According to X-ray diffraction analysis (XRD), cobalt ferrite was in a single cubic phase after being calcined at 1000 °C for 3 h. Moreover, the lattice constant decreased with increase in aluminum substituents. When the sample was analyzed by Scanning Electron Microscopy (SEM), we found that uniformly sized, well-crystallized grains were distributed in the sample. Furthermore, we confirmed that Al3+ ion-substituted cobalt ferrite underwent a transition from ferrimagnetic to superparamagnetic behavior; the superparamagnetic behavior was completely correlated with the increase in Al3+ ion concentration at room temperature. All these findings were observed in Mössbauer spectra. For the cobalt ferrite CoAlxFex2−O4, the coercivity and saturation magnetization decrease with an increase in aluminum content. When the annealing temperature of CoAl0.1Fe1.9O4 was steadily increased, the coercivity and saturation magnetization initially increased and then decreased.A sol-gel autocombustion method was used to synthesize Al C (520 °C), large anisotropy constant (2.65 × 105–5.1 × 105 J/m3) .  x = 0–1. ferrites(0.64 Å) :(1)ρx=8Me sample .0.1Fe1.9O4 were sintered at different temperatures. An average CoAl0.1Fe1.9O4 crystallite size increase by increasing the calcining temperature is observed in 0.1Fe1.9O4 increased with an increase in calcination temperature [As shown in perature .xFex2−O4  samples, which were annealed at 1000 °C for 3 h. Uniformly-sized, well-crystallized grains of CoAlxFex2−O4 were obtained. xFex2−O4  ferrites. The average grain size of CoFe2O4 and CoAl0.1Fe1.9O4 was about 137.5 nm and 130.5 nm, respectively. The average grain size decreased when aluminum substituents were increased. The XRD pattern confirms that the average crystallite size tends to decrease with increasing Al content. The average grain size was greater than a nanoparticle (100 nm), and the sintering temperature of the sample was very high because grain size increased with increasing annealing temperature [xFex2−O4 acquired at room temperature. The hyperfine parameters, isomer shift (I.S.), magnetic hyperfine field (Hhf), quadrupole shift (Q.S.), relative area (A0), and line width (Г), were obtained by fitted spectra using Mösswinn 3.0 software , and calibration was relative to a 25 μm thick sample of high-purity alpha iron. The characteristic features of the spectra were as follows: there were two Zeeman-splitting sextets; one sextet was assigned to Fe3+ ion at the tetrahedral site, while the other sextet was attributed to Fe3+ ions at the octahedral site. This proved the ferromagnetism of the samples. The first sextet had a larger value of isomer shift, and it was assigned to octahedral B site. The second sextet had a smaller value of isomer shift, and was assigned to tetrahedral A site. Compared to the tetrahedral A-site ions, the bond separation of Fe3+ ions was greater in the octahedral B site of the Fe3+-O2− complex  ions; the values of isomeric shift are in the range of 0.1–0.5 mm/s for Fe3+ ions [3+ state. By increasing the aluminum content, the values of the magnetic hyperfine field decreased at tetrahedral A and octahedral B sites. This is because magnetic ions (Fe3+ ions) are substituted by nonmagnetic ions (Al3+ ions), affecting the supertransferred hyperfine fields [xFex2−O4 (0.6 ≤ x ≤ 0.8) included the magnetic sextet of B site; the magnetic sextet of A site vanished. This indicates that Fe3+ ions existed only in the octahedral B site. When the spectrum of CoAlxFex2−O4 (composition with x = 0.9 and 1.0) was analyzed, a single sextet and a central paramagnetic doublet were observed; this indicates relaxation effects. When the nonmagnetic Al content was increased in CoAlxFex2−O4, the samples changed into a superparamagnetic character. The behavior of the sample went from a completely magnetic state to a mixed state of magnetic and superparamagnetic order [x = 1.5, Mössbauer spectra consisted only of a central doublet; this exhibits a superparamagnetic character. The central doublet was attributed to the nearest nonmagnetic neighbors of magnetically isolated Fe3+ ions. This leads to the deficiency of long-range magnetic ordering [It is well known that the values of isomeric shift are in the range of 0.6–1.7 mm/s for Fe/2) ions . As showic order ,12. For ordering ,14.xFex2−O4 ferrite can be written as follows:The cation distribution of CoAlA sites to B sites can be written as follows [Af and Bf are the recoil-free fractions of Fe3+ ions in tetrahedral A sites and octahedral B sites, respectively. The Mössbauer absorption area is proportional to the distribution of iron ions of A sites and B sites. In the current study, we assumed that Af and Bf are equal [Based on the above cation distribution, the absorption-area ratio of  follows :(3)SASB=re equal . Table 4xFex2−O4 samples at room temperature. For all the samples, magnetization reached saturation when the strength of the magnetic field was 10,000 Oe. 3+ ion content. The saturation magnetization can be expressed with the following equation [Bn is the magnetic moment and M is the relative molecular mass. The relative molecular mass of CoAlxFex2−O4 decreased with an increase in Al content. The change in magnetic moment Bn was determined by Néel’s theory of magnetism. The magnetic moment of Al3+, Co2+, and Fe3+ ions was 0 μB, 3 μB, and 5 μB [Bn is expressed by Equation (5) [BM and AM are magnetic moments of the B and A sublattices, respectively. 3+ ion content.equation :(4)σs=55and 5 μB ,16,17, rtion (5) ,16:nB = x ≤ 0.1). According to Equation (4), calculated saturation magnetization decreased with an increase in Al3+ ion substitution. The change trend of experimental and calculated saturation magnetization was similar for x ≤ 0.1, and there was deviation between experimental and calculated saturation magnetization, which can be attributed to the actual situation of ion distribution being more complicated than that obtained from the Mössbauer spectra. For the substituents (x ≥ 0.5), there was a big difference between calculated saturation magnetization and experimental saturation magnetization, and the experimental value was smaller than the calculated value for saturation magnetization [3+ ion substituents was too high in cobalt ferrite samples. This led to a decrease in A–B interaction and an increase in B–B interaction, which subsequently decreased magnetization.tization ,19,20. Ttization . It is rxFex2−O4 decreased with an increase in Al3+ ion content (x). Based on the results of the Mössbauer spectroscopy, we inferred that Co2+ ions of CoFe2O4 samples were located at the tetrahedral A sites and octahedral B sites. The magnetocrystalline anisotropy is primarily attributed to Co2+ ions of octahedral sites, which are present in pure cobalt ferrite CoFe2O4 [2+ ions is 3d7 [2+ ions in the octahedral site, causing frozen orbital angular momentum and spin coupling [3+ ions elicit zero angular momentum (l = 0), which does not affect magnetic anisotropy [3+ ions were replaced with Fe3+ ions, the spin-orbit coupling weakened and magnetocrystalline anisotropy decreased.  CoFe2O4 . The eles is 3d7 . The anicoupling . The Al3isotropy ,24,25. WCH, magnetic anisotropy K1, and saturation magnetization SM [Equation (6) describes the relationship between the following parameters: coercivity ation MS :(6)HC=2K3+ ions, it led to a decrease in coercivity.When magnetic anisotropy decreased with an increase in Al0.1Fe1.9O4 sample at room temperature; magnetic hysteresis curves of CoAl0.1Fe1.9O4 sample were also obtained after sintering them at 600 °C and 1000 °C, respectively. 0.1Fe1.9O4 sample increased with an increase in sintering temperature; these changes were attributed to an increase in particle size [0.1Fe1.9O4 sample; moreover, the CoAl0.1Fe1.9O4 sample did not show any significant change even after being annealed at 600 °C. This confirms that the uncalcined sample has good crystallinity, which was further established by XRD.cle size . There i0.1Fe1.9O4 sample initially increased and then steadily decreased. This may be attributed to variation in grain size. The coercivity of the single-domain region is given by the following equation: HC = g–h/D2. In the multidomain region, the relationship between coercivity and grain size is established by the following equation: HC = (a + b)/(D). Here, ‘D’ is the diameter and ‘g, h, a, and b’ are constants of the particle [0.1Fe1.9O4 samples that were calcined at different temperatures; the grain size of CoAl0.1Fe1.9O4 samples varied from the single-domain region to the multidomain region. With an increasing annealing temperature, the coercivity of CoAl0.1Fe1.9O4 sample increased initially and then decreased.With a steadily increasing sintering temperature, the coercivity of CoAlparticle ,26. Hencparticle ,28. In oxFex2−O4 samples that were calcined at 1000 °C. The lattice constant decreased when smaller Al3+ ions were replaced with larger Fe3+ ions. The XRD spectra of CoAl0.1Fe1.9O4 samples were obtained after sintering them at different temperatures; these samples were prepared with a sol-gel autocombustion method, so they had good crystallinity. SEM results indicate that well-crystallized particles of uniform size were present in the sample. We obtained the Mössbauer spectra of CoAlxFex2−O4 samples, which were calcined at 1000 °C. The Mössbauer spectra reveal that with an increase in aluminum concentration, CoAlxFex2−O4 samples undergo a transition from ferrimagnetic behavior to superparamagnetic behavior. Cation distribution was estimated from the Mössbauer data. The coercivity and saturation magnetization of CoAlxFex2−O4 samples decreased with an increase in Al content (x). The changes in saturation magnetization can be attributed to Néel’s theory and the Yafet-Kittel model. Coercivity decreased with an increase in aluminum content, which is attributed to the weakening of magnetocrystalline anisotropy. The coercivity and saturation magnetization of CoAl0.1Fe1.9O4 sample initially increased and then steadily decreased. Particle size increased with an increase in annealed temperature.XRD analysis reveals the single-phase structure of CoAl


This allows IP3Rs to evoke regenerative Ca2+ signals, the smallest of which are Ca2+ puffs that reflect the coordinated opening of a few clustered IP3Rs. We use total internal reflection microscopy (TIRF) microscopy to record Ca2+ signals in HEK cells expressing all three IP3R subtypes or a single native subtype. Ca2+ puffs are less frequent in cells expressing one IP3R subtype, commensurate with them expressing fewer IP3Rs than wild-type cells. However, all three IP3R subtypes generate broadly similar Ca2+ puffs with similar numbers of IP3Rs contributing to each. This suggests that IP3R clusters may be assembled by conserved mechanisms that generate similarly sized clusters across different IP3R expression levels. The Ca2+ puffs evoked by IP3R2 had slower kinetics and more prolonged durations, which may be due to IP3 binding with greater affinity to IP3R2. We conclude that Ca2+ puffs are the building blocks for the Ca2+ signals evoked by all IP3Rs.All three subtypes of inositol 1,4,5-trisphosphate receptor (IP Summary: All IP3 receptor subtypes can generate Ca2+ puffs, suggesting that these coordinated openings of clustered IP3Rs are the building blocks of all IP3-evoked Ca2+ signals. IP3 binding to IP3R initiates its activation by promoting the Ca2+ binding that leads to channel opening . Each forms a Ca2+ channel that is co-regulated by IP3 and Ca2+, but the subtypes differ in their expression patterns  in human embryonic kidney 293 (HEK) cells expressing one or all three IP3R subtypes. We demonstrate that all three IP3R subtypes generate Ca2+ puffs with broadly similar properties. We conclude that Ca2+ puffs are the fundamental building blocks for Ca2+ signals evoked by all IP3Rs.Here, we use total internal reflection fluorescence microscopy (TIRFM) to record Ca3R subtypes  .We used wild-type (WT) HEK cells, which express three IPsubtypes , and HEKsubtypes . Identicine-IP3) . We conf3R subtype (Ab1–3) confirmed that WT HEK cells express three IP3R subtypes , we established that overall IP3R expression levels were similar in each cell line expressing a single IP3R subtype , photolysis of ci-IP3 evoked Ca2+ puffs after a short latency. The Ca2+ signal then propagated after a few seconds to produce a global increase in cytosolic free Ca2+ concentration ([Ca2+]c)  A–D. Thes[Ca2+]c) . However[Ca2+]c) , the sitcordings .Fig. 2.3 also evoked Ca2+ puffs in HEK cells expressing single IP3R subtypes. In these cell lines, all of which were exposed to the same stimulus, the latency before the first Ca2+ puff was longer than in WT cells, Ca2+ puffs were less frequent, we detected fewer Ca2+-release sites and it was rare for Ca2+ signals to propagate into a global increase in [Ca2+]c . Fortuitously, the average number of Ca2+ puffs detected during the entire recording interval was not significantly different between the four cell lines, but we detected significantly more Ca2+ release sites in WT HEK cells . The number of new sites identified in each successive interval declined mono-exponentially with half-times (t1/2=3.76–5.53 s)  that were fast enough to ensure that, within the 40-s recording interval , we should identify >94% of all Ca2+ release sites. The analysis of WT cells used a shorter time-bin (2 s) to accommodate the increased frequency of Ca2+ puffs (2+ release sites (t1/2=2.01 s), indicating that we should again detect >93% of sites . Hence, for all cell lines our analyses are likely to detect most Ca2+ release sites. The fewer sites identified in cells with single IP3R subtypes are, therefore, not due to inadequate sampling. We conclude that the cell lines with single IP3R subtypes express about one-third the total number of IP3Rs of WT cells   before the Ca2+ signal invaded the cell, whereas – for the other cell lines – it entailed recording less frequent Ca2+ puffs for longer periods (40 s) .There is considerable variation in the shape and duration of Caig. S1E) . For Ca2baseline C. To secs (40 s) F,G. To v2+ puffs in the four cell lines . The rise times (∼20 ms), decay times (∼30 ms) and durations at half-maximal amplitude (∼30 ms) were similar in WT cells and in cells expressing only IP3R1 or IP3R3. However, the rise and decay times  were significantly longer in cells expressing only IP3R2, as was the mean duration of Ca2+ puffs (62.6±5.6 ms) . We conclude that all three IP3R subtypes generate Ca2+ puffs with broadly similar characteristics, although Ca2+ puffs evoked by IP3R2 typically last about twice as long as those in cells expressing IP3R1, IP3R3 or all three subtypes.We compared mean rise times, decay times and durations of the Call lines A–C and t±5.6 ms) . The fre2+ puff, it was sometimes possible to observe stepwise closures of individual IP3Rs   and the peak amplitude of each Ca2+ puff, to estimate the number of IP3Rs contributing to a Ca2+ puff . In WT cells, the mean number of IP3Rs per puff was 2.77±0.10  . The ampig. S1E) B, suggesductance . We useda2+ puff . The res.77±0.10 C,D. Our a2+ puff . Collectht IP3Rs . The meaWT cells C,D, but ectively D. We con subtype D, Ca2+ pell line .Fig. 4.2+ puffs occurring in the most-active cells differed in their properties. There were no clear differences in the frequency distribution for rise time, decay time or Ca2+ puff duration, or in the estimated number of IP3Rs per puff for the more-active and less-active cells . We also compared the mean peak amplitude of Ca2+ puffs evoked in each cell with mean rise and fall times. Again, there was no significant correlation between puff amplitude and kinetics; the only exception was a positive correlation between amplitude and decay time for cells expressing IP3R2 .We considered whether Ca3R subtypes generate Ca2+ puffs with broadly similar properties. Ca2+ puffs have hitherto been observed only in cells that express all three IP3R subtypes, albeit with one subtype usually predominating. Ca2+ puffs originate at fixed intracellular sites, at each of which a loose cluster of a few IP3Rs is anchored near the plasma membrane, where an additional signal licenses the cluster to respond to IP3 . These results suggest that, first, for each subtype, IP3Rs are similarly distributed across the ∼90-nm depth of the TIRF field, consistent with each coming close to the plasma membrane. Second, across a range of IP3R expression levels, IP3Rs are assembled into similarly sized clusters, but with more clusters in cells with more IP3Rs . We conclude that the clusters of IP3Rs that generate Ca2+ puffs are probably not random associations. Instead, IP3Rs appear to be assembled through mechanisms that are conserved for all IP3R subtypes, and which ensure that similarly sized clusters are assembled irrespective of IP3R expression levels.We show, for the first time, that all three IPd to IP3 . Neithersubtypes D, yet ea2+ puffs evoked by cells expressing all IP3R subtypes or by those expressing only a single subtype are striking. However, Ca2+ puffs evoked by IP3R2 were more long-lasting than those evoked by other subtypes. We have not determined the mechanisms responsible for the slower kinetics of Ca2+ puffs evoked by IP3R2. One possibility is that the greater affinity of IP3 for IP3R2  and Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 (50:50) supplemented with GlutaMAX were from ThermoFisher . Pluronic acid F-127, dimethyl sulfoxide (DMSO), poly-l-lysine and foetal bovine serum were from Sigma-Aldrich . EGTA-AM was from Merck Millipore . Caged-IP3-PM  was from SiChem GmbH . Custom-made rabbit anti-peptide antibodies against IP3R2  and to a sequence conserved in all three IP3R subtypes  were provided by Poccono Rabbit Farm and Laboratory Inc. . Additional primary antibodies were: IP3R1 , IP3R3  and β-actin . Secondary antibodies conjugated to horseradish peroxidase  were from Insight Biotechnology Ltd .Cal520-AM was from Stratech Scientific . Bovine serum albumin (BSA) was from Europa Bio-Products . Gibco TrypLE Express, NP-EGTA-AM   were grown on poly-l-lysine-coated (10 µg/ml) glass-bottomed dishes . They were used after 48 h, when they were sub-confluent.WT HEK cells, from which the gene-edited lines were generated, were provided by Dr D. Yule . HEK cells in which CRISPR/Cas9 had been used to generate lines expressing a single IP(HEK-KO)  were frog, 4 min, 4°C) were separated  and transferred to a polyvinyl difluoride (PVDF) membrane using an iBLOT gel-transfer system (ThermoFisher). Membranes were blocked in Tris-buffered saline  and incubated  with primary antibody. After three 5-min washes in TBS, membranes were incubated  with secondary antibody and washed (3×5 min). Bands were visualised with ECL Primer Western blotting detection reagent  and a Syngene PXi chemiluminescence detection system . We confirmed that, within the range of loadings used for analysis, IP3R band intensities scaled linearly with the amount of protein loaded.Cells were lysed in cold RIPA buffer  with protease inhibitors . Proteins in the supernatant  relative to the common antibody (e.g. Ab1:AbC for IP3R1). These ratios were used to convert intensities of IP3R subtype-selective bands in WT cells to AbC intensities, from which we estimated the relative expression of the three IP3R subtypes  and ci-IP3. Cells were washed with HEPES-buffered saline  and then incubated (20°C in darkness) in HBS containing pluronic acid F-127 (0.02% w/v), Cal520-AM (5 µM) and ci-IP3-PM (1 µM). After 1 h, cells were washed and incubated in HBS containing pluronic acid F-127 and EGTA-AM (5 µM). After 45 min, the medium was replaced with HBS. After a further 30 min, the cells in HBS at 20°C, were used for imaging. The same method, but with NP-EGTA-AM (1 μM) replacing ci-IP3 PM, was used to load cells with caged EGTA .To record local Caiffusion , a Ca2+ 2+ signals by using total internal reflection fluorescence microscopy (TIRFM). Excitation (488 nm) was provided by a diode-pumped solid-state laser  and a band-pass filter . The angle of the excitation beam was adjusted to achieve TIRF with a penetration depth of ∼90 nm. Emitted light was captured through a band-pass filter  using an Andor iXon 897 EMCCD camera . To achieve high temporal resolution, images were captured (188 Hz) from only a part of each cell  by streaming directly into RAM. After visualisation using Metamorph , images were exported as tif files. Images were captured for 40 s before and after flash photolysis of ci-IP3.An Olympus IX83 inverted microscope equipped with 100× oil-immersion TIRF objective  was used for measurements of local Ca3 or NP-EGTA within the entire imaging field was achieved using a light-emitting diode , with the duration of exposure  determined by a shutter controlled within Metamorph. The conditions used for flash photolysis of ci-IP3 were optimised to allow identical stimulation of all cell lines, with each line providing sufficient Ca2+ puffs for quantitative analysis within a practicable recording interval (≤40 s).Photolysis of ci-IPFig. S2C). Images were processed in Fiji  that identifies clusters of pixels within which fluorescence changes exceed a critical threshold. From each recording, the last 500 frames (∼2.7 s) before photolysis were averaged to provide an F0 value and its standard deviation (s.d.) for each pixel. Image stacks of [(F/F0 )−1]/s.d. were then created and Gaussian-filtered, and pixels that exceeded a critical value  were located. A threshold-cluster algorithm , we measured the unitary steps during the decay phase of 40 Ca2+ puffs , where ΔF = Fpeak−Fpre. Fpeak and Fpre are, respectively, the F/F0 values determined at the peak of the Ca2+ puff and the average from the same region for ten frames before and after the puff.To estimate the number of active IPn usually referring to the number of cells analysed. Statistical analyses used two-tailed Mann–Whitney tests or, for multiple comparison, the Kruskal–Wallis test with Dunn's correction for multiple comparisons . Statistical significance is denoted by: *P<0.05, **P<0.01, ***P<0.001.Results are presented as means±s.e.m. unless stated otherwise, with


Lactobacillus kefiranofaciens is the only dominant and stable bacterial species in TKGs regardless of culture conditions and time. FISH and SEM showed that L. kefiranofaciens exhibited two distinct morphotypes of short rod (3.0 μm in length) and long rod (10.0 μm in length) upon colonization of either the outer surface or inner component of TKGs, providing evidence for its trophic adaptation to the hollow globular grain structure of TKGs. These findings pave ways for further study of the specific symbiotic interaction between L. kefiranofaciens and the dominant Saccharomyces cerevisiae yeast in TKGs in vivo.Tibetan kefir grains (TKGs), natural starters for milk fermentation, are believed to comprise diverse microflora of lactic acid and acetic acid bacteria. In order to better understand the bacterial community in TKGs, TKGs that had been cultured continuously either naturally or aseptically for 10 months were subject to analysis using both culture-dependent and various culture-independent methods. Results of DGGE, metagenomics, FISH, qPCR and isolation all demonstrated that  They have been used to ferment cow milk or goat milk into kefir, which is a kind of beneficial beverage. It has been shown that kefir might possess higher nutritional value as well as more health benefits, including enhancing immune system, improving digestive health, and performing antimicrobial, antitumoral and antioxidant activities , than otLactobacillus family  and two different sampling time-points (before and after 10 months of continuous cultivation).In our previous work, the diversity of yeasts in TKGs cultured in our laboratory was investigated . InteresLactobacillus kefiranofaciens is the dominant bacterial species in the same TKGs as analyzed for the diversity of yeasts  that was renewed every 24 h. Two distinct cultivations as described in  were per2.2TKGs (approximately 14 mm in diameter) growing in renewed milk for 24 h were sampled and washed thoroughly with sterile water to remove any adhered kefir and attached microbes prior to DNA extraction. Approximately 40 mg of biomass from each grain (n = 3) was taken aseptically, pestled with TissueRuptor  and then suspended in 600 μl of 1.2 M sorbitol buffer. After addition of 50 U lyticase , the samples were allowed to react for 30 min at 30 °C. The samples were centrifuged at 1500×g for 10 min, and the deposits were resuspended in buffer  with 20 mg/ml lysozyme  for 50 min at 37 °C. Subsequently, total DNA was extracted according to the instructions of the Marine Animals DNA Kit  with a modification involving the addition of 50 μl of (20 mg/ml) Proteinase K solution .2.3Taq PCR MasterMix , 1 μl of 10 μM of each primer (V3-F and V3-R) . PCR was performed in a total reaction volume of 50 μl, containing 25 μl of 2× nd V3-R)  (Table 1DGGE was performed under the following conditions: a linear 40–60% denaturant gradient (100% denaturant contained 7 M of urea and 40% of deionized formamide), a constant voltage of 60 V for 16 h , and a temperature of 60 °C. The gels were stained in 1× SYBR I in TAE buffer  for 20 min, and then visualized by using Bio-Rad Gel Doc XR+ imaging system.2.4The TKGs  16S rRNA gene sequence data of the V1-V2 region  Table 1Table 1 wArchaea, Eukaryota, chloroplasts, or mitochondria were discarded. Sequences affiliated with Lactobacillus were compared with NCBI bacterial 16S ribosomal sequence database using BLASTn  to identify the specific species.Sequence classification was performed using a naive Bayesian classifier trained against Ribosomal Database Project (RDP) training set (version 9)  with 80%Finally, sequences were clustered into operational taxonomic units (OTUs) based on pairwise identity of 97% and abundance of more than 0.005%. Similarities among samples were evaluated with collector's curve of OTUs, Chao1 richness and inverse Simpson index.2.5TKGs (A-2) were sampled and washed with sterile water to remove clotted milk from the grain surfaces. Approximately, 0.1 g grains were grinded aseptically with TissueRuptor  and then suspended in 1 ml of 0.9% saline. One hundred microliter of aliquot of the suspension was spread onto six MRS agar plates (pH 5.0 and 1.5 g of agar per 100 ml), which were then incubated at 30 °C for 7 days under an anaerobic condition .Taq PCR MasterMix , 1 μl of 10 μM each primer (http://blast.ncbi.nlm.nih.gov/Blast.cgi).Eighteen colonies (three per plate) were randomly selected and then subjected to colony PCR assay. The primers used were 27-F and 1492-R  as showeh primer  and the L. kefiranofaciens cells was performed according to the instructions described in the Bacterial Genomic DNA Extraction kit . The V3 region of the 16S rRNA gene was amplified for DGGE analysis using the same method described above.Isolation of the genomic DNA from 2.6L. kefiranofaciens strains were cultured in MRS broth (pH 5.0) for 5 or 7 days under anaerobic condition . One milliliter of L. kefiranofaciens cells was centrifuged at 6000× g for 1 min. The cell pellets were re-suspended in PBS solution and was fixed in 4% of formaldehyde for 14 h. The fixed L. kefiranofaciens cells were filtered through a 0.2 μm GTTP membrane (Merck Millipore), stained with 1 × SYBR Green I  for 15 min, and observed under an epifluorescence microscope .2.7L. kefiranofaciens cells were the same as described above, except that the cells were fixed in 2.5% of glutaraldehyde solution for 2 h at 4 °C. The cells were then washed in 0.1 M of phosphate buffer for 3 times, each 15 min, and post-fixed in 1% of osmium tetroxide for 1 h at 4 °C, followed by 3 washes with 0.1 of M phosphate buffer and then with a series of ethanol  containing anhydrous sodium sulfate) for 15 min at room temperature.Procedures for preparing L. kefiranofaciens cells were desiccated in a mix solution (isoamyl acetate: ethanol = 1:1) and in isoamyl acetate for 30 min, respectively, then dried in a critical-point dryer  for 5 h. The dehydrated samples were displayed on specimen stubs with a double-sided adhesive tape. The samples were observed using a SEM  after being coated with gold using a sputter coater (Cressingtom 108 auto).The fixed Methods for sample preparation and SEM observation of TKGs were the same as described in our previous work .2.8L. kefiranofaciens, specific probe was designed based on the 16S rRNA gene sequence obtained in this study and its specificity was verified using BLASTn program (http://blast.ncbi.nlm.nih.gov).Probes used in this study are shown in Preparation of frozen thin sections of TKGs for FISH was performed according to the methods described previously . After t2.9L. kefiranofaciens were designed based on its full length 16S rRNA gene sequence using Geneious 6.1.8 software (Biomatters). Specificity of primer set was confirmed with the GenBank database using the Primer-BLAST program (http://www.ncbi.nlm.nih.gov/tools/primer-blast).Primers for real-time qPCR are shown in L. kefiranofaciens was extracted according to the methods described above. The primer set of 27F/1492R , 1 μl of 10 μM of each primer, and approximately 100 ng of DNA template. The PCR program was as follows: 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and a final extension step of 72 °C for 10 min. The amplified PCR products were purified using the PCR Fragment Purification Kit  and then cloned with the pGM-T Cloning Kit . The recombinant plasmids were transformed into competent Escherichia coli T0P 10 cells following supplier's instructions. Positive clones were identified by colony PCR, 2.0% agarose gel electrophoresis and sequencing. Sequence similarity of obtained sequences was analyzed by BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The positive clones carrying the 16S rRNA gene fragments of L. kefiranofaciens were cultivated. The recombinant plasmids were extracted using the QIAprep Spin Miniprep Kit  and quantified by BioTek Synergy 2.0  and Quant-iT PicoGreen dsDNA Reagent and Kit .Plasmid standards used in this study were prepared as described in . Briefly7F/1492R  was usedTaq II (2×) , and 0.4 μl of Rox Reference Dye (50×). The following qPCR thermal program was used: 95 °C for 10 min, followed by 40 cycles at 94 °C for 15 s, 59 °C for 30 s, and 72 °C for 30 s. The amplification reaction was performed in an ABI 7500 Fast real-time PCR system along with version 2.0.1 of the software . Quantification of total bacteria  contained 2.0 μl of template (plasmid standard or kefir grain genomic DNA), 0.4 μl of each of the specific primers, 10 μl of SYBR Premix Ex bacteria  and L. kofaciens  were per2.10High-throughput sequencing reads were deposited into the SRA database under their corresponding accession numbers (SRR5039623–SRR5039626).33.1DGGE was performed in order to identify the bacterial community in TKGs. As shown in 3.2To get a thorough understanding of the bacterial diversity in TKGs, samples of N-1, N-2, A-1 and A-2 were further analyzed with 16S metagenomics. Given that no differences in DGGE profiles were observed for three parallel samples, only one of them was subjected to sequencing analysis. The number of obtained sequences and the sequencing coverage for each sample were presented in Lactobacillus (≈97%). BlastN similarity search showed that L. kefiranofaciens was the only predominant species assigned to the Lactobacillus-associated sequences  as well as that between the time span of 10 months (N-1 and A-1 vs. N-2 and A-2) were not statistically significant , indicatTaken together, we conclude that culture condition and time span had little impact on the bacterial diversity in TKGs. This finding is in concordance with the DGGE results presented above.3.3L. kefiranofaciens was performed to verify its presence in TKGs. Since the bacterial community in the four samples of TKGs were almost identical based on the results of DGGE and 16S metagenomics analyses, only the samples of A-2 were subjected to bacterial isolation. No morphological differences were observed among the obtained colonies on MRS agar plates (n = 6). The colony PCR products were sequenced and analyzed with BLASTN. All 18 sequences shared 99–100% of similarity with the 16S rRNA gene of L. kefiranofaciens. In addition, the DGGE profiles of the L. kefiranofaciens cells were almost identical to that of TKGs   A and B. –0.6 μm) C and 10.–0.6 μm) D.Fig. 6S3.5in silico as well as through hybridization with pure culture of L. kefiranofaciens. Both BLASTN similarity search and SILVA database check confirmed that the probe Lbk is 100% specific for L. kefiranofaciens only. However, whole cell hybridization initially failed for both probes, EUB338 and Lbk. Nevertheless, after treatment of the bacterial cells with lysozyme, all DAPI-stained cells showed positive signals with both bacteria-specific and L. kefiranofaciens-specific probes  vs. (0.91 ± 0.29) × 1010 (N-1) and (1.05 ± 0.04) × 1010  vs. (0.75 ± 0.31) × 1010 (N-2); (2.75 ± 0.12) × 1010  vs. (1.62 ± 0.08) × 1010 (A-1) and (1.72 ± 0.02) × 1010  vs. (1.63 ± 0.04) × 1010 (A-2). These results further supported that L. kefiranofaciens is the only dominant bacterial species in TKGs and it remains stable under different culture conditions and time spans.Furthermore, no significant variation in copy number per gram of TKGs was observed between total bacteria and tivation B: . Furthermore, the short L. kefiranofaciens cells on grain surface revealed almost no signals after hybridization with these two probes, however, most of the long cells in the inner parts of grains demonstrated positive hybridization  were found in TKGs cultured either naturally or aseptically .This work was partially supported by the The authors declare no conflict of interest.Data associated with this study (high-throughput sequencing reads) has been deposited at into the SRA database under the accession numbers SRR5039623–SRR5039626.


NMR and X-ray crystallography reveal that Leishmania has a unique type of N-terminally-extended cap-binding protein (eIF4E4) that binds via a PAM2 motif to PABP1. This relies on the interactions of a combination of polar and charged amino acid side-chains together with multiple hydrophobic interactions, and underpins a novel architecture in the Leishmania cap4-binding translation factor complex. Measurements using microscale thermophoresis, fluorescence anisotropy and surface plasmon resonance characterize the key interactions driving assembly of the Leishmania translation initiation complex. We demonstrate that this complex can accommodate Leishmania eIF4G3 which, unlike the standard eukaryotic initiation complex paradigm, binds tightly to eIF4E4, but not to PABP1. Thus, in Leishmania, the chain of interactions 5′cap4-eIF4E4–PABP1-poly(A) bridges the mRNA 5′ and 3′ ends. Exceptionally, therefore, by binding tightly to two protein ligands and to the mRNA 5′ cap4 structure, the trypanosomatid N-terminally extended form of eIF4E acts as the core molecular scaffold for the mRNA-cap-binding complex. Finally, the eIF4E4 N-terminal extension is an intrinsically disordered region that transitions to a partly folded form upon binding to PABP1, whereby this interaction is not modulated by poly(A) binding to PABP1.Trans-splicing of trypanosomatid polycistronic transcripts produces polyadenylated monocistronic mRNAs modified to form the 5′ cap4 structure (m Trypanosoma brucei (African sleeping sickness), Trypanosoma cruzi (Chagas disease) and Leishmania species (responsible for multiple forms of leishmaniasis), and many more people are at risk of infection  recognition, as well as polypeptide elongation and termination ,2. Whilenfection . It is sic mRNAs ,6, a proic mRNAs , and othic mRNAs .7Gpppm36,6,2′Apm2′Apm2′Cpm23,2′U) that is added to the 5′end of the monocistronic mRNAs during processing -3′mRNA. At least inasmuch as eIF4E can influence the accessibility of the 5′cap to proteins involved in the catalysis or modulation of decapping, the relationship between the 5′ and 3′ ends of mRNA potentially also plays a role in the control of mRNA degradation  binding protein PABP . This isradation ,27.Leishmania, and to characterize key structural features underpinning its assembly. Techniques of NMR, X-ray crystallography, fluorescence anisotropy, microscale thermophoresis and surface plasmon resonance have been used to develop a quantitative understanding of the structures and interactions underpinning this unique type of interaction chain. In particular, we demonstrate the uniquely pivotal structural role of the trypanosomal eIF4E4–PABP1 interaction mediated by the eIF4E4 N-terminal region centered on the PABP-interacting motif 2 (PAM2).Taking as our starting point earlier evidence that N-terminally extended versions of eIF4E (i.e. isomers 3 and 4) can interact with PABP , grown in Terrific Broth medium supplemented with 25 μM ampicillin. Once the culture had reached OD600 = 0.6, expression was induced by the addition of 0.5 mM IPTG and then each expression strain was incubated with shaking for 17–22 h at 16°C. The cells were pelleted by centrifugation at 10 000 rpm for 45 min at 4°C in a Beckman-Coulter Avanti J-26 XP centrifuge using a JLA 8.1 rotor, then re-suspended in lysis buffer , using 2 ml of buffer for each gram of cells. The cells were lysed by sonication (4 × 45 seconds on 70% power interspersed with 1min periods on ice). After clarification by centrifugation at 16 000 rpm for 45 min at 4°C in a Beckman–Coulter Allegra 64R centrifuge using F0685 rotor, the supernatant containing soluble proteins was incubated overnight at 4°C with 2 ml of the affinity resin  that had been equilibrated with the lysis buffer. All affinity purifications were performed in batch mode. After incubation with lysate, the resin was first washed 3 × 20 minutes with 12 ml of lysis buffer (without Triton X-100 and protease inhibitors), and then 3 × 20 min with 12 ml of wash buffer . The proteins were eluted from the resin using elution buffer (wash buffer containing 0.5 M imidazole and 0.1% DDM). Depending on the protein yield, between 4 and 10 elutions were made, each using 2ml of elution buffer incubated for 15min with the resin. Gel filtration was used to separate protein complexes from single proteins and aggregates. Selected elution fractions derived from affinity purification were pooled and concentrated to 0.5ml, using Sartorius Vivaspin 3000 MWCO PES 20 ml concentrators (3000rpm in an Eppendorf 5810R centrifuge). For the highly-produced proteins, and for proteins prone to aggregation, 0.5ml volumes of the best elution fraction(s) were collected for further use. These were loaded onto a Superdex®200 Increase 10/300 GL column , using a GE AKTA FPLC Purifier UPC100, and 0.6 ml fractions were collected. Fractions were analysed via SDS-PAGE and those containing the purest proteins were selected for further studies. The protein concentrations in the isolated fractions were determined by running at least four different dilutions of the proteins on a stained  SDS gel against BSA standards, followed by densitometric analysis using ImageQuant software (GE). The identities of all proteins and protein domains were confirmed using mass spectrometry.All proteins (and protein complexes) were encoded by synthetic reading frames in the −1 after which peptides were eluted onto the analytical column at 300 nl min−1 by increasing the mobile phase B concentration from 4% B to 25% over 22 min then to 90% B over 3 min, followed by a 10min re-equilibration at 4% B. The eluted peptides were then subjected to electrospray ionization and analyzed on a Thermo Orbitrap Fusion system . Precursor peptides scanning from 375 to 1500 m/z were analyzed at 120 K resolution (at 200 m/z) with a 2 × 105 ion count target. Tandem MS was performed by isolation at 1.2Th using quadrupole HCD fragmentation with a normalized collision energy of 30 and rapid scan MS analysis in the ion trap. The MS2 ion count target was set to 3 × 103 and the maximum injection time was 200 ms. Precursors with charge state 2–6 were selected and sampled for MS2. The dynamic exclusion duration was set to 30 s with a 10 ppm tolerance around the selected precursor and its isotopes. Mono-isotopic precursor selection was turned on. The instrument was run in top speed mode with a 1 s cycle. Raw MS data were processed using MSConvert in the ProteoWizard Toolkit (version 3.0.5759). The MS2 spectra were searched using the Mascot engine  against the available Leishmania database and the common Repository of Adventitious Proteins Database (http://www.thegpm.org/cRAP/index.html). Peptides were generated from tryptic digestion with up to two missed cleavages, carbamidomethylation of cysteines as fixed modifications, and oxidation of methionines as variable modifications. Precursor mass tolerance was 10 ppm and product ions were searched at 0.8 Da tolerances. Scaffold  was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at >95% probability by the Scaffold algorithm and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm . Proteins sharing significant peptide evidence were grouped into clusters.The identification of proteins was performed via mass spectrometry using nanoLC-ESI–MS/MS. The appropriate protein bands were excised from SDS-PAGE gels and digested with trypsin. The resulting peptides were separated by reversed phase chromatography using an Acclaim PepMap μ-pre-column cartridge 300 μm i.d. × 5 mm 5 μm 100 Å and an Acclaim PepMap RSLC 75 μm × 25 cm 2 μm 100 Å (Thermo Scientific), installed on an Ultimate 3000 RSLCnano system (Dionex). Two mobile phases were used: mobile phase A comprised 0.1% formic acid in water; mobile phase B comprised 0.1% formic acid in acetonitrile. In the next step, the samples were loaded onto the μ-pre-column equilibrated in 2% aqueous acetonitrile containing 0.1% trifluoroacetic acid for 8 min at 10 μl min2O:TIS:EDT, 92.5:2.5:2.5:2.5 (5 ml × 3 h) and peptides were precipitated using cold ether. Pure peptides were obtained after preparative HPLC purification on a Jupiter Proteo 90Å 21.2 × 250 mm reverse phase column using a gradient of 20–60% acetonitrile with 0.1% TFA and lyophilization. High-resolution mass spectrometry (HR-MS) data were recorded using electrospray ionization in positive mode (ESI+) with a Bruker MaXis Impact spectrometer. Analytical HPLC experiments were performed using an Agilent 1290 Infinity LC series system equipped with an Ascentis Express Peptide ES-C18 100 × 2.1 mm column, 2.7 μm particle size on a 5–95% gradient of acetonitrile in water (with 0.1% TFA) over 10 min. The 14mer peptides  used to test the effects of mutations in the PABP1 binding site on eIF4E4 were synthesized and HPLC-purified (to 98% purity) by Thermo Fisher Scientific  and ammonium chloride . Protein samples were prepared in HEPES buffers  at concentrations in the range 50–200 μM. Experiments were performed on cryoprobe-equipped Bruker 600 and 950 MHz spectrometers in 5mm Shigemi tubes using 300 μl of solution in the presence of 5% D2O at a temperature of 300 K. Backbone assignments were accomplished via 1H–15N-HSQC and the triple resonance experiments HNCA/HNCOCA, HNCACB/HNCOCACB and HNCO/HNCACO. TROSY-based variants were used for experiments involving larger complexes. Data analysis was performed using the software packages Bruker TopSpin 3.5pl7 and CCPN Analysis V2.4.2.Isotopically labelled proteins were prepared by growing the appropriate strains in an Applikon ez- Control Bioreactor. The minimal medium was supplemented with D-glucose  and PABP1(J) complexed with peptide were grown at 22°C by vapour diffusion in 96-well plates (Swiss-Sci) using a Mosquito liquid handling robot (TTP LabTech) with 100 nl protein mixed with 100 nl well liquor. Crystals of PABP1(J) were harvested from a drop with 0.2 M ammonium sulphate, 0.1 M sodium cacodylate at pH 6.5, 30% PEG8000 (Structure Screen) and those of the protein in complex with the peptide were grown in 0.2 M ammonium sulphate; 0.1 M MES; 20% PEG 8000 (ProPlex). Both were cryo-cooled by plunging the crystals directly into liquid nitrogen. Data were collected at Diamond Light Source beamlines IO3 (PABP1(J)) and I24 (PABP1(J) + peptide) and processed at the beamline with XDS through the Xia2 pipeline. Further processing was carried out in CCP4. The structure of PABP1(J) was solved by molecular replacement in Phaser using the structure of an MLLE domain from human PABP1 (PDB accession 3PTH) as a search model. Refinement was carried out using the PHENIX package. The model was first rebuilt using PHENIX.autobuild and thereafter refined using a combination of Phenix.refine and manual intervention using Coot. The structure of the complex with peptide was also solved by molecular replacement using the refined structure of the PABP1(J) domain alone as the search model. The structure was refined as above. Individual chains were defined as TLS groups in the refinement of both structures. Interactions between amino acid residues in the crystal structure were displayed using EMBL/EBI software LIGPLOT+ . Both structures contain two PABP1(J) domains in the asymmetric unit, which are covalently linked through a disulphide bridge between cysteine 552 of each molecule. Data collection and refinement statistics are shown in For crystallization, the protein was concentrated to 20 mg mlhttp://us.expasy.org/tools/pscale/Hphob.Eisenberg.html (Electrostatic surface modelling was performed using the PyMOL V2.0.5 – APBS (Adaptive Poisson–Boltzmann Solver) plugin ,29. Hydrerg.html .kON and kOFF values), two types of model were fitted to the raw data: a homogeneous 1:1 binding model, and a heterogeneous 1:1 two-ligand model. The heterogeneous model was found to give better fits. This is not unexpected, since the mode of protein coupling used tends to result in a population of protein molecules that are heterogeneously oriented with respect to the SPR chip surface ::PABP1(J), and the data were fitted using the general equation y = a-b*c∧x with the help of the OriginLab model Asymptotic 1.Microscale thermophoresis experiments were performed using eIF4E4(iv) or eIF4G3 labelled with a fluorescent tag , or the eIF4G3 peptide VEQIRSVRNNYLEPPYPGFSLDEVVR labeled with fluorescein-5-isothiocyanate. The concentration of the fluorescently labelled protein (eIF4E4(iv)) was held constant, while the concentration of non-labelled ligand protein was varied. The experiment was conducted by preparing 16 tubes with various ratios of the interacting labelled/non-labelled proteins. The solutions were introduced into capillaries, which were then loaded into a Nanotemper Monolith NT.115 instrument for measurement. For the SPR experiments, eIF4E4(iv) was immobilized via amine coupling to a Biacore CM5 sensor chip  and titrations against PABP1(J) were performed in a Biacore T200 instrument. In order to maximise confidence in the calculated dissociation constant values  was introduced at the N-terminal or C-terminal end of each protein domain used in this study (Leishmania PABP1 and eIF4E4 by modeling MLLE and PAM2 domains from other (human) proteins  and eIF4E4(iv) and used these protein domains, together with the non-labelled versions, for NMR analysis. By this means, we were able to identify the amino acids comprising the respective binding motifs  to be folded  to 15N-13C- labeled PABP1(J)  domain (15N–13C-eIF4E4(iv) caused major chemical shift perturbations (CSPs) that map to the region N141-G150 of eIF4E4   Figure  enabled ) domain . In the 4 Figure , which mPABP1(J) . The resLeishmania PABP1  of eIF4E4, which follows the same pattern of PAM2 motifs found in other proteins, including Paip1 and eRF3 . The eIV)-A—F-P , C, E, GLeishmania PABP1 (KITGMFLE) represents a variation on the MLLE motif generally found in other poly(A)-binding proteins; this residue is located on α-helix 3 and forms part of a hydrophobic pocket that interacts with M140 in the eIF4E4 sequence  in parallel with eIF4E4(v) (E. coli.Given that the eIF4E4 factor carries both the PABP1-binding (PAM2) site in its N-terminal extension Figure  and eIF4IF4E4(v) . Co-exprLeishmania. Since eIF4E4(v) included the putative binding motifs for both PABP1 and eIF4G3  and 15N-,13C- labeled eIF4E4(v), and found that the addition of a synthetic 28mer peptide that includes the eIF4G3 motif  = 3.3 × 10−7 M; KD = 5.5 × 10−7 M; KD value is 25 times greater than that of the corresponding non-mutated PABP1(J) value.We wanted to establish a quantitative understanding of the relative significance of interactions between the y Figure . MicroscKD values = 3.4 × 10−7M) also occurs between Alexa-647-labelled eIF4G3 and the complex eIF4E4(v):PABP1  binding protein in eukaryotic cells. In many other organisms, strong binding between eIF4G and PABP promotes translation ,20,23,25d assays ,36,37. OLeishmania eIF4E3 N-terminal region possesses a sequence that is only partly related to the PAM2 motif found in eIF4E4  species  have been described as essential proteins that are present in at least the promastigote stage of the trypanosomatid developmental cycle . These tn eIF4E4 . Moreovepanosoma . Overall species . We conc species  and, as  species . Future Leishmania PABP1 that eIF4E4 interacts with is the MFLE motif, which is a variant of the standard MLLE motif, and using a mutated PBAP1(J) protein domain we have confirmed the importance of F525 as a key part of the hydrophobic pocket that accommodates M140 of eIF4E4.From the X-ray and NMR structural data in this study we have learned that there are a large number of atomic interactions underpinning binding between eIF4E4 and PABP1. By performing microscale thermophoresis binding assays using FITC-14mer peptides carrying sequence variants, we have deepened understanding of the relative significance of the respective amino acids around the PAM2 motif in eIF4E4. These experiments have demonstrated the significant roles of E146 and F147, and the at most minor roles of N143 and of M148, in binding. In the case of N143, the NMR data indicated that binding between eIF4E4 and PABP1 has an effect on the environment of this residue, but the X-ray results indicate that it does not engage in significant interactions. In addition, the peptide binding results Table , underpiLeishmania eIF4E4. Since there is evidence that phosphorylation of disordered PAM2-containing protein regions can modulate binding to PABC domains -3′mRNA (Leishmania eIF4G3–eIF4E4(cap4)-PABP1-poly(A) biomolecular chain lacks direct binding between eIF4G3 and PABP1, and this raised the question whether the binding of poly(A) ribonucleotides to PABP1 might instead modulate the binding affinity between PABP1 and eIF4E4. Our results suggest that the changes in the roles of the respective translation factors in Leishmania have created a biomolecular chain that functions without poly(A)-mediated modulation of protein-protein interactions. This outcome is consistent with the observation that the PABP1 PABC domain forms a stable fold in solution that is only minimally altered by binding of the eIF4E4 peptide.Previous studies of initiation complexes in yeast, mammalian and plant systems have revealed evidence of cooperativity of the interactions between components in the molecular chain mRNA5′-m)-3′mRNA . Of part)-3′mRNA ,43. As wLeishmania cap-binding-complex proteins reported here are weaker than those reported previously for eukaryotic eIF4E and eIF4G  binding to the eIF4E4(iv) protein domain (KD = 0.22 × 10−7M) is very similar to that observed for PABP1(J) binding to the 14mer eIF4E4 peptide (KD = 0.33 × 10−7 M). This suggests that the primary determinants of eIF4E4 binding are contained in the PAM2 motif.It is also noteworthy that, although still classifiable as of relatively high affinity, the interactions between the Leishmania eIF4G3–eIF4E4(cap4)–PABP1–poly(A) biomolecular chain that links the 5′ and 3′ ends of trypanosomatid mRNAs. We anticipate that future work will build on these insights to help expand this picture by exploring a wider network of interactions involving other translation factors, such as the DEAD-box RNA-binding protein eIF4A. We note that such future work could reveal further interaction interfaces that may be worthy of consideration as potential drug targets.Finally, this quantitative study has focused on key interactions and structures underpinning the unique architecture of the Supplementary DataClick here for additional data file.


Their dynamic characteristics, as well as the key environmental affecting factors, were also analyzed. The results showed that the flux variation ranges of CO2, CH4, and N2O were 7.92 ± 1.30~650.93 ± 28.12 mg·m−2·h−1, −57.71 ± 4.65~32.51 ± 13.03 ug·m−2·h−1, and −3.87 ± 1.35~31.1 ± 2.92 ug·m−2·h−1, respectively. The three greenhouse gas fluxes showed significant seasonal variations, and differences in soil CO2 and CH4 fluxes between different forest types were significant. The calculation fluxes indicated that the permafrost soil of the Daxing’an Mountains may be a potential source of CO2 and N2O, and a sink of CH4. Each greenhouse gas was controlled using different key environmental factors. Based on the analysis of Q10 values and global warming potential, the obtained results demonstrated that greenhouse gas emissions from forest soil ecosystems in the permafrost region of the Daxing’an Mountains, northeast China, promote the global greenhouse effect.With global warming, the large amount of greenhouse gas emissions released by permafrost degradation is important in the global carbon and nitrogen cycle. To study the feedback effect of greenhouse gases on climate change in permafrost regions, emissions of CO The average surface temperature of the earth has increased by 0.85 °C compared to that before the industrial revolution. This trend is expected to continue and reach an increase of 0.3~4.8 °C by the end of this century ,2. The l2, CH4, and N2O), which has a positive feedback effect on the global carbon or nitrogen cycle [Permafrost is the result of energy exchange between the lithosphere and the atmosphere and is also an important part of the cryosphere. Global climate change directly affects both the evolution and development of permafrost. Permafrost soils are large pools of carbon and nitrogen. Jorgenson et al.  estimateen cycle  and climen cycle . Therefo2 flux was significantly higher than that of broadleaf forests. In contrast, Leckie et al. [2 flux. Wang et al. [4 absorption capacity than broadleaf forest soil. Castro et al. [4 fluxes between both forest types were not significant. The conclusions for soil N2O flux in broadleaf forests and coniferous forests were also different. Butterbach-Bahl et al. [2O. Several scholars have suggested that the N2O emission of both forest types are similar, or that coniferous forests are slightly higher [2, CH4, and N2O in soil remain unclear and need further investigation.Forest ecosystems are important players in the global carbon and nitrogen cycle process, and maintain 86% of the plant carbon pool and 73% of the soil carbon pool . With gle et al.  believedg et al.  reportedo et al.  believedl et al.  held thay higher . In summ4 km2 [4 km2), is one of the main distribution areas of permafrost at high latitudes in China, and is to the south of the most prominent part of the Eurasian high-latitude permafrost region [2, CH4, and N2O emissions were investigated, and the global warming potential (GWP) and Q10 were evaluated. The purpose is to assess the greenhouse effect of greenhouse gases, released as a result of permafrost degradation in the Daxing’an Mountains, and provides a basis for the study of carbon and nitrogen budget in the Daxing’an Mountains’ forest ecosystem.China is one of the world’s three countries with permafrost regions. The permafrost area is about 220 × 104 km2 . Northeat region ,22 or pet region ,24. Rela−2, and ≥10 °C annual accumulated temperature of 1436~2062 °C. The zonal soil of the study site is dark brown forest soil. The vegetation in this area belongs to the southern extension of the Eurasian cold-temperate coniferous forest. The zonal vegetation is dominated species of Larix gmelinii. Other major species include Betula platyphylla, Pinus sylvestris var.mongolica, Populus davidiana, etc. Common plants include Ledum palustre var. dilatatum, Rhododendron dauricum, Vaccinium uliginosum, Vaccinium vitis-idaea, and Pyrola incarnate. Sample selection was based on representative and feasibility principles. After comprehensive investigation, the three most typical forest types in the permafrost region of the Daxing’an Mountains: Larix gmelinii forest (LF), Pinus sylvestris var.mongolica forest (PF), Betula platyphylla forest (BF) were selected. One 100 m × 100 m fixed experimental block with similar site conditions was set in each forest type , and a total of 3 experimental blocks were set. All acronyms such as LF, PF, and BF which applicable for all tables and all figures.The study site was situated in the experimental area of the Mohe Forest Ecosystem National Positioning Observation and Research Station in Heilongjiang province  . The exp2, CH4, and N2O gas fluxes in situ. The static chamber was mainly composed of the chamber and a stainless-steel base. Before sampling, the stainless-steel base with the water groove was buried in the quadrat one week in advance, and was kept fixed during the whole monitoring period to reduce interference by the surrounding environment. The outside of the chamber was affixed with insulation material to decrease the temperature disturbance by the environment inside the box. Three holes were reserved at the top of the chamber to connect the fan power cable, the thermometer probe, and the sampling port, respectively. A 12-V battery-powered fan was installed in the chamber to evenly mix the gas.Three 5 m × 5 m quadrats were randomly distributed along the diagonal of each fixed experimental block (100 m × 100 m) in each forest type, but each quadrat was separated by at least 20 m in order to collect greenhouse gas samples. A total of 9 quadrats were set in the three forest types. The static opaque chamber and gas chromatography method were adopted to monitor soil COIn sunny weather, gas samples were collected during mid-morning , which was used to represent one day of average flux , and onc10)  were measured simultaneously with soil sampling. All soil samples were shipped back to the laboratory in ziplock bags for the determination of soil moisture, bulk density (Bd), pH, ammonium nitrogen (NH4+-N), nitrate nitrogen (NO3−-N), total nitrogen (TN), and total organic carbon (TOC)  [In each quadrat (5 m × 5 m), five soil samples (0–15 cm) were randomly collected by removing the surface layer of the soil, and uniformly mixed into one subsample. Each time, 9 soil subsamples were collected from 3 fixed experimental blocks (100 m × 100 m), and total of 216 soil subsamples were collected during the observation period. The atmospheric pressure , air temperature (Ta), 10 cm soil temperature (Ton (TOC) . Soil moerlands) . Soil TNerlands) . Soil TOerlands) .F (mg·m−2·h−1) is the gas flux, dc/dt is the slope of the linear regression for the gas concentration gradient over time, M (g·mol−1) is the molecular mass of gas, P (Pa) is the atmospheric pressure in sampling site, T (k) is the temperature inside the chamber during sampling, H (m) is the height of chamber, V0 (m3·mol−1), P0 (Pa) and T0 (k), are the gas mole volume, atmospheric pressure under standard conditions, and absolute air temperature, respectively. A positive F-value means that there is a net emission of gas, and a negative value is the opposite.The gas flux was calculated according to the following equation :(1)F=dcdFCO2, FCH4 and FN2O represent the greenhouse gas emission flux during the monitoring period (t·hm−2), 25 and 298 are the conversion factors of CH4 and N2O, respectively (for a 100 timeframe), to present GWP in t CO2 Eq·ha−1.The global warming potential (GWP) was calculated according to :(2)GWP=10 [F is the gas flux, T is the soil temperature, coefficient a is the intercept of soil respiration when temperature is zero, coefficient b represents the temperature sensitivity of soil respiration, Q10 is the temperature sensitivity.The following equation was established to calculate the temperature sensitivity of gas flux to the changes of T10 ,32:(3)F=The differences in soil environment factors among all forests were compared using one-way ANOVA and Tukey’s tests. The relationships of the soil greenhouse gas fluxes with soil temperature and moisture were assessed by regression analysis. The correlations between soil greenhouse gas fluxes and physical and chemical properties were analyzed by Pearson correlation with two tails. SPSS 20.0  was used for statistical analysis. All figures were drawn using OriginPro 2016 software .2 fluxes in the three forest types in the permafrost region of Daxing’an Mountains were basically exactly the same , while emissions were relatively low during the non-growing season (average flux 57.92 ± 29.13 mg·m−2·h−1). PF soil CO2 emissions reached a maximum in July (650.93 ± 28.12 mg·m−2·h−1), and LF and BF reached the maximum emissions in August . PF, LF, and BF reached minimum fluxes of 7.92 ± 1.3 mg·m−2·h−1 (February), 10.27 ± 3.92 mg·m−2·h−1 (February), and 15.63 ± 2.31 mg·m−2·h−1 (March), respectively. The average annual flux of CO2 in three forest types followed PF (291.76 mg·m−2·h−1) > LF (273.84 mg·m−2·h−1) > BF (243.29 mg·m−2·h−1). The soil CO2 fluxes in coniferous forest were higher than in broad-leaved forest, and PF soil CO2 cumulative fluxes were significantly higher than BF , while it was August for BF (−57.71 ± 4.65 ug·m−2·h−1). The peak fluxes from PF and BF occurred in February , and in January for LF (32.51 ± 13.03 ug·m−2·h−1). The average annual flux of CH4 in LF, PF, and BF were −19.33 ug·m−2·h−1, −24.59 ug·m−2·h−1, and−30.46 ug·m−2·h−1, respectively. The soil CH4 absorption fluxes in broad-leaved forest were higher than in coniferous forest, and BF soil CH4 cumulative absorption fluxes were significantly higher than LF  and PF (31.1 ± 2.92 ug·m−2·h−1) were recorded in June, and those for BF (24.91 ± 2.8 ug·m−2·h−1) in October. In contrast, the minimum absorption of N2O fluxes were recorded in January  and March (BF: −2.17 ± 2.13 ug·m−2·h−1), respectively. The average annual flux of N2O for PF (13.33 ug·m−2·h−1) were higher than LF (11.8 ug·m−2·h−1) and BF (11.45 ug·m−2·h−1). The soil N2O fluxes in coniferous forest were higher than in broad-leaved forest.The soil N016~2018 c. The so2 accumulation fluxes of three forest types in the permafrost region of the Daxing’an Mountains dominated, followed by N2O and CH4 as absorption fluxes , the coefficient of variation of the regression model ranged between 0.22 and 0.89. The Q10 values of CO2 fluxes in LF, PF, and BF were 5.47, 3.67, and 4.06, while those for N2O were 2.23, 1.82, and 1.49, respectively.Soil temperature and moisture directly or indirectly affected the production and release of greenhouse gases. The correlations between gas fluxes and soil temperature were higher for CO2 fluxes had a significant negative correlation with TN (P < 0.05). The soil CH4 fluxes exhibited significant correlation with TOC in LF and BF (P < 0.01) and had a significant positive correlation with TN in LF (P < 0.05). The soil N2O fluxes showed a significant positive correlation with NH4+-N and NO3−-N in LF, NO3−-N in PF (P < 0.05), and significant positive correlation with soil pH, NO3−-N and TN in BF (P < 0.01).The Person’s correlation analysis between the greenhouse gas fluxes and various environmental factors is showed in 2 emission fluxes in typical forest LF, PF, and BF in the Daxing’an Mountains were similar, and reached a peak during the growing season , heterotrophic respiration , and mineralization decomposition of organic matter. The vegetation in this study area is mostly a shallow root system. During summer, with gradually increasing temperature, the appropriate combination of water and heat in the soil drives root respiration and soil microbial activity, which promotes soil CO2 to reach emission peaks. Previous studies reported that the seasonal change of CO2 emissions were dominant controlled by soil temperature [2 fluxes as the dominant environmental variables in affect CO2 emissions  are more sensitive than BF (Q10 = 4.06) and PF (Q10 = 3.67) to temperature changes, which is consistent with the research results of Zheng et al. [2 fluxes indicates strong positive short-term feedback between climate warming and soil CO2 fluxes.This study showed that the seasonal variation of COperature ,35. Thismissions a. During reduced , thus re changes . The Q10.33~5.53 . The LF g et al. . The sig4 is produced by a biogeochemical cycle. It is oxidized by methanotrophs at the soil–water interface or rhizosphere aerobic environment, and the remainder is released into the atmosphere [4 as trace gas has both absorption [4 fluxes; therefore, the difference in hydrothermal conditions under different ecosystems leads to different soil CH4 fluxes [4 showed overall absorption. Soil CH4 emission, with maximum emission fluxes, only occurred in winter (January or February), and the other seasons showed absorption with the maximum absorption fluxes appearing in June , soil N2O fluxes showed peak emission periods. Thomas et al. [2O, which is sequestered by the frozen soil [2O enclosed in the soil is burst out into the atmosphere after thawing. In summer, plants grow vigorously and absorb a large amount of nitrogen. Competition between vegetation and microbes causes soil microbes to utilize substrate reduction, and frequent rainfall during the summer causes the shallow soil to alternate between wet and dry conditions, all of which affects the soil N2O emission rate [2O fluxes were higher than LF and BF. The available nitrogen (NH4+-N and NO3−-N) contents of different forest types showed that BF was significantly higher than both PF and LF (P < 0.05), and the NH4+-N content was significantly higher than the NO3−-N content (P < 0. 05)   . Therefo < 0.05) . Accordi(1)2 and N2O and a “sink” of CH4. The three greenhouse gas fluxes showed strong temporal variety, while the fluxes varied depending on the different sites. The PF soil CO2 fluxes were significantly higher than BF. At the same time, the soil absorption fluxes of CH4 in BF were significantly higher than that in LF.The typical forest soil of the Daxing’an Mountains permafrost region was a “source” of CO(2)2 and CH4 fluxes of different forest types in this high-latitude permafrost region. Q10 values showed that LF soil greenhouse gas fluxes were more sensitive to temperature. The N2O fluxes were mainly correlated by the soil nitrogen content.Soil temperature and moisture were key environmental factors that correlated the CO(3)2 and N2O emission rates of the three forest types increased with increasing temperature, and the CH4 absorption rate decreased, thus enhancing the atmospheric greenhouse effect. On a 100-year time scale, the greenhouse gas GWP of the three forest soil systems in the Daxing’an Mountains permafrost region was positive, which had positive feedback on global warming.Against the background of climate warming, the CO2, CH4, and N2O fluxes in the study area had significant emission potential. Temperature alteration leads to complex changes in hydrothermal conditions that either directly or indirectly affect forest ecosystems in the cold region, which transforms ground-gas exchange ratios to affect greenhouse gas fluxes. In the future, stable isotope tracing and microbial high-throughput sequencing technologies will be comprehensively used to analyze the mechanism of soil greenhouse gas accumulation, conversion, and transmission in permafrost region.The Daxing’an Mountains permafrost region is extremely sensitive to climate change. The CO


The effects of cupric oxide dosage, reaction time and initial pH value were investigated in batch experiments and a plausible mechanism was proposed. The results showed that LA decarboxylation over cupric oxides at around 300 °C under acidic conditions produced the highest yield of butanone (67.5%). In order to elucidate the catalytic activity of cupric oxides, XRD, AFM, XPS and H The surface roughness (Rms) of CuO(I) and CuO(II) was 123.32 nm and 40.75 nm, respectively. The thermal desorption of hydrogen (H2) adsorbed on CuO was investigated first to determine their reduction temperatures. A color change from black (CuO) to dark yellow (Cu2O) to metallic-brown (Cu) was observed during their reduction process. The TPR profiles are illustrated in 2+ occurred by a two-step mechanism:CuO(I) was prepared by precipitation of copper nitrate  solution with sodium hydroxide (NaOH). The precipitate was washed with deionized water and then dried in a vacuum oven at 50 °C for 4 h. The dried sample was calcined in a muffle furnace at 800 °C for 2 h. CuO(II) was prepared by mixing stoichiometric amounts of oxalic acid and copper acetate in distilled water. The resultant blue solid (CuC2O4) was washed with deionized water and then dried in a vacuum oven at 70 °C for 4 h. The prepared sample was ground and subsequently calcined in a muffle furnace at 800 °C for 2 h. CuO(I) and CuO(II) were used in LA decarboxylation and further analyses.CuO(I) was prepared by precipitating copper nitrate [Cu(NO2PO4-NaOH (0.2 mol/L) solution and the initial pH of the reaction solution was adjusted with NaOH (0.2 mol/L). The total volume was 60 mL. Then a metal oxide or metal-oxides mixture was added into the prepared reaction solutions. After the reactions were completed, the samples were taken with 5 mL pipette and directly transferred to a 20 mL headspace vial for GS-GC detection. In view of the volatility of products, three samples were taken and the average value of the data was recorded. And the samples were filtered through a 0.25 μm membrane filter for ion chromatography detection.All experiments were carried out in a 4576-HP/HT Pressure Reactor . Reaction solutions were obtained by adding levulinic acid (0.01 mol) into KHHead space (HSS 86.50)-gas chromatography  (HS-GC) was used extensively in the separation and identification of mixtures of volatile compounds. LA decarboxylation samples were analyzed with a HS-GC using a DB-5 column (30 m × 0.25 mm i.d. × 0.25 μm film) at a column temperature of 120 °C, a flow rate of 4.7 mL/min and with a flame ionization detector at the temperature of 220 °C. The analysis was performed at 60 °C for 5-min. The component concentrations were calculated by the area normalization method. The sample vials was shaken for 20 min at 75 °C. The temperatures of sampling probe and tube were 85 °C and 95 °C, respectively. Automatic sampling was employed. The sample analysis was confirmed by the head space-gas chromatograph-mass spectrometer technique (HS-GC-MS). Some samples were detected with a HS-GC-MS (Agilent 6890-5975) equipped with a DB-5 column (30 m × 0.25 mm i.d. × 0.25 μm film). The temperature program was used: 60 °C –(5 min) −10 °C/min −120 °C. The carrier gas was helium with a flow rate of 1.0 mL/min and the mass spectrometer used an electron impact (EI) ionization mode with 70 eV of electron energy. The HS-GC of samples shown in Ion chromatography  was used for the analysis of levulinic acid in the aqueous phase to evaluate the conversion of levulinic acid. Liquid reactant after the reactions was filtered and diluted before testing. The analysis was conducted in an anion-exchange column (AS11-HC) with 2 mol/L NaOH leacheate in a conductivity cell of 35 °C. The flow rate was 1.0 mL/min and the sample size was 50 μL. The calibration was performed using aqueous solutions. An ion chromatogram of a sample is shown in The grain size of CuO powders was characterized by X-ray diffraction (XRD)   using the standard reflection mode with Cu/Ka (λ = 0.154056 nm) in the angle 20° < 2θ < 70° and step angle was 0.02°.The surface morphologies of CuO powders were observed by atomic force microscope (AFM)  using tapping mode with an RTESP cantilever.To determine the surface compositions of CuO, X-ray photoelectron spectroscopy (XPS) was carried out with an Axis Ultra (DLD) spectrometer  using Al Kα source and Mg Kα source. Binding energy was calibrated with hydrocarbon- contaminated C1s peak at 284.6 eV at ambient temperature.2 (10 vol.%)/Ar. The amount of H2 uptake during the reduction was measured by a thermal conductivity detector (TCD). The water produced during the reduction was trapped in a 5Å molecular sieve column.The temperature programmed reduction (TPR) measurement of CuO powders was carried out using an AutoChem II 2920 Chemisorb system from room temperature to 500 °C at a heating rate of 10 °C/min by flowing 50 mL/min of Hetc). An acidic system helped to facilitate the oxidative decarboxylation of LA. At pH 3.2, the butanone yield reaches 67.5%.It is shown based on above discussions that butanone can be produced through oxidative decarboxylation of biomass–derived levulinic acid with CuO. CuO itself was reduced to its elemental form (Cu). The LA decarboxylation is the result of a combination of CuO’s oxidability, structure  and external conditions (temperature, pH,


They both crystallize in the common RbFe(HPO4)2 structure type (Rc).The crystal structures of hydrothermally synthesized  ammonium gallium bis[hydrogen arsenate(V)], (NH4)Ga(HAsO4)2, and thallium aluminium bis[hydrogen arsenate(V)], TlAl(HAsO4)2, were solved by single-crystal X-ray diffraction. Both compounds crystallize in the common RbFe(HPO4)2 structure type (Rc) and share the same tetrahedral–octahedral framework topology that houses the M+ cations in its channels. One of the two Tl sites is slightly offset from its ideal position. Strong O—H⋯O hydrogen bonds strengthen the network.The crystal structures of hydrothermally synthesized ( These result in topologies with several interesting properties such as ion exchange (Masquelier M+–M3+–O–(H)–As5+ , which led to an unusually large variety of new structure types 2 type , was found to exhibit a large crystal–chemical flexibility, which allows the incorporation of a wide variety of M+ and M3+ cations. Previously, it was also known for the phosphate members RbAl(HPO4)2 and RbGa(HPO4)2 Ga (this work), RbIn, RbGa, RbAl, RbFe, CsIn and CsFe 2 structure type Fe(HPO4)2 type (P4)3M+(HTO4)2  compounds crystallize 6 type 2 type 2 has the smallest unit cell of all the arsenates of this structure type published to date. Still, the size of the M+-hosting voids seems to be too large for the Tl+ cation, since Tl1 is slightly offset from the ideal position at 0, 0, 3/4  and there are minor, but distinct negative and positive residual electron densities close to the Tl2 atom. The latter is severely underbonded, with a very low bond-valence sum (BVS) of only 0.54 valence units (v.u.) 6  Å, Table 2TlAl(HAsO4)Ga(HAsO4)2, the bond-valence sum values for the M3+ cations and As are quite similar  and numbers for As that are close to the expected values . The NH4+ cations (average N⋯O = 3.268 Å for N1 and 3.336 Å for N2) seem to fill the M+-hosting voids much better, and the BVSs 2, the vessels were filled with distilled water to about 70% of their inner volumes, which led to initial and final pH values of 1 and 0.5, respectively, and the synthesis was allowed to proceed at 493 K for 9 d. (NH4)Ga(HAsO4)2 was grown over a period of 7 d and the initial and final pH values were 3 and 1, respectively. The reaction products were washed thoroughly with distilled water, filtered, and dried at room temperature. (NH4)Ga(HAsO4)2 formed large colourless pseudo-octahedral crystals 2 formed small pseudo-hexagonal platelets. Both compounds are stable in air.The compounds were grown by hydrothermal synthesis at 493 K  using Teflon-lined stainless steel autoclaves with an approximate filling volume of 2 cmls Fig. 3, while T4)Ga(HAsO4)2 was deposited at the International Centre for Diffraction Data under PDF number 00-059-0055 Ga(HAsO4)2 were undertaken to discriminate between H3O+ and NH4+. They confirmed the suspected formula and revealed no impurities.Semiquantitative SEM–EDX analysis (15 kV) of carbon-coated, horizontally oriented crystals of 2 Ga(HAsO4)2, several electron-density peaks between 0.4 and 0.75 e Å−3 were recognizable that could be attributed to the H atoms of the NH4+ cation. These peaks are located at the following coordinates for the N1 atom: 0.0170, 0.1329, 0.7450; 0.0641, 0.0560, 0.7414 and −0.0910, 0.0000, 0.7500. For the N2 atom, the coordinates are: 0.0478, −0.0330, 0.6635; −0.0655, −0.1106, 0.6786; 0.1301, 0.0094, 0.6695 and −0.0521, −0.0657, 0.6513. However, despite the use of restraints, no sensible coordination geometry for the H atoms around the N atoms could be found. Therefore, they were omitted from the model. As a result of the fact that there are 12 possible N—H⋯O bonds for each N atom, with only two symmetry-equivalent positions for N1 and four for N2, it seems reasonable to assume that the H-atom positions around the N atoms are, in both cases, highly disordered. The final residual electron density in (NH4)Ga(HAsO4)2 is < 1e Å−3.For the refinement of both compounds, the coordinates of RbFe(HPO4)2 revealed a considerable residual electron-density peak of 2.2 e Å−3 1.28 Å away from As and 1.61 Å away from the O1 site. The corresponding position can be generated by a mirror plane in (110) and therefore could be an alternative flipped As position (sharing the same O1 atom). Since the inclusion of the alternative position led to a considerable drop in R1 and weighting parameters and the highest residual electron density dropped to < 1 e Å−3, this position was kept in the model. The occupancy of the alternative position AsB  refined to only 2.1%, which makes it impossible to locate the alternative O ligand positions that should comprise the coordination sphere of the AsB position. For the final refinement, the displacement parameters of the AsB position were restrained to be the same as for the main As position and the sum of As was restrained to give a total occupancy of 1.00. We note that a similar alternative position was also found for isotypic CsIn(HAsO4)2 6 , possibly in part or in whole due to the stereochemical activity of the lone electron pair on the Tl+ cations. So, although the Tl1 site is slightly offset from its ideal position , we unfortunately did not manage to get rid of the negative residual electron density of about −2 e Å−3 next to Tl2. The most positive residual electron density peak, however, dropped to < 1 e Å−3.There was also considerable residual electron density of ±2 e Å 10.1107/S2056989018013567/pk2608sup1.cifCrystal structure: contains datablock(s) NH4GaHAsO42, TlAlHAsO42. DOI: 10.1107/S2056989018013567/pk2608NH4GaHAsO42sup2.hklStructure factors: contains datablock(s) NH4GaHAsO42. DOI: 10.1107/S2056989018013567/pk2608TlAlHAsO42sup3.hklStructure factors: contains datablock(s) TlAlHAsO42. DOI: 1869299, 1869298CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


We found that BDMC33 significantly inhibited PGE2 synthesis in a concentration-dependent manner albeit at a low inhibition level with an IC50 value of 47.33 ± 1.00 µM. Interestingly, the PGE2 inhibitory activity of BDMC33 is not attributed to inhibition of the COX enzyme activities, but rather BDMC33 selectively down-regulated the expression of COX-2. In addition, BDMC33 modulates the COX expression by sustaining the constitutively COX-1 expression in IFN-γ/LPS-treated macrophage cells. Collectively, the experimental data suggest an immunodulatory action of BDMC33 on PGE2 synthesis and COX expression, making it a possible treatment for inflammatory disorders with minimal gastrointestinal-related side effects.Our preliminary screening had shown that the curcumin derivative  or BDMC33 exhibited improved anti-inflammatory activity by inhibiting nitric oxide synthesis in activated macrophage cells. In this study, we further investigated the anti-inflammatory properties of BDMC33 on PGE BDMC332,6-biscyclohexanoneCOXcyclooxygenaseDMSOdimethyl sulfoxideDXMdexamethasoneFBSfoetal bovine serumHRPhorseradish peroxidaseIFN-γinterferon-gammaLPSlipopolysaccharideNOnitric oxide2PGE2prostaglandin ENSAIDnon-steroidal anti-inflammatory drugRAW 264.7murine macrophage cells2, which regulates several responses in the humans such as the reproductive, gastrointestinal, neuroendocrine, and immune systems ..13].L-glutamine (2 mM), streptomycin (50 μg/mL) and penicillin (50 U/mL) at 37 °C and 5% CO2. When RAW 264.7 cells reached confluency of 80–90%, the cells were scraped out and centrifuged at 110 × g in 4 °C for 10 min. The cell viability of cultured cells used in the assay was always >95% as determined by trypan blue dye exclusion.The murine macrophages-like cell line (RAW 264.7) from the European Collection of Cell Cultures  were maintained in DMEM supplemented with 10% FBS, 4.5 g/L glucose, sodium pyruvate (1 mM), 5 cells/well) were seeded into a tissue culture grade 96-well plate except for blank and incubated for 2 h at 37 °C, 5% CO2 for cell attachment. The attached cells were activated with 100 U/mL of recombinant IFN-γ and 5 μg/mL of LPS with or without presence of BDMC33 at a final volume of 100 μL/well. DMSO was used as vehicle to add BDMC33 into the culture medium and the final concentration of DMSO was 0.1% in all cultures. Cells were then incubated at 37 °C, 5% CO2 for 17–20 h. The level of PGE2 was determined by using PGE2 EIA kit.RAW 264.7  were added into each well. The cells were incubated in 37 °C for 4 h and the formazan salts were dissolved by adding 100% DMSO. The absorbance was then measured at 570 nm on a SpectraMax Plus Microplate reader .2 secretion PGE2 EIA kits . The protocols provided by the manufacturers were followed to the detail. The data was acquired using a SpextraMax Plus microplate reader . The concentration of PGE2 for each sample was calculated from their respective standard curves.The cell culture supernatants were collected and analyzed for PGE5 cells/well) were seeded into a tissue culture grade 96-well and triggered with IFN-γ/LPS for 6 h. Then, the spend media were removed and washed three times with 200 µL of media without IFN-γ/LPS. The 50 µL of serial concentration diluted BDMC 33 was added into the plate and incubated for a further 30 min in 37 °C. The exogenous substrate of arachidonic acid (50 µL) added for 15 min to produce sufficient substrate for measuring COX-2 enzyme activities. Lastly, the PGE2 concentration of supernatants was determined by PGE2 EIA kit.RAW 264.7 cells  and incubated in ice for 30 min. Following incubation, the cells were sonicated at 20 Hz for 30 s. Then the cells were incubated for 20 min on ice and centrifuged at 25, 150 × g at 4 °C for 30 min. The supernatants collected and protein content was measured using Bradford assay. In order to determine the expression of COX, equal amounts of protein (20 μg) were electrophoresed in a 10% SDS-PAGE and blotted onto a PVDF membrane (Amersham). Membrane were blocked for 1 h at room temperature in blocking buffer (0.3% BSA in PBST) and then incubated with primary antibodies for 1 h. The primary antibodies were used: rabbit polyclonal antibodies against COX-1 , COX-2  and HRP conjugated β-actin . After several washes with PBST, HRP-conjugated donkey anti-rabbit IgG  were added for further 1 h incubation. The proteins were detected by using ECL blotting reagent according to the manufacturer’s instruction. The image captured using VersaDoc Imaging Device  and relative volumes of bands were measured by Quantity One software.RAW 264.7 cells were induced with IFN-γ/LPS as described earlier and treated with different concentrations of BDMC33. To prepare the whole cell extract of RAW 264.7, the cells were scraped out from culture flask and washed three times with ice-cold Tris-sucrose washing buffer. Then, the cell pellets were resuspended in lysis buffer . Statistical significance of differences between groups was accepted at P < 0.05.Statistical analysis were performed using one–way analysis of variance (ANOVA) followed by Dunnett test 2 inhibition and differential expression of COX isoforms in IFN-γ/LPS-treated macrophage cells. The immunomodulatory effect of BDMC33 denotes a possible use as a future anti-inflammatory agent with minimal gastro-related side effects. However, further studies are needed to identify the molecular target(s) and signaling pathway that contribute the anti-inflammatory activities of BDMC33 on differential expression of COX isoform in macrophage cells. In conclusion, we have demonstrated the immunomodulatory action of BDMC33 on PGE