis. 1–3) and two known compounds  from the extracts of Chaetomium sp 88194. All the compounds were evaluated for the cytotoxicity against MCF-7 cell line. The results indicated that Chaetocochins G (1) displayed much more potent inhibitory activity than the common chemotherapeutic agent 5-fluorouracil, with IC50 values of 8.3 μg/mL, and induced cell cycle arrest at the G2/M phase in MCF-7 cells, accompanied by initiation of apoptosis. Further study demonstrated that Chaetocochins G induced cellular apoptosis via the activation of the Caspase 3, PARP, and Bax up-regulation as well as Bcl-2 inhibition. This paper is the first report for apoptosis-inducing effects of tryptophan–derived epidithiodioxopiperazines, and provides an insight into their anticancer mechanism. These results suggest that indole diketopiperazines from endophytic Chaetomium sp 88194 may be potential resource for anticancer drug research and discovery.In conclusion, the current study describes the isolation and identification of three new indole diketopiperazines  or CDCl3 (δH 7.26/δC 77.16). The CD spectra were obtained on a JASCO J-810 spectrometer. X-ray crystallographic data were collected from Bruker Smart APEX-II CCD diffractometer equipped with graphite-monochromatized Cu Kα radiation (λ = 1.54178 Å). HPLC isolation was performed on waters 2535 pump and a waters 2998 detector using a C18 column  and MeOH−H2O or CH3CN−H2O as the mobile phase at 2.0 mL/min flow rate. MPLC was carried out with a QuikSep-50 chromatography system . TLC was carried out using glass-precoated silica gel GF254  and visualized under UV light. Silica gel , ODS , and Sephadex LH−20  were used for column chromatography.Melting points (uncorrected) were determined on a Beijing Tech X-5 microscopic melting point apparatus . Optical rotations were determined in CHClCymbidium goeringii, which was collected from Xinning, Hunan Province of China, in November 2008. The strain was purified and identified belonging to the Chaetomium sp by molecular identification. The DNA sequence data from the fungus were deposited at GenBank with accession number KP171518, and the strain was deposited at Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.The strain of the fungus obtained from China Forestry Culture Collection Center  was isolated from the 2O (200 mL). After autoclaving at 121°C, 15 psi for 30 min, each flask cooling to room temperature was inoculated with the fresh mycelium and incubated at 25°C for 28 days.The fermentation was carried out in 40 fernbach flasks (1000 mL), each containing 200 g of rice and distilled H2 gradient to give four fractions, A–D. Fractions C (2.7 g) was chromatographed over ODS by MPLC and eluted with a MeOH−H2O gradient to yield four fractions (CA–CD).The fermented solid rice medium (8.0 kg) was soaked with 95% aqueous EtOH  at room temperature. The solvent was evaporated in vacuo to afford a residue (22.0 g), which was then partitioned between ethyl acetate (3 L × 3) and aqueous (3 L). On evaporation, the organic phase (250 g) was subjected to silica gel (200–300 mesh) column chromatography and eluted with a MeOH−CHClA was further separated by semipreparative HPLC  to yield compounds 1 . Subfraction CB was further separated over Sephadex LH-20 eluting with CHCl3–MeOH (1:1) to give three subfractions (CBA–CBC). Subfraction CBB was further purified by semi-preparative HPLC eluted with MeOH–H2O  to give compounds 3 , compounds 2  was obtained from CBC through semi-preparative HPLC eluted with CH3CN–H2O . Subfraction CC was purified by Sephadex LH-20 column chromatography eluting with CHCl3–MeOH (1:1) to give two subfractions (CCA–CCB). Then, CCB was chromatographed over ODS by MPLC and eluted with a MeOH−H2O gradient to yield compounds 4 (1.95 mg) and 5 (5.8 mg).Subfraction C1) were obtained from CHCl3 containing a small amount of MeOH at room temperature. Crystal data were obtained on a Bruker Smart APEX-II CCD single-crystal X-ray diffractometer with the Cu Kα (λ = 1.54178 Å). Structure solution and refinement were performed with the SHELXL-97. Crystal data of 1: C35H42N6O6S4·H2O (M = 789.00); needle crystal (0.20 × 0.20 × 0.10 mm); space group P21; unit cell dimensions a = 13.3191(3) Å, b = 13.7927(3) Å, c = 21.8874(5) Å, α = 90.00°, β = 96.0450(10)°, γ = 90.00°, V = 3998.50(15) Å3; Z = 4; T = 296(2) K; ρcalcd = 1.311 Mg/m3; absorption coefficient 2.623 mm−1, F(000) = 1664, A total of 10529 reflections were collected in the range 2.03° < θ < 58.42° with 10529 independent reflections [R(int) = 0.0000]; completeness to θmax was 97.3%; full-matrix least-squares refinement on F2; the number of data/parameters/restraints were 10529/1029/47; goodness-offit on F2 = 1.067; final R indices [I > 2σ(I)] R1 = 0.0437, wR2 = 0.1226; R indices  R1 = 0.0578, wR2 = 0.1312; absolute structure parameter 0.015(12).Colorless needle crystals of Chaetocochins G (3) were obtained from CH3OH at room temperature. Crystal data were obtained on a Bruker Smart APEX-II CCD single-crystal X-ray diffractometer with the Cu Kα (λ = 1.54178 Å). Structure solution and refinement were performed with the SHELXL-97. Crystal data of 3: 2(C16 H20 N3 O4 S)·H2 O (M = 716.82); monoclinic crystal (0.71 × 0.53 × 0.40 mm); space group P21; unit cell dimensions a = 8.8404(2) Å, b = 14.4203(3) Å, c = 13.5625(2) Å, α = 90.00°, β = 92.8240(10)°, γ = 90.00°, V = 1726.86(6) Å3; Z = 2; T = 298(2) K; ρcalcd = 1.379 Mg/m3; absorption coefficient 1.925 mm−1, F(000) = 756, A total of 21876 reflections were collected in the range 3.26° < θ < 68.72° with 6000 independent reflections [R(int) = 0.0328]; completeness to θmax was 96.9%; full-matrix least-squares refinement on F2; the number of data/parameters/restraints were 6000/452/1; goodness-offit on F2 = 1.042; final R indices [I > 2σ(I)] R1 = 0.0284, wR2 = 0.0785; R indices  R1 = 0.0286, wR2 = 0.0787; absolute structure parameter 0.025(10).Colorless cubic crystals of Chetoseminudin E (1) and Chetoseminudin E (3) have been deposited in the Cambridge Crystallographic Data Centre . Copies of these data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html  1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk).Crystallographic data for the structures of Chaetocochins G (R and 3S isomers of Oidioperazines E (2) were explored using both BALLOONσ of 0.45 eV. The calculated spectra for each conformation were combined after Boltzmann weighting according to their population contribution.The conformational spaces for the 3μg/mL streptomycin. Cultures were maintained in an incubator  with 5% CO2 at 37°C.MCF-7 human breast cancer cells, purchased from the cell bank of the Basic Medical College of Huazhong University of Science and Technology, were cultured in DMEM medium , supplemented with 10% fetal bovine serum , 100 U/mL penicillin, and 100 3 per well) and allowed to adhere for 12 h before treated with Chaetocochins G at a series of concentration  for 48 hours. Add 20 μL MTT (5 mg/mL) per well was added prior to 4 h incubation at 37°C. Upon medium removal, formazans depositing on the plate were dissolved in 150 μL DMSO, then, the cell viability was detected by a Thermo Multiskan MK3 microplate reader  at λ = 492 nm, and the IC50 values were obtained from the MTT viability curves using GraphPad Prism 4.0.The viability of cells was performed using an MTT   method in 96-well microplates, as reported previouslyμg/mL) for 48 h. a staining method using AO  and EB  was performed and photographed using a fluorescence microscope .After incubation with various concentrations of Chaetocochins G  and incubated for 48 h. Then, the cells were harvested and washed with ice-cold PBS buffer, fixed with 70% alcohol at 4°C for 12 h, and stained with propidium iodide (PI) in the presence of 1% RNAase A. After 15 min incubation at 37°C, the cells were analyzed by a flow cytometry . The gated data were analyzed by modfit softwares .The cell cycle analysis was detected with a cell cycle and apoptosis analysis kit . MCF-7 cells were exposed to Chaetocochins G  for 48 h, cells were collected, washed with annexin-binding buffer, incubated with annexinV-FITC/PI for 15 min and analyzed by a flow cytometry .Apoptosis assay was performed using an apoptosis detection kit  according to the manufacturer's instructions. Briefly, after exposure to Chaetocochins G   membranes. After incubated overnight at 4°C with the respective primary antibodies, the horseradish peroxidase-conjugated secondary antibodies were added. Finally, the reactive band was identified using an enhanced chemiluminescent substrate to horseradish peroxidase.Western blotting analysis was performed as describedp < 0.05 were considered statistically significant.Statistical analysis of the data was processed with GraphPad Prism 4.0 software. Statistical analysis of the data was expressed as mean ± SD. Values were analyzed by one-way analysis of variance (ANOVA) using SPSS version 12.0 software. Y.H.Z., G.Z. and Y.B.X. designed experiments. J.J.L. complete the computation section. W.M. and J.W.Z. wrote the paper. Q.Y.T. and H.R.M. conducted experiments. F.Q.W. isolated and identified of the compounds. H.P.S. analyzed data. J.P.W., H.F.X. and S.H. prepared the article in the journal's format. All authors reviewed the manuscript.Supporting informationCIF check of Chaetocochins G (1)CIF check of Chetoseminudin E (3)


Clinically sufficient quantities of TCRδ1, TCRδ2, and TCRδ1negTCRδ2neg have been produced following co-culture on aAPC, and these subsets displayed differences in memory phenotype and reactivity to tumors in vitro and in vivo. Gamma delta T cells are also amenable to genetic modification as evidenced by introduction of αβ TCRs, chimeric antigen receptors, and drug-resistance genes. This represents a promising future for the clinical application of oligoclonal or polyclonal γδ T cells in autologous and allogeneic settings that builds on current trials testing the safety and efficacy of Vγ9Vδ2 T cells.γδ T cells hold promise for adoptive immunotherapy because of their reactivity to bacteria, viruses, and tumors. However, these cells represent a small fraction (1–5%) of the peripheral T-cell pool and require activation and propagation to achieve clinical benefit. Aminobisphosphonates specifically expand the Vγ9Vδ2 subset of γδ T cells and have been used in clinical trials of cancer where objective responses were detected. The Vγ9Vδ2 T cell receptor (TCR) heterodimer binds multiple ligands and results in a multivalent attack by a monoclonal T cell population. Alternatively, populations of γδ T cells with oligoclonal or polyclonal TCR repertoire could be infused for broad-range specificity. However, this goal has been restricted by a lack of applicable expansion protocols for non-Vγ9Vδ2 cells. Recent advances using immobilized antigens, agonistic monoclonal antibodies (mAbs), tumor-derived artificial antigen presenting cells (aAPC), or combinations of activating mAbs and aAPC have been successful in expanding gamma delta T cells with oligoclonal or polyclonal TCR repertoires. Immobilized major histocompatibility complex Class-I chain-related A was a stimulus for γδ T cells expressing TCRδ1 isotypes, and plate-bound activating antibodies have expanded Vδ1 and Vδ2 cells  They TRD) lies within the TCRα locus (TRA). Three unique Vδ alleles, TRDV1, TRDV2, and TRDV3, code for TCRδ1, TCRδ2, and TCRδ3, respectively. Additionally, shared Vδ and Vα variable regions exist in TRDV4/TRAV14, TRDV5/TRAV29, TRDV6/TRAV23, TRDV7/TRAV36, and TRDV8/TRAV38-2 loci. Recombination of these shared V alleles with a TRA junction region (TRAJ) results in TCRα14, TCRα29, TCRα23, TCRα36, and TCRα38-2, respectively, but recombination of these shared V alleles with TRD junction (TRDJ) and diversity (TRDD) regions results in TCRδ4, TCRδ5, TCRδ6, TCRδ7, and TCRδ8, respectively  region of the TCRδ chain. In humans, the TCRδ locus  heterodimers have specificity for MHC Class-I chain-related A (MICA) , 22, a mwith Vγ2 –32. Vγ9Vrculosis –37. Morerculosis , and in  for EBV , 39. In  for EBV , 41. Givin vivo propagation of γδ T cells, and the use of aminobisphosphonates has been subsequently translated into laboratory practice to grow γδ T cells ex vivo  and activating CD3 antibody (OKT3), commonly used for the propagation of αβ T cells directly from peripheral blood mononuclear cells (PBMC), do not reliably expand γδ T cells without further manipulation and so alternative approaches are needed. Aminobisphosphonates, e.g., Zoledronic Acid (Zol), used in the treatment of bone-related diseases, e.g., osteoporosis, resulted in o Figure A  but no complete responses (CR) as anti-tumor therapies. Six clinical trials have used either Zol, BrHPP, or 2M3B1PP to expand autologous Vγ9Vδ2 T cells ex vivo and these cells were directly infused  for treatment of RCC (N = 71) were detected, including one CR  (ex vivo expanded Vγ9Vδ2 T cells followed by Zol administration to boost their in vivo proliferation. Multiple myeloma (N = 29) were observed, two of which were CRs  treating intermediate-stage RCC , 5049, 5lymphoma , 52, mellymphoma , hormonelymphoma , breast lymphoma , and HIVlymphoma . These tt of RCC –59, non-t of RCC , 61, andt of RCC . Direct  N = 71) . Three t myeloma , RCC  with 36 durable CRs   –69. ThesN = 153) , 70. Mosnfusions . Three oex vivo from epithelial ovarian cancer and CRC tumor infiltrating lymphocytes (TILs) and resulted in high frequencies of Vδ1 cells  and antibody-dependent cellular cytotoxicity (ADCC), respectively, to neuroblastoma tumor cells (as predicted by whether or not they expressed Fc receptor CD16). aAPC-expanded polyclonal γδ T cells could be used for anti-tumor therapies because aAPC are currently available as a clinical reagent. However, human application of aAPC/mAb-expanded γδ T cells could depend on interest in the use of the current MCB of aAPC, generation of new MCB of aAPC at institutions where there are currently none, and production of γδ T cell agonistic antibodies in good manufacturing practice (GMP) conditions. Clinical testing of these cells could potentially lead to more widespread acceptance and use of γδ T cells as adoptive cellular therapies.Highly polyclonal γδ T cells have been generated through co-culture of patient or healthy donor γδ T cells with irradiated artificial antigen presenting cells (aAPC), IL-2, and IL-21 –80. The eg > Vδ2 . These ps Figure . This stneg and CD27+ γδ T cells are associated with IL17 and IFNγ production, respectively . Mice can now be immunized to generate mAb specific for desired TCRγδ isotypes where commercial and academic use of these detection antibodies can have tangible outcomes, including diagnostic and/or prognostic profiling of γδ T cells resident within tumors. γδ T-cell clones could be generated through co-culture of single γδ T cells with aAPC, and this can facilitate studies to determine Vδ/Vγ pairing, corresponding TCRγδ ligands, and pathogenic reactivity. The ligands on the K562-derived aAPC that TCRγδ binds are not currently known. Likely candidates include IPP and MICA/B for TCRδ2 and TCRδ1, respectively , 35. Eluectively –87, lead+ and CD8+ T cells reactive to multiple tumor cell lines  transposition transfers genes into quiescent T cells and allows manipulation of cells that are difficult to culture ex vivo  because their TCR ligands  are not MHC-restricted, γδ T cells (with or without genetic modification) could be generated from healthy donors in a third party manufacturing facility and given in the allogeneic setting as an “off-the-shelf” therapeutic. Additionally, a “universal” bank of polyclonal γδ T cells could be established that was known to have high anti-tumor immunity or contain a particular set frequency of Vδ1, Vδ2, and Vδ1efficacy . This cohe tumor –109. If he tumor , 111, anhe tumor . Tumor lhe tumor . Indeed,g., PAX5 , 115. Ung., PAX5 . In summDrew C. Deniger, Judy S. Moyes, and Laurence J. N. Cooper wrote the manuscript.Dr. Cooper founded and owns InCellerate, Inc. He has patents with Sangamo BioSciences with artificial nucleases. He consults with Targazyme, Inc. , GE Healthcare, Ferring Pharmaceuticals, Inc., and Bristol-Myers Squibb. He receives honoraria from Miltenyi Biotec. Other authors declare no other competing financial interests.


Previous studies on microbial production of 2,3-BDO has focused on sugar fermentation. Alternatively, biodiesel-derived crude glycerol can be used as a cheap resource for 2,3-BDO production; however, a considerable formation of 1,3-propanediol  and low concentration, productivity, and yield of 2,3-BDO from glycerol fermentation are limitations.Klebsiella oxytoca M3 in which pduC (encoding glycerol dehydratase large subunit) and ldhA (encoding lactate dehydrogenase) were deleted to reduce the formation of 1,3-PDO and lactic acid. In fed-batch fermentation with the parent strain K. oxytoca M1, crude glycerol was more effective than pure glycerol as a carbon source in 2,3-BDO production (59.4 vs. 73.8 g/L) and by-product reduction . When the double mutant was used in fed-batch fermentation with pure glycerol, cell growth and glycerol consumption were significantly enhanced and 2,3-BDO production was 1.9-fold higher than that of the parent strain (59.4 vs. 115.0 g/L) with 6.9 g/L of 1,3-PDO and a small amount of lactic acid (0.7 g/L). Notably, when crude glycerol was supplied, the double mutant showed 1,3-PDO-free 2,3-BDO production with high concentration (131.5 g/L), productivity (0.84 g/L/h), and yield (0.44 g/g crude glycerol). This result is the highest 2,3-BDO production from glycerol fermentation to date.Here, we report a high production of 2,3-BDO from crude glycerol using the engineered pduC and ldhA genes in K. oxytoca M1 and 1,3-PDO-free 2,3-BDO production was achieved by using the double mutant and crude glycerol. 2,3-BDO production obtained in this study is comparable to 2,3-BDO production from sugar fermentation, demonstrating the feasibility of economic industrial 2,3-BDO production using crude glycerol.2,3-BDO production from glycerol was dramatically enhanced by disruption of the The online version of this article (doi:10.1186/s13068-015-0336-6) contains supplementary material, which is available to authorized users. In the last few years, considerable effort and progress have been made in the production of bio-based bulk chemicals from renewable resources because of the decrease in fossil fuel availability and increasing concern for global warming . 2,3-ButJatropha hulls [Previous studies on biological production of 2,3-BDO have focused on sugar fermentation using glucose –10 and sha hulls , and sugha hulls .Klebsiella species, such as K. pneumoniae and K. oxytoca, have been found to utilize glycerol as the sole carbon source and produce 1,3-propanediol  and 2,3-BDO [2 under aerobic conditions) or via dihydroxyacetone in the absence of oxygen  ; however, 1,3-PDO was still generated up to 16.3 g/L, and this result was obtained using pure glycerol, not crude glycero1. Yang et al. [Bacillus amyloliquefaciens by supplying beet molasses as a co-substrate in addition to biodiesel-derived crude glycerol; however, when biodiesel-derived glycerol was supplied as the sole carbon source, much lower 2,3-BDO concentration (43.1 g/L) and productivity (0.45 g/L/h) were obtained by B. amyloliquefaciens. Moreover, considering that the 2,3-BDO titer and productivity levels obtained from glucose fermentation by K. pneumoniae (101.5–150.0 g/L and 2.54–4.21 g/L/h) [K. oxytoca (113–130 g/L and 1.63–2.1 g/L/h) [Several reports have investigated the effect of dissolved oxygen level  and pH , 21 on 2g et al.  demonstr1 g/L/h)  were mucK. oxytoca M1 was reported to be able to produce 2,3-BDO and acetoin selectively as the main products depending on temperature [K. oxytoca M3, in which pduC  and ldhA  were deleted to reduce the formation of by-products, such as 1,3-PDO and lactic acid. The double deletion mutant showed a significantly improved 2,3-BDO production from pure glycerol and crude glycerol in fed-batch fermentation compared to the parent strain. Notably, 1,3-PDO-free 2,3-BDO production by the double deletion mutant was observed when crude glycerol was used as the carbon source in fed-batch fermentation. To our knowledge, the final titer (131.5 g/L), the productivity (0.84 g/L/h), and the yield (0.44 g/g crude glycerol) of 2,3-BDO from crude glycerol obtained by the double deletion mutant were the highest recorded in 2,3-BDO production from glycerol as the sole carbon source to date.Previously, a newly isolated perature . In thisK. oxytoca M1, flask batch fermentation was performed with pure glycerol and crude glycerol as the sole carbon sources at the initial concentration of 35–40 g/L. As shown in Table K. oxytoca M1 was similar regardless of the type of glycerol  in a 3 L-bioreactor. Fed-batch fermentation using crude glycerol was also performed to investigate whether the accumulation of impurities in crude glycerol would inhibit the cell growth and 2,3-BDO production of K. oxytoca M1. When fed-batch fermentation using pure glycerol was carried out for 114 h, K. oxytoca M1 successfully produced 2,3-BDO at concentrations up to 59.4 g/L with the productivity of 0.52 g/L/h . Thus, further fed-batch fermentation was conducted with pH control at 6.Lactic acid and 1,3-PDO productions during the fed-batch fermentation Table  were difn source , 21, 29.n source . In the K. oxytoca M1 could produce 2,3-BDO using crude glycerol at a higher concentration and productivity levels than those achieved in previous studies, by-product formation needed to be decreased for efficient conversion of glycerol to 2,3-BDO. To reduce the formation of 1,3-PDO and lactic acid, the genes encoding glycerol dehydratase large subunit (PduC), which is responsible for the first step of 1,3-PDO synthesis from glycerol, and lactate dehydrogenase (LDH) converting pyruvate to lactic acid were chosen for deletion.Although pduC, the gene encoding PduC, on cell growth and 2,3-BDO production. Thus, to investigate whether the deletion of pduC exhibited adverse effects on cell growth and 2,3-BDO production, K. oxytoca M1 pduC deletion mutant was first constructed using the λ Red recombination system [pduC gene of K. oxytoca M1 was successfully deleted  in comparison with the 1,3-PDO production of the parent strain  in which the ldhA gene was abrogated from the pduC mutant  . The maximum DCW of K. oxytoca M3 was also greater than that of K. oxytoca M1 (8.6 g/L vs. 4.7 g/L) , cell mass, and by-products .To investigate the effect of the deletion of L) Figs. , 4a. ThepduC and ldhA, probably because increased NADH availability would induce the NADH-consuming ethanol synthesis pathway for NADH/NAD+ balance. The production of 1,3-PDO (6.9 g/L) by K. oxytoca M3 was lower than that of K. oxytoca M1 (8.9 g/L), but it was higher than that of K. oxytoca M2 (0.8 g/L) , the deletion of pduC was still effective to decrease carbon flux to 1,3-PDO.Regarding the formation of by-products, the concentration of ethanol was increased (4.0 vs. 9.3 g/L) Table  by the dK. oxytoca [K. oxytoca M3 was higher than K. oxytoca M1 (1.04:1 vs. 0.79:1), implying that K. oxytoca M3 consumed more NADH than K. oxytoca M1 per mole of 2,3-BDO production (1.51 vs. 1.44 mol of NADH). In fact, assuming that the aerobic route was involved to convert glycerol to pyruvate  . However, the percentage of NADH consumption toward 2,3-BDO production out of total NADH consumption in K. oxytoca M3 was much higher than in K. oxytoca M1 . This higher NADH consumption due to the higher -2,3-BDO portion by K. oxytoca M3 would explain how the redox balance was maintained despite the deletion of pduC and ldhA.It has been known that a mixture of meso-2,3-BDO  and -2,3-BDO  is produced by K. oxytoca M1 was 0.11:1 and 1.06:1 from glucose and glycerol, respectively, in flask cultures  [K. oxytoca M1 and K. oxytoca M3 from glycerol compared to other Klebsiella strains might be partially attributed to the regulation of redox balance by significantly increasing the -2,3-BDO portion.In the meantime, we found that the ratio of -2,3-BDO to meso-2,3-BDO produced by toca M1)  was testK. oxytoca M1. Therefore, fed-batch fermentation using crude glycerol was carried out with K. oxytoca M3 to investigate whether a higher 2,3-BDO production would be achieved in comparison to the results in Fig. As seen in Fig. K. oxytoca M3, which stopped after 114 h, 2,3-BDO production from crude glycerol occurred until 156 h. When crude glycerol was used as the sole carbon source for K. oxytoca M3, DCW was much higher than those obtained with pure glycerol . Because of this additional NADH consumption, the production of net NADH-consuming by-product might be decreased. Further investigation would be needed to elucidate the effect of crude glycerol on metabolism.As expected, only a small amount of lactic acid (0.8 g/L) was produced. Notably, in contrast to the fed-batch fermentation using pure glycerol by  M3 Fig. a, 1,3-PD M3 Fig. b. Moreov M3 Fig. . This deK. pneumoniae G31 using pure glycerol as the sole carbon source with a yield of 0.39 g/g and productivity of 0.47 g/L/h through the application of forced pH fluctuation. Yang et al. [B. amyloliquefaciens using beet molasses as a co-substrate in addition to biodiesel-derived glycerol; however, using crude glycerol as the sole carbon source, B. amyloliquefaciens produced only 43.1 g/L of 2,3-BDO with a yield of 0.38 g/g and productivity of 0.45 g/L/h. In this study, the efficient 2,3-BDO production from biodiesel-derived glycerol was fulfilled by the deletion of the pduC and ldhA genes in K. oxytoca M3 to reduce the formation of 1,3-PDO and lactic acid, and consequently the carbon flux was mainly redirected to 2,3-BDO. More importantly, use of the double deletion mutant and crude glycerol resulted in 1,3-PDO-free 2,3-BDO production, involving no concern to separate 1,3-PDO from 2,3-BDO-containing cell broth.Table g et al.  obtainedK. oxytoca strains [Overall, a new record of the highest 2,3-BDO concentration from crude glycerol (131.5 g/L) as the sole carbon source was achieved with the highest productivity 0.84 g/L/h) and yield (0.44 g/g crude glycerol) without 1,3-PDO production. It is worth noting that 2,3-BDO concentration obtained using crude glycerol in this study is comparable to the 2,3-BDO production from glucose reported by Ji et al. (130 g/L) and Park et al. (113 g/L) using  strains , 8. Furt4 g/L/h aK. oxytoca M3. Enhanced 2,3-BDO production from crude glycerol was achieved by disruption of the pduC and ldhA genes, which resulted in a nearly abolished lactic acid and 1,3-PDO production. To the best of our knowledge, 2,3-BDO concentration (131.5 g/L), productivity (0.84 g/L/h), and yield (0.44 g/g) achieved in this study are the highest levels in glycerol-based 2,3-BDO production reported to date, demonstrating that biodiesel-derived glycerol could be used to produce 2,3-BDO cost-effectively by the metabolically engineered strain K. oxytoca M3.In this study, biodiesel-derived glycerol was used as the sole carbon source for 2,3-BDO production by the engineered strain K. oxytoca M1 was deposited in the Korean Culture Center of Microorganisms (KCCM) as KCCM 1177P. K. oxytoca KCTC1686  was purchased from the KoreanAll bacterial strains and plasmids used in this study are listed in Table K. oxytoca M1 and its mutants were pre-cultured in Luria–Bertani (LB) medium at 30 °C. The defined medium used for flask fermentation contained (per L of distilled water): K2HPO4 13.7 g, KH2PO4 2 g, (NH4)2HPO4 3.3 g, (NH4)2SO4 6.6 g, MgSO4·7H2O 0.25 g, FeSO4·7H2O 0.05 g, ZnSO4·7H2O 0.001 g, MnSO4·H2O 0.01 g, CaCl2·2H2O 0.01 g, and EDTA 0.05 g. Pure glycerol or crude glycerol were added to the defined medium as needed. Crude glycerol provided by GS Caltex Corporation (South Korea) contained (wt/wt): 81.7 % of glycerol, 10.5 % of water, 5 % of MONG (matter organic non-glycerol), 2.9 % of ash, 2.4 % of sodium, and less than 0.01 % of methanol, magnesium and potassium. The medium used for pH-controlled fermentation was the defined medium supplemented with 5 g/L yeast extract and 10 g/L casamino acid [Collection for type culture . ino acid .For flask fermentation without pH control, pre-culture cultivation was carried out overnight in LB medium at 30 °C and 200 rpm. Then, the seed culture was inoculated in a 100 mL Erlenmeyer flask  containing 20 mL of the defined medium. Pure glycerol and crude glycerol (35–40 g/L) were added to the defined medium to investigate metabolite production patterns . All flask experiments were performed in triplicate.K. oxytoca M1 and the derivatives were inoculated into 100 mL of LB medium and cultivated on a rotary shaker at 200 rpm at 30 °C for 10 h. The seed culture  was then inoculated into the defined medium supplemented with 10 g/L casamino acid and 5 g/L yeast extract. All cultivations were carried out at 30 °C, and the pH level was maintained at 6 by automatic addition of 5 N NaOH. The aeration rate was controlled at 1.0 volume of air per volume of liquid per minute (vvm) with the agitation speed of 400 rpm.All trials for 2,3-BDO fermentation with pH control in this study were conducted in a 3 L stirred fermenter  with a working volume of 1 L. For seed culture preparation, The batch cultivation was carried out at 30 °C using fermentation medium containing 90 g/L of glycerol. Fed-batch fermentation was carried out with an initial glycerol concentration of 90–100 g/L, and then a concentrated solution containing 800 g/L of pure glycerol or crude glycerol was fed into the fermenter as required.pduC gene (accession number AIE72369) deletion mutant, K. oxytoca M2, was developed from K. oxytoca M1 using the λ Red recombination method [K. oxytoca M1 (CP008841) was provided by Macrogen Inc.  [pduC in genomic DNA were generated using the primers pduUp1 and pduUp2 to get 1031 bp of fragments and using the primers pduDown1 and pduDown2 to obtain 843 bp of fragments, respectively (Table R-FRT (930 bp), and downstream regions (843 bp) were amplified by the primers pduCDown2 and pduCUP1, using PCR products as a template by the overlap extension PCR method.The n method , 31 (Addh Korea) . Two PCRly Table . In addily Table  to get 9K. oxytoca M1 by electroporation at 12.5 kV/cm, 200 Ω, 25 μF . The 2639 bp linear fragments containing CmR were transformed to K. oxytoca M1 haboring pRedET, and the chloramphenicol-resistant colonies were selected on LB agar plates containing 25 μg/mL of chloramphenicol at 37 °C  obtained using the pduCcon1 and pduCcon2 primer pair  were transformed into the cells and the tetracycline-resistant transformants were selected at 30 °C. After cultivation at 42 °C overnight, the desirable antibiotic-susceptible transformants which lost the FRT flanking CmR gene and the 707-FLPe were selected. The resulting mutant was confirmed by PCR  double deletion mutant, K. oxytoca M3, was constructed from K. oxytoca M2 (K. oxytoca M ΔpduC) using the λ Red recombination method as described above. Two PCR products including the upstream and downstream regions of ldhA in genomic DNA were generated by using the primers ldhAUp1 and ldhAUp2  using the calibration curve of OD600 and the dry cell weight of K. oxytoca M1. The OD600 of the broth was measured by UV–visible spectrophotometry  with appropriate dilution.Dry cell weight  was calculated from optical density at 600 nm .The concentrations of meso-2,3-BDO, -2,3-BDO, -2,3-BDO, 1,3-PDO, and ethanol were measured using a gas chromatograph  equipped with a flame ionized detector (FID) and an HP-Chiral-20B GC column (30 m X 0.32 mm X 0.25 μm) (Agilent) under the conditions described previously , 27. Gly


Extensive intermolecular O—H⋯O hydrogen bonding supplements this bonding network.The anion has an open-chain structure in which one of the oxygen atoms of the sulfate residue, the S atom, the C atoms of the sugar chain and the O atom of the hydroxymethyl group form an essentially planar zigzag chain. A three-dimensional bonding network exists in the crystal structure involving hexacoordination of sodium ions by O atoms, three of which are provided by a single  +·C5H11O8S− , is formed by reaction of d-lyxose with sodium bisulfite (sodium hydrogen sulfite) in water. The anion has an open-chain structure in which one of the oxygen atoms of the sulfonate residue, the S atom, the C atoms of the sugar chain and the O atom of the hydroxymethyl group form an essentially planar zigzag chain with the corresponding torsion angles lying between 179.80 (11) and 167.74 (14)°. A three-dimensional bonding network exists in the crystal structure involving hexacoordination of sodium ions by O atoms, three of which are provided by a single d-lyxose–sulfonate unit and the other three by two sulfonate groups and one hydroxymethyl group, each from separate units of the adduct. Extensive intermolecular O—H⋯O hydrogen bonding supplements this bonding network.The title compound, Na Both of the d-lyxose adducts were stable on storage in a sealed container at room temperature.Crystallization of the sodium bisulfite adduct of S-configuration -1,2,3,4,5-pentahydroxypentane-1-sulfonate. The anion has an open-chain structure in which one of the oxygen atoms, O13, of the sulfonate residue, the S atom, the C atoms of the sugar chain and the oxygen atom, O5, of the terminal hydroxymethyl group form an essentially planar zigzag  chain with the corresponding torsion angles lying between the absolute values of 179.80 (11) and 167.74 (14)°. The atoms O13–C4 form a plane, with C5 and O5 displaced 0.229 (3) and 0.525 (2) Å, respectively, from that mean plane. All of the hydroxyl groups form medium-strength to weak intermolecular hydrogen bonds which connect molecules in an extensive three-dimensional network  to 2.5265 (16) Å. The sodium atoms are linked in planes parallel to the ab plane through coordinating sulfonate groups supported by H–O⋯Na coordination and hydrogen bonds  hexa-coordination of a sodium cation with oxygens from four different lyxose bisulfite residues, three of those oxygens coming from one such residue, and (ii) intermolecular hydrogen bonds from each of the five hydroxyl groups to acceptor oxygens in four different residues.5H11O8S1]−) at m/z 231.0108 but a significant peak was observed at 213.0075 ([C5H11O8S1– H2O]−). The mono-anion of d-lyxose gave a peak at m/z 149.0458 ([C5H9O5]−) and the base peak of the spectrum, observed at m/z 299.0982 ([C10H19O10]−), was assigned to a dimer ion ([2M – H] −) produced by association of a d-lyxose molecule (M = C5H10O5) with the mono-anion of d-lyxose ([C5H9O5]−) under the electrospray ionization conditions of the mass spectrometric measurement.High resolution mass spectrometry in negative ion mode showed no peak for  was dissolved in water (2 ml) and sodium metabisulfite (0.633 g) was added, Fig. 1ca 313 K). Crystallization did not occur on prolonged standing, so the solution was evaporated at ca 303 K until the volume was ca 1 ml. On further storage, crystals (0.313 g) were deposited, m.p. 417.6–420.1 K (decomp.) and after concentration of the mother liquors, a further crop (0.204 g) was obtained, m.p. 414–420 K; [α]D21 +8.9 (12 min.) . 1H NMR : δH 5.00 , 4.86 ; signals for the major acyclic sulfonate: δH 4.71 , 4.19 , 3.99 , 3.63 ; for the minor acyclic sulfonate: δH 4.63 ; ratio of major to minor sulfonate = 9.2 : 1. 13C NMR : δC 94.81 , 94.68 ; signals for the major acyclic sulfonate: δC 82.17 (C1), 70.43, 69.85, 69.32 , 63.78 (C5); the minor acyclic sulfonate showed a peak at δC 84.21 (C1).1H NMR spectrum indicated the species α-pyranose, β-pyranose, major acyclic sulfonate and minor acyclic sulfonate were present in the % ratios of 11.62 : 5.47 : 74.78 : 8.14. In the 13C NMR spectrum, based on peak heights, the corresponding ratios were: 15.69 : 5.23 : 71.90 : 7.19.Integration of the various signals for H-1 in the 2O/MeOH, solution) gave a peak at m/z 149.0458 ([C5H9O5]−), a significant peak at 213.0075 ([C5H11O8S1 – H2O]−), and the base peak at 299.0982 ([C10H19O10]−). No significant peak was observed for ([C5H11O8S1]−) at the calculated m/z of 231.0180.HRESMS .Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016005375/sj5496sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016005375/sj5496Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016005375/sj5496Isup3.cmlSupporting information file. DOI: 1471425CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Here we show a new ground state of this compound at ambient conditions, Cmcm-B6O (β-B6O), which in all quantum-mechanical treatments that we tested comes out to be slightly but consistently more stable. Increasing pressure and temperature further stabilizes it with respect to the known α-B6O structure. β-B6O also has a slightly higher hardness and may be synthesized using different experimental protocols. We suggest that β-B6O is present in mixture with α-B6O, and its presence accounts for previously unexplained bands in the experimental Raman spectrum.Boron suboxide B The hardness of α-B6O8910Superhard materials are used in many applications, from cutting, grinding and drilling tools to wear-resistant coatings12Ih) bear a special fascination because of incompatibility of fivefold symmetry with crystalline periodicity. The discovery of multiply-twinned particles B6O, an icosahedral packing of B12 icosahedra with Ih symmetry, had aroused interest6O, with space group Cmcm, which we name β-B6O. This structure is energetically almost degenerate with α-B6O (and slightly more stable), is predicted to have a higher hardness, and actually corresponds to twinned α-B6O structure.Objects with icosahedral symmetry  and oxygen-deficient versions of B6O with B6O-like structures and compositions between B and B6O. To our surprise, Cmcm-B6O 712136O are shown in 6O and two related forms of pure boron.Our variable-composition evolutionary searches expectedly find B6O and α-B6O, we calculated their enthalpies as a function of pressure, as shown in 6O is lower than that of α-B6O at ambient pressure, but the energy difference is only about 1.8 meV/formula within the GGA . As pressure increases, β-B6O becomes progressively more favorable than α-B6O, indicating that β-B6O might be more easily synthesized under pressure. The enthalpies of the two structures are so close that it makes us think: will the two structures coexist? what is their relationship? how to synthesize β-B6O? In order to answer these questions, we perform a detailed comparison of their structure, Raman spectra and phonon densities of states (PDOS).In order to further compare the stability of β-B6O structure has hexagonal close packing of B12 icosahedra (ABAB… stacking), while α-B6O is based on the cubic close packing (ABCABC… stacking) of B12 icosahedra, as shown in 6O can produce local β-B6O stackings. It may also be possible to obtain β-B6O-like stacking faults by deformation of α-B6O, through plane slips. Most properties of these two phases are very similar: e.g. predicted volume per formula  = 51.69 Å3/formula), hardness (Hv (α-B6O) = 38 GPa; Hv (Cmcm-B6O) = 39 GPa), elastic moduli .β-Bc moduli , DFT ban6O and Cmcm-B6O, they turn into α-BCmcm-B, respectively  is formed in shocked cubic diamond. To obtain multiple polytypes, methods like physical vapor transport (PVT), also known as seeded sublimation growth, can be used: e.g., different polytypes of SiC were obtained using the PVT method6O and α-B6O are energetically almost degenerate at zero pressure, structurally related and can coexist. To test the latter, we calculated their Raman spectra and compared with the experimental results1213et al.6O and α-B6O. The experimental data correspond to normal isotopic abundance, and so do our calculations. Correspondingly, the atomic mass of boron in the calculations adopt the weighted average value, i.e. 10.811, which is based on the isotopic abundance of boron. As one could expect, the Raman spectra of β-B6O and α-B6O are similar and match perfectly with the experimental spectra. For example, the Raman modes at 374, 499, 541, 737, 785, 833, 1088, 1119, 1141 cm−1 are consistent with Solozhenko’s results6O. The first two are computed to be at 516 and 533 cm−1 (marked by letter A in −1. The third mode is predicted to be at 630 cm−1 (marked with letter B), and Werheit and Kuhlmann have indeed observed a Raman-active phonon at 627 cm−1. We note that α-B6O does not have Raman-active modes between 570 and 700 cm−1, thus the one observed by Werheit and Kuhlmann at 627 cm−1 cannot come from α-B6O, but β-B6O. The fourth mode has the theoretical Raman frequency of 896 cm−1 (marked with letter C), while α-B6O has no Raman-active modes between 850 and 1000 cm-1. Thus, this mode is also unique to the β-B6O structure, and again seen in experiments: Solozhenko et al. observed Raman active phonon at 889 cm−1, and Werheit and Kuhlmann observed a Raman-active phonon at 902 (909) cm−1. Moreover, Wang et al. also observed similar Raman spectra in their B6O samples  of α-Bpressure , we once6O should be more stable than α-B6O, and demonstrated that the two phases actually coexist in experimental samples, we ask a question: why is the synthetic compound mostly α-B6O, instead of the more stable β-B6O? While there can be no definitive answer at this point, we suggest that this may be because of the use of α-rhombohedral-B (Rm)Rm)2O3) for synthesis of rhombohedral-B6O. Hubert et al.6O but also B6O twinning particles and some amorphous phases. And they observed a stacking of ABAB… around the planar defect, which is the same as β-B6O (We speculate it probably is β-B6O). To obtain β-orthorhombic-B6O, one would need to crystallize it from the melt , or use other phases of boron as precursors. Using Cmcm-B would be ideal, but this phase , energies (slightly lower for β-B6O), band gaps , hardnesses (β-B6O is slightly harder) and phonon densities of states, but have important differences in Raman spectra. By comparing calculated and experimental Raman spectra, we demonstrated that the experimental samples are actually a mixture of α-B6O and β-B6O. The discovery of β-B6O opens up new possibilities, in view of its greater stability, hardness and indirect band gap. Our findings also indicate possibilities of tuning the properties of B6O by obtaining phase-pure samples (probably not obtained to date), and the possibility of metastable oxygen-deficient compounds based on α-B6O or β-B6O - these can be obtained at high temperatures (where disordered oxygen vacancies will stabilize the structure) and low chemical potentials of oxygen.In summary, to our big surprise, ab initio evolutionary algorithm USPEX242526−1 resolution within structure search, and 2π*0.04 Å−1 for subsequent highly precise relaxations and properties calculations. In order to confirm the relative stability of α-B6O and β-B6O, we used local density approximation (LDA)We used the How to cite this article: Dong, H. et al. Prediction of a new ground state of superhard compound B6O at ambient conditions. Sci. Rep.6, 31288; doi: 10.1038/srep31288 (2016).


Association of particulate matter with adverse health effects has been established in epidemiological studies and animal experiments. Epidemiological studies are difficult to undertake while animal studies are impractical for high-throughput toxicity testing. The ease and rapidity of in vitro tests emphasizes their potential for use in risk assessment of chemicals and particles. We examined the association between in vitro and in vivo responses to ambient particles, to determine the potential of cell-based assays as standalone toxicity screening tools.Assays of cytotoxicity and key inflammatory mediators were applied to determine the in vitro biological potency of a panel of urban and mineral particles in J774A.1 macrophages and A549 lung epithelial cells. The particles were also screened for the presence of AhR agonists using the Ah receptor-dependent gene induction assay and for endotoxin using the Limulus amebocyte lysate assay. A subset of the particles with a contrasting in vitro toxicity profile was delivered intratracheally in BALB/c mice to assess their in vivo biological potency. Results from various bioassays were combined within the in vitro and in vivo models. The combined potency measures were examined for associations.p = 0.052) in a correlation within in vitro and in vivo models, with a ranking of fine particle (DWR1), minerals  and coarse particles  from low to high potency.Overall, J774A.1 cells were more sensitive to particle effects than A549 cells. Whereas the combined cytotoxicity estimates were highly correlated between the two cell lines, the combined in vitro inflammatory potency estimates were not, emphasizing functional differences of the two cell types. Secretion of inflammatory markers by J774A.1 cells was correlated with AhR ligand binding profile and endotoxin levels of particles. Particle instillation led to an acute toxicity response in BALB/c mice, with neutrophilia and release of inflammatory mediators. While the combined toxicity estimates were not correlated between in vitro and in vivo models, the combined inflammatory and integrated potency estimates (toxicity and inflammation) approached the threshold for significance  contains supplementary material, which is available to authorized users. Air pollution continues to exert a societal burden by impacting multiple aspects of human health, especially through its association with cardiopulmonary morbidity and mortality . ExposurUrban air particles are complex mixtures of chemical and biogenic constituents associated with natural and anthropogenic particle sources and their modification in the atmosphere. Increasing evidence shows that in addition to size, chemical composition of the particles plays an important role in determining their hazard potential. Mitigation of specific constituents of air pollution mixtures and particles with highest hazard potential may present a reasonable regulatory strategy for reduction of adverse population health effects.Both, in vitro and in vivo approaches are widely utilized for the study of the adverse effects of chemicals and inhaled environmental pollutants by employing individual endpoint assays to determine the adverse effects of particulate materials or to examine specific biological mechanisms of toxicity. Animal studies have been generally accepted as a major approach in the regulatory risk assessment regimes –14. DespFew attempts have been made to conduct more comprehensive in vitro/in vivo evaluations of panels of particles and particle fractions including gasoline and diesel engine emission organic fractions , 18, pol10 emissions from a local steel mill source [The usefulness of in vitro studies is highlighted by demonstrations of coherence of the particle-induced biological effects in cultured cells with in vivo responses from a controlled human exposure study  and withl source . Particll source .In the present study, we assessed the toxicity and inflammatory potential of a panel of airborne particulates and reference particles in cell lines in vitro and in BALB/c mice exposed in vivo to particles at 24 h post-intratracheal instillation. An approach was developed to summarize large datasets to facilitate a comparison between the in vitro and in vivo responses to particles and to evaluate the level of concordance between the two models for toxicity testing.2 reference material) and SRM-154b  were obtained from the National Institute of Standards and Technology . Cristobalite particles will be referred to as CRI throughout the manuscript. The TiO2 particles were washed in methanol and phosphate buffered saline (PBS) to remove potential naphthalene contaminant [2.5 DWR1 particles were collected in Toronto as described before [The particulate preparations SRM-1648 , SRM-1649a , SRM-1879 , iron (1 mg/g), magnesium (1.4 mg/g), lead (0.05 mg/g), titanium (0.06 mg/g). SiO2 and TiO2 are mineral particles containing a variety of trace elements. Available physicochemical information from in-house analysis and NIST Certificates of Analysis are presented in supplemental information , SRM-1648 (St. Louis) and SRM-1649 (Washington) are TSP matter from urban environments, characterized by enrichment in elements including aluminium (20–30 mg/g), calcium (26–128 mg/g), iron (16–30 mg/g), magnesium (9–16 mg/g), lead (5–14 mg/g), titanium (2–3 mg/g) and zinc (2–11 mg/g), in the milligram per gram range. DWR1 is a PM2 well) plates at cell densities of 4 × 104 (A549) and 8 × 104 cells/cm2 (J774A.1). For BrdU incorporation, cells were seeded at 1 × 104 (A549) and 2 × 104 cells/cm2 (J774A.1). For the LDH assay, A549 and J774A.1 cells were cultured in serum-free media supplemented with 2 mM L-glutamine, 4.5 g/L glucose (J774A.1 cells only) and 6 mg/mL BSA. H1L1.1c2 cells were seeded in 24-well (0.5 cm2 well) plates at a density of 1 × 104 cells per well, in 1 mL of complete medium. The cells were then incubated for 24 h at 37 °C in 5 % CO2 and 100 % relative humidity for attachment.A549 human alveolar type II epithelial  and J774A.1 murine monocyte/macrophages  cell lines were from ATCC . H1L1.1c2 murine hepatoma cells containing a stably transfected Ah receptor-dependent luciferase reporter gene were described elsewhere . A549 we2) in a final volume of 200 μL/well, 5 % FBS. The A549 and J774A.1 cell culture plates were incubated at 37 °C for 24 h before the cytotoxicity measurements were carried out. Culture plates containing H1L1.1c2 cells were incubated at 37 °C for 4 h before they were assayed for induction of aryl hydrocarbon response.Particle suspensions were thawed to room temperature and sonicated for 20 min in ice-cold, ultrasonic water bath, and diluted (1–2 mg/mL) in complete media devoid of serum for dosing cells. The diluted particle suspensions were sonicated for 5 min in an ice-cold, ultrasonic water bath prior to dosing the cells. A549, J774A.1 and H1L1.1c2 cell monolayers were dosed with a 100 μL suspension of particles resulting in 0, 10, 20, 40, 80 and 100 μg/well , ATP assay, BrdU incorporation assay and lactate dehydrogenase (LDH) assay, and in H1L1.1c2 cells for Ah receptor-dependent gene induction, as briefly outlined below. Assays were performed using aliquots from cell culture supernatants and lysates that were clarified by centrifugation to remove the remnants of particles that could potentially interfere with the assay reagent chemistry and detection methods. The approach utilized has been previously described . ATP ass2). Rate of reduction is the delta fluorescence at 2 h – 5 min. Five independent experiments were conducted (n = 5), with triplicate wells within each experiment.The spent medium was collected and replaced with 100 μL of fresh complete medium containing a 1:10 dilution of Resazurin (Alamar Blue) reagent  and fluorescence was measured at λEx 530/25 nm and λEm 590/35 nm using Cytofluor 2350 fluorometer , 5 min an = 3), with triplicate wells within each experiment.Cellular ATP content was assessed using the ViaLight Plus assay . Cells were lysed with 10 μL of the assay cell lysis reagent for 10 min, followed by 25 μL of ATP-monitoring reagent (AMR) containing firefly luciferase, luciferin, pyrophosphate, BSA and magnesium ions. After 2 min incubation in the dark, the cellular ATP content was determined by top reading luminescence measurements using LMax luminometer. Luminescence (rate of photon emission) is directly proportional to concentration of ATP substrates). The ATP assay was performed in three independent experiments (n = 3), with triplicate wells within each assay were conducted.Following exposure to particles, 5-bromo-2-deoxyuridine (BrdU) was added to each well at 100 μM and the cells were returned to the incubator for 2 h. The culture medium was removed by inversion of the 96-well plates, and the cells were fixed for 30 min at room temperature using the FixDenat solution . The cells were washed with blocking buffer (1 % BSA in PBS), followed by an incubation with 100 μL of anti-BrdU antibody conjugated with peroxidase  for 90 min at room temperature. After extensive washing, cells were incubated with 100 μL of the 3,3’,5,5’-tetramethylbenzidine (TMB) chromogenic substrate for 10 min at room temperature in the dark, and the reaction product was quantified at 370 nm using SpectraMax Plus spectrophotometer. Three independent assays (n = 3) or four  independent assays, with triplicate wells within each experiment were conducted.Culture supernatants were recovered and the cells were lysed by a rapid freeze-thaw cycle. LDH activity was measured in the clarified supernatants and the cell lysates by spectrophotometry at 450 nm (SpectraMax Plus) from production of NADH from lactate coupled with the instantaneous conversion of tetrazolium to a red formazan by NADH diaphorase . Release of cellular LDH was determined from activity in supernatant over total LDH (supernatant plus lysate). Three , with triplicates within each experiment.After dosing with particles, H1L1.1c2 cell plates were incubated at 37 °C for 4 h, followed by two washes in ice-cold PBS, and 15 min lysis on ice using 50 μL/well of luciferase assay system (Promega Corporation) lysis buffer, on a plate shaker (250 rpm). The lysates were transferred to v-bottom plates and centrifuged at 3000 × g  using a Sorvall Legend RT centrifuge. Clarified cell lysates (25 μL) were transferred into a white, clear-bottomed 96-well plate, 50 μL of luciferase substrate was added by injection using an L-Max luminometer, followed by 15 s integrated luminescence reading per well. Three independent experiments were conducted (Cell culture supernatants of A549 cells were assessed for Interleukin (IL)-1β , IL-1ra , IL-2 (n.d.), IL-4 (n.d.), IL-5 (n.d.), IL-6 (430 ± 111), IL-7 (n.d.), IL-8 (200 ± 4), IL-9 (n.d.), IL-10 (12.5 ± 1.9), IL-12 (p70) (n.d.), IL-13 (n.d.), IL-15 (n.d.), IL-17 (n.d.), basic Fibroblast Growth Factor (FGF) (n.d.), Eotaxin (n.d.), Granulocyte Colony-Stimulating Factor (G-CSF) (n.d.), Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) (2.8 ± 0.4), Interferon (IFN)-γ (n.d.), IFN-γ Inducible Protein (IP)-10 (n.d.), Monocyte/Macrophage Chemoattractant Protein (MCP)-1 (701.5 ± 44.8), Macrophage Inflammatory Protein (MIP)-1α (n.d.), MIP-1β (0.9 ± 0.5), Platelet-Derived Growth Factor (PDGF)-BB (n.d.), Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) (n.d.), Tumor Necrosis Factor (TNF)-α (0.1 ± 0.1), and Vascular Endothelial Growth Factor (VEGF) (n.d.), while cell culture supernatants of J774A.1 were assessed for IL-1α (1.2 ± 0.1), IL-1β (5.9 ± 1.8), IL-2 (n.d.), IL-3 (n.d.), IL-4 (n.d.), IL-5 (n.d.), IL-6 (9.2 ± 1.0), IL-9 (n.d.), IL-10 (12.5 ± 1.9), IL-12 (p40) (n.d.), IL-12 (p70) (n.d.), IL-13 (n.d.), IL-17A (n.d.), Eotaxin (n.d.), G-CSF (n.d.), GM-CSF (9.4 ± 1.3), IFN-γ (n.d.), Keratinocyte Chemoattractant (KC) (n.d.), MCP-1 (n.d.), MIP-1α (above assay limit), MIP-1β (n.d.) and RANTES (239 ± 28) using Bio-Plex cytokine panels , with a Bio-Plex 200 array system.n = 5 animals per dose). Mice were euthanized after 24 h, bronchioalveolar lavage (BAL) fluid and blood plasma were obtained as described before [Seven weeks-old, pathogen-free BALB/c male mice ~25 g) were from Charles-River . Mice were housed in shoe-box plexiglass cages, on woodchip bedding, provided with mouse chow and water ad libitum, maintained under HEPA air filtration at 22 +/− 2 °C, ~50 % humidity and were held to a 12-h light/dark cycle. The animals were acclimatized for at least 1 week prior to experimentation. The animal treatment protocol was reviewed and approved by the Animal Care Committee of Health Canada. The particle preparations were thawed at room temperature, sonicated for 10 min, diluted in saline and instilled intratracheally (50 μL volume) using the MicroSprayer aerosolizer  at doses of 0, 50, 100, 250 μg into anaesthetized mice in supine position on an angled stand ( g were f8-Isoprostaglandin-2a (Isoprostane) was quantitated from blood plasma samples by competitive Enzyme Immuno-Assay (EIA) following manufacturer-recommended procedures . LDH in BAL was analyzed essentially as described for cell cultures. Total protein was measured by the Coomassie Plus (Bradford) assay as per manufacturer’s protocol .The profile of IL-1α , IL-1β (1.3 ± 0.2), IL-4 , IL-5 (0.5 ± 0.5), IL-6 (0.1 ± 0), IL-10 (0.3 ± 0.13), GM-CSF (1.3 ± 0), KC ((3.8 ± 0.9), MIP-1α (25.4 ± 8.8), RANTES (0.3 ± 0.2) and TNF-α (3.7 ± 1.0) was determined by Bio-Plex analyses of BAL samples using Bio-Plex cytokine panels, with a Bio-Plex 200 array system. Basal levels in control cells are in parentheses above and represent pg/mL ± standard error mean measured in cell culture supernatants and n.d. denotes cytokine/chemokine not detected.The dose–response data for cell viability, cytokine secretion and Ah receptor-dependent gene induction , were normalized within an experiment for all doses (including 0 μg dose control), to the grand mean value of all controls (0 μg dose of particles), to obtain fold-effect (FE) for each particle dose. Potency (β) was derived fromwhere β is the rate of change of dose with respect to the logarithm of fold-effect for a given endpoint . The dosV) in a given assay, potency for Ah receptor-dependent gene induction (βAhR), and potencies for secretion of a cytokine (βI). βI was adjusted for cell viability according toThe following potencies were calculated: potency for viability R) of the particles was defined as any deviation from baseline in cytotoxicity assays. βRCELLS was calculated by averaging the absolute values (to capture any deviation from baseline control levels) of the βV according to:Biological reactivity (β3)I-Vfor chemokines and pro-inflammatory cytokines and −1*βI-V for anti-inflammatory cytokines. Two different estimates of inflammatory potency were calculated.I-VHI, an average considering the anti-inflammatory effect of IL-10 (−1*βI-V for IL10).βI-VLO, an average considering anti-inflammatory effects of IL-6 and IL-10.βAn average potency for inflammation was calculated within each cell line using +1*β4)RCELLS and one of the two estimates of βI-V were further integrated to calculate cell-specific Integrated Beta potencies (Iβ). For example,β5)R and βI-V were integrated across the cell lines as follows.β6)R and one of the two estimates of βI-V across cell lines were further integrated to a grand Integrated potency (Iβ). For example,βThe potencies were combined as follows;T BALB/c was determined from the average of potencies for BAL endpoints , while a potency estimate for inflammation, βIBALB/c was determined from the potencies of particles for inducing the release of cytokines and chemokines. As described for the in vitro inflammatory potency estimates, βI HI, an average considering the anti-inflammatory effect of IL-10 (−1*βI for IL10) and βI LO, an average considering anti-inflammatory effects of IL-6 and IL-10 were determined. An integrated potency estimate that incorporates all in vivo data, Iβ BALB/c was obtained from the average of βTBALB/c and βI BALB/c.In a similar fashion, potency estimates were determined for each in vivo endpoint. A potency estimate for acute lung toxicity, βDOSE  for in vitro data, or  for in vivo data, and Particulate Matter (PM) as factors. Datasets not meeting normality and equal variance were subjected to log10, inverse or square root transformations (in the order given) until the assumptions were met, or else rank transformed prior to analyses. Pairwise multiple comparisons were carried out using Tukey’s procedure to elucidate the pattern of significant effects (α = 0.05). The analyses were conducted using SigmaPlot, version 12.5 .To ensure that assays were sensitive to the dose of particles, fold effect (FE) data were first analyzed by two-way ANOVA with http://www.broadinstitute.org/cancer/software/genepattern) [http://jtreeview.sourceforge.net) [Hierarchical clustering of the pattern of cytokine secretion by A549 and J774 in response to particle exposure were conducted using the GenePattern webtool (pattern) , and visrge.net) .Linear regression between corresponding individual or combined potency estimates in vitro and in vivo was conducted using Sigmaplot v12.5 and depicted using Microsoft Excel 2010 . The strength of the relationship between every two variables was described by a correlation coefficient R and the significance of the hypothesis test by the p-value of 0.05 (two-tailed test), or one-tailed test, where applicable .2, SiO2) or for five particles tested in vivo  for Table The correlations presented are performed between in vitro and in vivo matched endpoints across all particles, based on individual particle potencies Table  or combihttp://www.socscistatistics.com/tests/spearman/default2.aspx; Social Science Statistics).In addition, a selection of key potency estimates were ranked and the strength of their relationship was reassessed using Spearman’s rank-order correlation test (one-tailed test), as the selected variables were observed to have a consistent positive relationship, with a significance level of 0.05, using Spearman’s Rho calculator (p < 0.001), most prominent at the highest doses of the particles. The LDH assay also revealed a significant decrease in LDH release in J774A.1 cells exposed to lower and intermediate doses of urban particles in comparison to baseline levels, but not in response to mineral particles  measured by AhR-mediated luciferase reporter gene activity in H1L1.1c2 cells. A small but significant decrease in AhR-mediated gene activity below baseline levels was detected in cells exposed to the mineral particles  indicated that the aggregated cytotoxic responses of A549 and J774A.1 cells were significantly correlated  and the other cytokines . Cell viability-adjusted inflammatory potency  of the particles in the cells was visualized by a heatmap. In both cell lines, the majority of the cytokines were increased in response to particle exposures, except IL-10, which generally remained unchanged or decreased in J774A.1 cells  and remained unchanged or showed an increasing trend in A549 cells  and clustered separately from the rest of the cytokines  and clustered separately from the remaining cytokines , but not with A549-produced cytokines.Notably, we observed a significant correlation between AhR activity in H1L1.1c2 cells with IL-1β, IL-6 and TNF-α release in J774A.1 cells  and A549 cells (βI-V A549) revealed a significant correlation with the inflammatory potency estimate in J774A.1 cells , but not A549 cells . SRM-1649 particles had noticeably less endotoxin (3.0 EU/μg particles) while mineral particles did not contain endotoxin  by intratracheal instillation (IT). Acute pulmonary toxicity was observed in mice exposed to the high dose (250 μg) of the particles, as shown by a general increase in BAL total protein  and BAL LDH . A particle dose-dependent increase in 8-isoprostane levels in blood plasma  indicated a systemic oxidative stress after exposure to particles. Neutrophil influx  and a drop in lung-resident alveolar macrophages  were consistent with a particle challenge and clearance after 24 h. Based on the in vivo toxic potency estimates , particles elicited an increase in acute toxicity and cell injury marked by an increase in neutrophils and a decrease in macrophages. SRM-1649, EHC-2000 and CRI clustered separately from less toxic particles DWR1 and TiO2  were measured in BAL fluid collected from mice exposed to particles for 24 h, except IL-10 which was not altered , or IL-6 only (βI HI BALB/c). The inflammatory potency estimates βI BALB/c ranked identically to toxicity estimates βT BALB/c, with no impact of the inflammatory potency scenarios on the ranking of particles . Endotoxin content in particle preparations administered to mice was not correlated with the combined in vivo inflammatory potency estimates (βI BALB/c) of the particles .Majority of the individual particle potency estimates for the corresponding endpoints were not correlated, with the exception of a single association; the cellular LDH content in J774A.1 cells, significantly correlated with LDH content measured in BALB/c BAL fluid, ; βI-V CELLS versus βI BALB/c  and Iβ CELLS versus Iβ BALB/c . However, a consistent increasing trend between these in vitro and in vivo estimates was observed across the particles, where DWR1 was ranked low, CRI and TiO2 were ranked intermediate and SRM-1649 and EHC-2000 were ranked high in potency , correlations of βI-V CELLS versus βI BALB/c and the integrated potency estimates Iβ CELLS versus Iβ BALB/c approached the threshold for significance α = 0.05 . Finally, improved correlation coefficients for the associations of the combined particle potency estimates were observed when mineral particle responses were excluded from the estimates, albeit they were not statistically significant due to low power ; βI-V CELLS versus βI BALB/c  and Iβ CELLS versus Iβ BALB/c . Remarkably, CRI contributed most to the decreased association of in vitro and in vivo potency estimates, as exclusion of CRI only revealed improved correlation comparable to the associations excluding both mineral particles ; βI-V CELLS versus βI BALB/c  and Iβ CELLS versus Iβ BALB/c . When the Spearman’s rank-order correlation analysis was conducted on the particle ranks while excluding CRI, a statistically significant rank-order was observed for βR CELLS versus βT BALB/c, while βI-V CELLS versus βI BALB/cand the integrated potency estimates Iβ CELLS versus Iβ BALB/c did not show a significant trend in a correlation due to low power .When the combined particle potency estimates in vitro and in vivo were assessed by Pearson product–moment correlation analysis for all particles tested, no statistically significant correlations were observed between the corresponding pairs of the key estimates, including the key estimates; βIn this work we examined the level of concordance between in vitro and in vivo responses to particulate matter, to determine the potential of cell-based cytotoxicity assays and cytokine/chemokine mediators in screening the toxicity of ambient particles. The dose response results from the bioassays conducted in cell lines and from intratracheal exposures of mice to particles were summarized as slope-based measures of particle potency for each endpoint. The potency values were combined within each exposure model to obtain model-specific consensus potency estimates of toxicity and inflammatory potential of the particles, which were further combined into an overall integrated potency estimate. The key derived particle potency estimates were then assessed for correlation across the in vitro and in vivo models.2. In vivo toxicity of the same sample however was high, in accordance with our historical observations and literature data on toxicity of silica. Identity of the CRI samples was reconfirmed by ICP-MS/AES analysis of the archived, diluted working particle stocks that were used in the experiments. We have also carefully validated the datasets, but could not explain this atypical behavior of CRI in the series of in vitro assays presented here, e.g. from computational errors or material switching in assays, and therefore we report the data as observed.We note that in the present series of experiments we observed an atypically low in vitro cytotoxicity of the CRI sample by comparison to TiOUrban and mineral particles induced toxicity in vitro and in vivo Figs.  and 6c. 10particles [The greater susceptibility of the macrophages to particle exposures compared to the epithelial cells observed in this work Fig.  highligharticles . Similararticles .The impact of increased sensitivity of J774A.1 cells to particle exposures was also revealed by a small decrease of LDH release below control levels in cells exposed to the intermediate doses of the urban particles Fig. . A combiNevertheless, when the panel of in vitro toxicity endpoints (viability assays) was combined, the overall particle potency was correlated across the cell lines Table  regardleIn the present study, however, inflammatory potential determined from the release of cytokines did not correlate between the two cell lines, or with the overall cytotoxic potency of the particles Table . This re2-induced acute lung inflammation is more severe, while fibrotic response is attenuated in AhR−/− mice compared with C57Bl/6 mice, suggesting a complex role for AhR in regulation of inflammation [It has been shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-activated AhR signaling participates in the up-regulation of IL-1β, IL-6 and IL-8 expression through activation of the nuclear factor (NF-kB) and extra-cellular stimulus-activated kinase (ERK) signaling cascades. Dioxins and PAHs are major components of cigarette smoke. The AhR protein expression was up-regulated in the presence of TNF-α and smoking and exposure to TCDD enhanced rheumatoid arthritis (RA) inflammatory processes in synovial tissue from RA patients . Interesammation . The cor2 particles  in macrophage and lung epithelial cells exposed to coarse and fine particles for 20 h correlated with in vivo inflammation endpoints  measured in BAL of rats at 24 h after intratracheal exposure to the same particles. In contrast, poor correlation was observed between different endpoints from in vivo allergy models and inflammatory parameters, suggesting that a variety of endpoints are needed to assess the potential hazard of particles .In the present study, we have observed a significant correlation of LDH content in J774A.1 macrophages with LDH in BALB/c lung lavage  were not significantly correlated. However, the combined potency estimates for inflammatory potential  and the integrated particle potency estimates (Iβ) that further combined the toxicity and inflammatory bioassay response potency estimates approached the threshold for significance α = 0.05 in a correlation . The lack of strong associations may be due to insufficient power to obtain statistical significance. Although eight particles (with six urban particles) were screened in vitro, for workload reasons a subset of five particles only (including three urban particles) was tested in vivo, an issue that highlights the impracticality of in vivo assays for high-throughput testing. A better correlation may also have been achieved with additional in vivo time points, capturing different stages of the transient particle-induced in vivo toxicity and inflammation profile. The use of a single species could also have potentially led to higher in vitro/in vivo correlations. However, employing different, routinely used species (mouse and human) enabled the assessment of whether integrating toxicity endpoints originating from widely varied cell types  would constitute a robust in vitro surrogate that can be useful in predicting in vivo responses of biological and health relevance. As previously seen, the ranking of potencies of a series of particles was comparable between the mouse macrophage cell line J774A.1 and the human cell line THP-1 (unpublished observations). There are other variables beyond species that affect potency estimates, such as cell seeding or monolayer density at time of exposure, etc.Our combined estimates of particle potency in vitro and in vivo for toxicity;  show promise in capturing additional factors that may narrow the gap between in vitro and in vivo testing , 54, theAhR, aryl hydrocarbon receptor; AMR, ATP-monitoring reagent; ANOVA, analysis of variance; ATCC, American type culture collection; ATP, adenosine triphosphate; BRDU, 5-bromo-2'-deoxyuridine; BSA, bovine serum albumin; CRI, cristobalite; DMEM, Dulbecco’s modified Eagle’s medium; DWR1, Downsview reference 1; EHC, Environmental Health Centre; EIA, enzyme immunoassay; EPA, Environmental Protection Agency; ERK, extra-cellular stimulus-activated kinase; EU, endotoxin units; FBS, fetal bovine serum; FE, fold effect; FGF, fibroblast growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte macrophage colony-stimulating factor; HEPA, high-efficiency particulate arrestance; ICP-MS/AES, inductively coupled plasma-mass spectrometry/ atomic emission spectroscopy; IFN, interferon; IP-10, IFN-γ Inducible Protein 10; KC, keratinocyte chemoattractant; LDH, lactate dehydrogenase; M199, medium 199; MCP-1, monocyte/macrophage chemoattractant protein; MEM, minimum essential medium Eagle; MIP-1, macrophage inflammatory protein; NF-kB, nuclear factor kB; NIST, National institute of standards and technology; NLR, nucleotide oligomerization domain-like receptor; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; PM, particulate matter; RA, rheumatoid arthritis; RANTES, regulated on activation, normal T cell expressed and secreted; SiNPs, silicon dioxide nanoparticles; SRMs, standard reference materials; TCDD, 2,3,7,8-Tetrachlorodibenzodioxin; TLR, Toll-like receptor; TMB, 3,3',5,5'-tetramethylbenzidine; TNF-α, tumor necrosis factor alpha; TSP, total suspended particulates; VEGF, vascular endothelial growth factor; XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilideAdditional file 1: Table S1.Table S2. Pearson correlations for cytotoxic potency and cytokine inductions in cell lines versus BALB/c mice exposed to particles. Table S3. Pearson correlations for the combined average in vitro (cell lines) and in vivo (BALB/c mice) particle potency estimates. (DOC 77 kb)Elemental composition and size of the urban particulate matter and mineral particles. Additional file 2: Figure S1.n = 5). ATP content of J774A.1 (C) and A549 (D) cells exposed to particles. (n = 3). Proliferation in J774A.1 (E) and A549 (F) cells exposed to particles. (n = 3). Two way ANOVA; Resazurin assay, J774A.1, PM main effect, p < 0.001, SRM-1648 vs. DWR1, EHC-98 or EHC-2000 (†), SRM-1649 vs. DWR1, EHC-93, EHC-98, EHC-2000, TiO2 or CRI (‡), Tukey test, p < 0.05, Dose main effect, p < 0.001, Doses 0, 10 or 20 vs. 40, 80 or 160 (#), Dose 40 vs. 80 or 160 (not shown), Dose 80 vs. 160 (not shown), Tukey test, p < 0.05; Resazurin assay, A549, PM × Dose interaction, p = 0.001, asterisks (*) represent effects significantly different from control, Tukey test, p < 0.05. ATP assay, J774A.1, PM main effect, p < 0.001, EHC-98 vs. SRM-1648, SRM-1649 or EHC-2000 (†), SRM-1649 vs. DWR1, TiO2 or EHC-93 (‡), Tukey test, p < 0.05, Dose main effect, p < 0.001, Dose 160 vs. all doses (#), Tukey test, p < 0.001; ATP assay, A549, PM main effect, p < 0.001, DWR1 vs. all particles (†), EHC-93 vs. SRM-1649, CRI or TiO2 (‡), Tukey test, p < 0.05, Dose main effect, p < 0.001, Dose 10 vs. 0, 40, 80 or 160 (not shown), Dose 20 vs. 80 (not shown), Dose 160 vs. 0, 20, 40 or 80 (#), Tukey test, p < 0.05. BrdU assay, J774A.1, PM main effect, p < 0.001, DWR1 or CRI vs. EHC-93, EHC-98, EHC-2000, SRM-1648, SRM-1649 or TiO2 (†), Tukey test, p < 0.05, Dose main effect, p < 0.001, Doses 0 or 10 vs. 20, 40, 80 or 160 (‡), Doses 20 or 40 vs. 80 or 160 (not shown), Dose 80 vs. 160 (not shown), Tukey test, p < 0.05; BrdU assay, A549, Dose main effect, p < 0.001, Dose 10 vs. 0, 20, 80 or 160 (†), Dose 40 vs. 80 or 160 (‡), Tukey test, p < 0.05. (DOCX 80 kb)Resazurin reduction by J774A.1 (A) and A549 (B) cells exposed to particles. Average fold-effect (FE) over control ± standard error values are shown . Two way ANOVA; GM-CSF, PM × Dose interaction, p = 0.001, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05; IL-1α, PM × Dose interaction, p < 0.001, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05; IL-β, PM × Dose interaction, p < 0.001, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05; IL-10, PM × Dose interaction, p = 0.026, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05; RANTES, PM × Dose interaction, p < 0.001, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05; TNF-α, PM × Dose interaction, p < 0.001, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05. (DOCX 73 kb)Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (A), interleukin (IL)-1α (B), IL-β (C), IL-10 (D), Regulated upon Activation Normal T cell Expressed and Secreted (RANTES) (E) and Tumor Necrosis Factor (TNF)-α (F) levels in cell culture supernatants of J774A.1 cells exposed to particles for 24 h. Values are presented as mean fold-effect (FE) ± standard error . Two way ANOVA; GM-CSF, Dose main effect, p < 0.001, Dose 160 vs. 0, 10 or 40 (†), Dose 10 vs. 40 (not shown), Tukey test, p < 0.05; IL-1β, PM main effect, p = 0.004, TiO2 vs. DWR1 or EHC-93 (†), Tukey test, p < 0.05, Dose main effect, p < 0.001, Doses 40 or 160 vs. 0 or 10 (‡), Tukey test, p < 0.05; IL-8, PM × Dose interaction, p = 0.016, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05; IL-10, not statistically significant; MCP-1, PM × Dose interaction, p = 0.001, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05; MIP-1β, PM main effect, p = 0.014, TiO2 vs. EHC-93 (†), Tukey test, p < 0.05, Dose main effect, p = 0.023, Dose 160 vs. 10 (‡), Tukey test, p < 0.05; TNF-α, PM main effect, p < 0.001, TiO2 vs. DWR1, EHC-93 or EHC-2000 (†), CRI vs. DWR1 (‡), Tukey test, p < 0.05, Dose main effect, p < 0.001, Dose 0 vs. 10, 40 or 160 (#), Dose 10 vs. 40 (not shown), Dose 160 vs. 10 or 40 (not shown), Tukey test, p < 0.05. (DOCX 85 kb)Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (A), interleukin (IL)-1β (B), IL-8 (C), IL)-10 (D), Monocyte Chemoattractant Protein (MCP)-1 (E), Macrophage Inflammatory Protein (MIP)-1β (F) and Tumor Necrosis Factor (TNF)-α (G) levels in cell culture supernatants of A549 cells exposed to particles for 24 h. Values are presented as mean fold-effect (FE) ± standard error . Two way ANOVA; LDH, PM × Dose interaction, p = 0.035, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05; 8-isoprostane, Dose main effect, p = 0.006, Dose 0 vs. 100 (†), Tukey test, p = 0.004; Macrophages, PM main effect, p = 0.018, TiO2 vs. SRM-1649 (†), Tukey test, p = 0.049, Dose main effect, p < 0.001, Dose 0 vs. 50, 100 or 250 (‡), Dose 50 vs. 250 (#), Tukey test, p < 0.05; Band cells, Dose main effect, p < 0.001, Dose 0 vs. 50 or 100 (†), Tukey test, p < 0.001; Lymphocytes, Not statistically significant. (DOCX 63 kb)Lactate dehydrogenase (LDH) levels in bronchioalveolar lavage (BAL) fluid (A), 8-isoprostane levels in blood plasma (B), Macrophage (C), Band cell (D) and lymphocyte (E) counts in BAL fluid from BALB/c mice exposed to particles by intratracheal instillation, at 24 h post-exposure. Values represent mean fold-effect (FE) ± standard error of the mean . Two way ANOVA; GM-CSF, Dose main effect, p < 0.001, Dose 250 vs. 0 (†), Dose 250 vs. 50 (‡), Tukey test, p < 0.05; IL-1α, PM main effect, p < 0.001, CRI, EHC-2000 or SRM-1649 vs. DWR1 or TiO2 (†), Tukey test, p < 0.05, Dose main effect, p < 0.001, Dose 0 vs. 50, 100 or 250 (‡), Dose 50 vs. 100 (not shown), Dose 250 vs. 50 or 100 (not shown), Tukey test, p < 0.05; IL-β, PM × Dose interaction, p = 0.015, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05; IL-5, PM × Dose interaction, p = 0.007, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05; IL-10, not statistically significant; KC, PM × Dose interaction, p = 0.001, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05; MIP-1α, PM × Dose interaction, p < 0.001, asterisks (*) represent effects significantly different from Dose 0 control, Tukey test, p < 0.05; RANTES, Dose main effect, p < 0.001, Dose 0 vs. 100 or 250 (†), Dose 250 vs. 50 or 100 (‡), Tukey test, p < 0.05; TNF-α, PM main effect, p = 0.003, TiO2 vs. CRI, EHC-2000 or SRM-1649 (†), Tukey test, p < 0.05, Dose main effect, p < 0.001, Dose 0 vs. 50, 100 or 250 (‡), Dose 250 vs. 50 (#), Tukey test, p < 0.05. (DOCX 100 kb)Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) (A), interleukin (IL)-1α (B), IL-1β (C), IL-5 (D), IL-10 (E), Keratinocyte-derived Chemokine (KC) (F), Macrophage Inflammatory Protein (MIP)-1α (G), Regulated upon Activation Normal T cell Expressed and Secreted (RANTES) (H) and Tumor Necrosis Factor (TNF)-α (I) levels in bronchioalveolar lavage fluid of BALB/c mice exposed to particles by intratracheal instillation, at 24 h post-exposure. Values represent mean fold-effect (FE) ± standard error of the mean (