In the central nervous system, cPLA2 activation is implicated in the pathogenesis of various neurodegenerative diseases that involves neuroinflammation, thus making it an important pharmacological target. In this paper, a new class of arachidonic acid (AA) analogues was synthesized and evaluated for their ability to inhibit cPLA2. Several compounds were found to inhibit cPLA2 more strongly than arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor that is commonly used in the study of cPLA2-related neurodegenerative diseases. Subsequent experiments concluded that one of the inhibitors was found to be cPLA2-selective, non-cytotoxic, cell and brain penetrant and capable of reducing reactive oxygen species (ROS) and nitric oxide (NO) production in stimulated microglial cells. Computational studies were employed to understand how the compound interacts with cPLA2.Cytosolic phospholipase A The mammalian PLA2 family comprises six main types of enzymes, namely the secreted small molecular weight sPLA2, the larger cytosolic Ca2+-dependent cPLA2, the Ca2+-independent iPLA2, the platelet-activating factor acetylhydrolases PAF-AH and the lysosomal PLA2. Of these PLA2 enzymes, the ubiquitous cPLA2 is found to be responsible for the release of arachidonic acid (AA), an important PUFA which serves as the precursor for the synthesis of eicosanoids and prostanoids that mediate a wide variety of inflammatory responses2. In the central nervous system, cPLA2 activation has been implicated in the pathogenesis of a number of neurodegenerative  and neuropsychiatric (schizophrenia and depressive disorder) diseases7. Effects of neuroinflammation and its involvement in nitrosative/oxidative signaling pathways for example have been frequently cited as a pathogenetic link to Aβ-amyloid production due to the actions of cPLA28. A direct effect of sustained and aberrant cPLA2 activation could result in enhanced neural membrane destruction eventually compromising functions on membrane ion receptors, leading to cell death7. Thus cPLA2 is an important target for drug discovery, and for the development of new therapeutics to treat neurodegenerative and neuropsychiatric disorders. An ideal cPLA2 inhibitor should not only reach the site where inflammatory processes are taking place (this may be achieved through improved drug delivery systems), they should also inhibit inflammation, block oxidative stress and cross the blood-brain barrier (BBB). To-date, various compounds have been shown to inhibit cPLA2 selectively16. However many of these compounds have not yet been shown to possess brain penetrability18. Thus as part of our efforts to develop cPLA2 inhibitors as potential drug candidates for the treatment of neurological disorders, we have synthesized a new series of AA analogues 1–2 and evaluated them for their PLA2 inhibitory activities and ability to cross the BBB. In our inhibitor design, we have focused on the arachidonyl scaffold as earlier studies have shown that this pharmacophore is strongly recognized by cPLA223. We herein present the synthesis of AA analogues 1–2 and the investigation of these compounds for their (i) inhibition of cPLA2, (ii) cytotoxicity, (iii) selectivity and anti-neuroinflammatory properties and (iv) ability to cross the blood-brain barrier; finally computational studies were performed to understand how the compounds bind to cPLA2.Phospholipases A2 inhibitor, AACOCF3. Being cell penetrant, AACOCF3 has been effective in reducing the undesirable effects of dysregulated cPLA2 systems, including those found in neurological diseases26. A series of compound with the CF3 moiety was synthesized and presented as compound 1.Using AA as a scaffold, our inhibitor design involved modification at different selected positions of the compound Fig. . In part1 was prepared by assembling the alkynes together through a series of coupling and bromination reactions according to the synthetic strategy shown in Fig. 30. A total of 3 coupling and 2 bromination reactions were performed to reach the skipped diynes 8a–e  and 10a–c . Compound 8a was initially hydrogenated with Brown’s P2 nickel catalyst in ethanol according to a published procedure27. However this did not provide the desired compound 9a. Optimization of the hydrogenation reaction by varying the equivalent of the catalyst and reaction conditions eventually provided 9a in 68% yield (Supplementary Table 9a by stirring TMSCF3 and CsF as a TMS activator with the ester in CHCl3 at room temperature did not yield 1a (AACOCF3)31. Replacing CsF with TBAF also failed to provide the desired compound32. We attempted to convert 9a to the corresponding carboxylic acid and then treating it with LDA and EtO2CCF333. However this too did not yield 1a. Finally we trifluoromethylated the intermediate carboxylic acid with pyridine and TFAA35 and gratifyingly this gave 1a in 48% yield (over 2 steps). Analogues 1b–1h were synthesized in the same manner by varying alkyne 3 in the first step of the reaction  was used as the additive but it gave 13b in only 20% yield. Hence we tried hexamethylphosphoramide (HMPA) which provided 13b in 86% yield. This may be attributed to the increased polarity of the P=O bond in HMPA as compared to the C=O in DMPU which stabilizes the acetylide to a greater extent. HMPA was thus used as an additive for the synthesis of intermediates 13a–d. To form the primary amide 1m, tetraene 9a was amidated by heating it overnight at 80 °C in a solution of ammonia dissolved in methanol and Mg(OMe)2. The chlorodifluoroketo and difluoroketo functionalities in 1n and 1o were respectively synthesized by hydrolyzing the ester 9a to the corresponding carboxylic acid followed by a reaction with the respective anhydrides in pyridine and dichloromethane at room temperature  at a standard 10 μM concentration. Potential inhibitors were determined by their ability to lower cPLA2 activity to a greater extent than 1a.Table 1a–1c and 1i–1l), varying the tail length did not provide a significant improvement in cPLA2 inhibitory activity as compared to AACOCF3. In fact, increasing the alkyl chain length from C5 (1i) to C13 (1l) resulted in a significant decrease in cPLA2 inhibition. Replacing the alkyl tail with a phenyl moiety provided 1d which showed the highest cPLA2 inhibitory activity in the trifluoromethylketone series of compounds. This could be attributed to the additional interactions with cPLA2 (vide infra).From Table 1a to be light and oxygen sensitive36. We hypothesized that reducing the number of skipped alkenes on the backbone could improve the compound’s stability. Hence analogues with a reduced number of double bonds (Table 1f–l) were synthesized . A plausible explanation could be due to the increased rigidity of the compound with more double bonds, making them less rotatable and hence improving their affinity for the enzyme pocket.The skipped alkene functionality in the arachidonyl backbone causes zed Fig. . However37. To better understand the effects of these methylene groups on the inhibitory properties of the compound, 1e with two methylene groups was synthesized. Results obtained showed that 1e had the same cPLA2 inhibitory activity as 1a . However, reducing the electrophilicity of the carbonyl group by reducing the number of fluorine atoms on the trifluoromethyl moiety did not result in a more potent cPLA2 inhibitor (Table 1n–1o). Similarly a decrease in cPLA2 inhibitory activity was observed in 1m when the carbonyl was converted into a primary amide. However all the compounds in Table 2) except 1m (43.1 Å2). Since the polar surface area measures the compound’s ability to permeate cells, we decided to explore other analogues of 1m by functionalizing the amide moiety41.Earlier studies have shown that the presence of a trifluoromethylketo moiety confers a very high electrophilicity on AACOCFes Table m–1o. Howet al.42 have shown that site-directed mutagenesis of Arg200 in cPLA2 severely affected its catalytic property while molecular docking and crystallography studies by other groups have indicated that Arg200 plays a stabilizing role by interacting with the phosphate group on phosphatidylcholine or the carboxylate groups on cPLA2 inhibitors44. Through a combination of deuterium exchange mass spectrometry and modeling, Burke and co-workers have also reported the interaction of Arg200 with cPLA2 inhibitors containing carboxyl groups45. We thus postulated that functionalizing the amide moiety of 1m with a negatively charged moiety that interacts with Arg200 could improve the compound’s cPLA2 inhibitory activity. Hence various analogues containing an alkyl extension ending with either a carboxylic acid, ester, sulfonamide or amide functionality were synthesized , suggesting that four methylene groups were optimal between the amide and carboxyl groups. To determine if the interaction between the carboxylic acid functionality and Arg200 was responsible for increasing the compound’s cPLA2 inhibitory activity, 20c containing an ester instead of a carboxylic acid was also prepared. A significant decrease in activity was observed in 20c, thus confirming the importance of the carboxylic acid moiety.We started the investigation by synthesizing 1 that this effect is insignificant, the presence of a carboxyl-containing extension in 2 would result in a longer molecule than 1, which could alter the interactions between 2 and the putative binding pocket in cPLA2. Comparison of the activities of 2b, 2g, 2k and 2q  between the first double bond and the carbonyl group being the optimal number.Next, we directed our efforts to determine the effect of the number of methylene groups between the first double bond and the carbonyl group. Although it was observed in our earlier studies of compound 2d), para- (2n) and ortho- (2o) carboxylic acid were synthesized. 2d exhibited the best cPLA2 inhibition, providing a 2.5-fold increase in activity. When the carboxylic acid moiety was replaced with an amide or sulfonamide .Varying the alkyl tail of 1a, 2g and 2i for 48 h and 72 h 46 was used as a reference and produced a 62% reduction of sPLA2 activity. 2i did not provide a significant change in sPLA2 activity, indicating that it could not inhibit sPLA2. However, 2g showed a significant increase of sPLA2 activity to 184%, suggesting that it could possibly be a sPLA2 activator, thereby disqualifying it as a selective cPLA2 inhibitor. With the elimination of 2g, only 2i was assessed for its ability to oppose microglia activation via cPLA2 inhibition .To further explore their selectivity towards cPLAsay Fig. . Thioeth48. Recent studies have shown that BV-2 microglial cell could serve as an in vitro model to mimic such neuroinflammatory states when stimulated with lipopolysaccharide (LPS)49. LPS activates BV-2 cells by triggering a cascade of inflammatory events which includes the production of NO. This event is characterized by the generation of the biomarker, inducible nitric oxide synthase (iNOS), as well as ROS. Chuang et al.50 have also shown that cPLA2 is a major contributor in this pathway and inhibiting this enzyme reduces the production of the neurotoxic mediators and provide neuroprotective effects. Therefore to investigate the ability of 2i in inhibiting cPLA2 in a neuroinflammatory model, BV-2 cells were treated with LPS (2 μg/mL), incubated for different time points, collected and lysed for protein extraction. Western blots conducted showed that iNOS level was highest between 8–14 h  were incubated together with LPS in the BV-2 cells for 14 h  and nitric oxide (NO), produced during microglia activation, contribute to inflammatory response and cytotoxic damage to the surrounding neurons and neighbouring cells4 h Fig. . Densito4 h Fig. . The cPLint Fig. . Next, 24 h Fig. . The LPS2i  were incubated with the BV-2 cells in the presence of 2 µg/mL LPS for 14 h, a dose-dependent decrease in ROS was observed  disorder is its ability to pass through the BBB to reach the therapeutic target. Hence screening for its ability to penetrate the BBB is of great importance.53 have demonstrated that the parallel artificial membrane permeation assay (PAMPA)53 assay provides good prediction of in vivo BBB permeability and is a useful tool to screen compounds for brain penetration. Thus to explore whether 2i is able to penetrate into the brain, we used PAMPA with porcine brain lipids as the lipid barrier. Commercially available and highly potent cPLA2 inhibitors, CDIBA 12 and pyrrophenone, were also evaluated for their ability to penetrate the BBB. The effective permeability (Pe) of 2i, CDIBA and pyrrophenone were found to be 12.34 ± 1.46 × 10−6, 3.98 ± 0.24 × 10−6 and 2.00 ± 0.05 × 10−6 cm/s . Pe values of reference compounds determined under similar conditions were of the order propranolol > carbamazepine > quinidine > caffeine > dopamine, which agreed with reported literature55. A minimum Pe of 7 × 10−6 cm/s has been cited as the threshold for permeability across the blood brain barrier56. As the Pe of 2i exceeded this value, we are optimistic that 2i has the potential to transverse the BBB. We also found good aqueous solubility  for 2i at pH 7.4. Taken together, the promising physicochemical profile of 2i warrants continued attention on this compound as an inhibitor of cPLA2.Earlier studies2g and 2i on the cPLA2 enzyme. The crystal structure of cPLA2 in its apo form  and with a few missing regions was obtained from the protein data bank57. The missing regions were modelled and the complete structure was subjected to molecular dynamics (MD) simulations (as outlined in Methods). The complete model of cPLA2 remained stable during the simulation. The conformations sampled during the last 50 ns of the MD simulations were clustered into conformational sub-states using the Kclust program from the MMTSB tool set, with an rmsd of 2 Å set as cutoff. The cluster centroids of the top 5 most populated clusters were used for docking calculations.A docking study was performed to rationalize the inhibitory activities and to identify the possible binding sites of 2 binding site around the catalytically important residue Ser22844. This binding site was shown to be highly negatively charged on one end and slightly positively charged on the other end. Both these charged ends are connected by a 22 Å long, narrow tunnel that is made up of hydrophobic amino acids . Analysis of the top scored solutions (lowest energy solutions) showed that all the compounds bind similarly in the active sites of cPLA2 with (i) the electrophilic carbon of the substituted ketone in 1 or amide in 2 being in close proximity to Ser228, and (ii) Gly197/Gly198 interacting with the carbonyl oxygen of 1 or the amide oxygen of 2. In addition, docking analysis of 2 shows the carboxylic acid moiety interacting with the side chains of Arg200. For the aminobenzoic acid analogues 2d and 2n, the carboxylic acid functionality is in closer proximity to Arg200 when it is in the meta position than the para position  and 1 μL of 1 mM of the respective compound dissolved in DMSO. The final concentration of the test compound achieved is 10 μM. The reaction was initiated by adding 50 μL of the substrate solution and incubating at room temperature for 1 h. Fluorescence was measured using a Varioskan plate reader  by exciting at 485 nm, while fluorescence emission was detected at 515 nm. Positive control was performed when 1 μL of test compound was replaced by vehicle DMSO. % activity was expressed as the mean value of duplicate wells from three experiments. % activity was calculated by the following formulaThe assay was conducted according to the manufacturer’s instruction  with slight modification. Briefly, substrate solution was separately prepared by mixing 5 μL of DOPG, 5 μL of DOPC and 5 μL of substrate respectively in 1.5 mL of 1X phospholipase A2 reaction buffer were added into each well of a 96-wells plate. This was followed by addition of 1 μL of recombinant cPLA2  and 1 μL of the respective concentration of the test compound dissolved in DMSO. The reaction was initiated by adding 50 μL of the substrate solution and incubating at room temperature for 1 h. Fluorescence was measured using a Varioskan plate reader by exciting at 485 nm, while fluorescence emission was detected at 515 nm. Positive control was performed when 1 μL of test compound was replaced by vehicle DMSO. IC50 was expressed as the mean value of duplicate wells from three experiments.The assay was conducted as described above. Instead of adding a fixed concentration of the test compound, various concentrations of compound were reconstituted in DMSO. 48 μL of 1X phospholipase A2, it was replaced by sPLA2. 48 μL of 1X phospholipase A2 reaction buffer was added into each well of a 96-wells plate. This was followed by addition of 1 μL of sPLA2, human recombinant type V  and 1 μL of 1 mM of the respective compound or thioetheramide-PC  dissolved in DMSO. The final concentration of the test compound achieved is 10 μM. The reaction was initiated by adding 50 μL of the substrate solution and incubating at room temperature for 1 h. Fluorescence was measured using a Varioskan plate reader by exciting at 485 nm, while fluorescence emission was detected at 515 nm. Positive control was performed when 1 μL of test compound was replaced by vehicle DMSO. % activity was expressed as the mean value of duplicate wells from four experiments. % activity was calculated by the following formula:The assay was conducted as described above. Instead of adding cPLA2 and in a water saturated environment. When performing LPS treatment, BV-2 cells were plated on a 10 cm dish in 10 mL culture medium at a density of 1 × 106 cells per dish one day prior to the experiment. After observing that cells were at 90% confluency, lipopolysaccharide  pre-dissolved in culture medium to achieve a concentration of 2 μg/mL was added to the dish. Different concentrations of test compounds in DMSO were added to the LPS containing medium which was then transferred to the 10 cm dish. DMSO was used as a vehicle control and added to the non-LPS treated control. The cells were incubated at 37 °C, under a 5% CO2 environment. After the respective timepoints, cells were washed once with 1xPBS (10 mL) and scrapped on ice with 8 mL 1xPBS. Collected cells were centrifuged at 800 rpm for 3 min at 4 °C, before discarding the supernatant. Cells were lysed by RIPA buffer supplemented with 1× protease inhibitor (Roche) and 1 mM PMSF and incubated under ice for 30 min. This was followed by pelleting the cell debris at 4 °C for 30 min at 13200 rpm before transferring the supernatant to a clean tube and storing the sample at −20 °C. Three independent biological repeats were performed.Murine BV-2 cells were cultured in DMEM  supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin and 100 units/mL penicillin. Cells were maintained in an incubator at 37 °C, under a 5% CO2 environment in 100 μL of culture medium. Thereafter, the compound of interest dissolved in culture medium at a concentration of 10 μM was replaced into each well. DMSO was added as vehicle in the control group. 3 biological replicates were performed. The plates were then incubated at 37 °C with 5% CO2 for 48 h and 72 h. At each individual time point, 20 μL of CellTiter 96® AQueous One Solution (MTS reagent) was added into each well and re-incubated at 37 °C with 5% CO2 without light for 3 h. Colorimetric readings were taken using a Varioskan plate reader at 490 nm and analyzed. Three independent biological repeats with four technical replicates were performed.Cells were plated on a 96-wells plate and grown for 24 h to a density of 8000 cells per well for both HEK293T and BV-2 cells. Cells were cultured at 37 °C under a 5% COvs. its respective concentration (µg/ml). This was then used to determine the protein concentration for unknown samples.BCA assay  was performed to quantify the concentration of proteins in the cell lysate. 10 µL of test samples (1 µL with 9 µL of lysis buffer) and BSA standards (range from 1.25 to 50 μg/ml) were mixed in a 96 well-plate and incubated with 190 µL of working reagent mixture (Ratio of Reagent A:B used was 50:1) at 37 °C for 20 min. The colour intensity that was developed was read at 562 nm using a spectrophotometer. A standard curve of different concentrations of BSA was plotted using the average blank-corrected readings for each standard 2 primary antibody , mouse monoclonal anti-GAPDH , with secondary horse-radish peroxidase-conjugated antibodies .25 μg of individual protein samples were mixed with an equal volume of 2x Laemmli buffer containing DTT and denatured at 90 °C for 10 min. The lysates were ran in a 10% tris-glycine SDS-PAGE gel at 100 V for 1.5 h in 1x tris-glycine running buffer. The protein samples on the gel were then transferred onto a PVDF membrane. The membranes were then blocked for 1 h at room temperature in 5% skim milk in 1x TBS-0.1% Tween 20 (TBST), followed by overnight incubation with the respective primary antibody dissolved in 5% milk in TBST at 4 °C. Secondary detection were conducted with a secondary horse-radish peroxidase-conjugated antibody (1:10000) dissolved in 5% skim milk in TBST for 1 h at room temperature. Enhanced chemiluminescence (ECL) detection of protein bands was performed using ECL Plus Western Blotting detecting system . Antibodies used in western blot include rabbit polyclonal anti-NOS2 , mouse monoclonal anti-cPLA2 environment. Medium were removed and washed once with 1x HBSS. 100 μL of 10 µM CM-H2DCFDA  in 1x HBSS solution was added into each well and incubated at 37 °C for 1 h. Fluorescence detection was done by exciting the dye at 485 nm and detection of emission at 525 nm. Five independent biological repeats with six technical replicates were conducted.20,000 BV-2 cells were seeded into a 96-well plate in 100 uL of culture medium and allowed to grow overnight. When a 90% cell confluency was observed, lipopolysaccharide pre-dissolved in culture medium to achieve a concentration of 2 μg/mL was added to each well of the 96 well. Different concentrations of test compounds in DMSO were added to the LPS containing medium and was then transferred to the cells. DMSO was used as vehicle control and added to the non-LPS treated control. The cells were incubated for 14 h at 37 °C, under a 5% COSolubility determinations were carried out on Multiscreen® Solubility filter plates (Millipore-MSSLBPC10) from Millipore Corporation  following the protocol (PC2445EN00) from the manufacturer.e) of 2i. The method reported by Di et al.53 was followed with modifications. Determinations were carried out on MultiScreen-IP PAMPA assay (donor) plates (MAIPNTR10) and MultiScreen Receiver Plates (MATRNPS50) from Millipore Corporation (USA) with porcine polar brain lipids  as the lipid barrier.The Parallel Artificial Membrane Permeability Assay (PAMPA) was used to determine the effective permeability  was dispensed into the donor well. A stock solution (5 mM) of 2i prepared in DMSO was diluted with 1x phosphate buffer  to give a 50 µM solution. An aliquot (300 µL) of the solution was dispensed into the donor well and an equal volume of buffer (1xPBS with 1% DMSO) was added to the corresponding acceptor well. The donor and acceptor plates were assembled, the unit was placed in a humidified box and gently agitated on a mini shaker at room temperature (25 °C) for 16 h. After this time, aliquots (200 µL) were withdrawn from the donor and acceptor wells, diluted separately to 1 mL with PBS, and quantified at λmax of 290 nm on a Shimadzu UV-1800 Spectrophotometer.An aliquot (5 µL) of 2% porcine brain lipids in dodecane :A and VD are the volumes of acceptor (cm3) and donor (cm3) wells respectively, A is the area of the surface area of the membrane (0.24 cm2), t is the permeation time (s). The Pe of each compound was obtained from at least 3 separate experiments using 2 different stock solutions. For each independent determination, triplicates (3 wells) were run for each compound.The PAMPA permeabilities of standards  were determined under similar conditions. 300 µL aliquots of stock solutions  were dispensed to donor wells as described earlier. Quantification was by UV at λmax of 272 nm (caffeine), 284 nm (carbamazepine), 330 nm (quinidine), 280 nm (dopamine) and 288 nm (propanolol). Calibration plots of standards were determined under similar analytical conditions. The effective permeability (Pe) of these compounds have been reported to vary in the sequence propranolol (most permeable) > carbamazepine > quinidine > caffeine > dopamine (least permeable)Details on protein modeling, ligand preparation and ligand docking can be found in the supplementary information, available in the online version of the paper.All data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary Information


The mutation Q39K alone was found to be neutral and hardly affected the catalytic properties of β‐lactamases. However, in combination with the key mutations, this substitution resulted in decreased KM values towards hydrolysis of a chromogenic substrate, CENTA. The ability of enzymes to restore catalytic activity after exposure to elevated temperature has been examined. All double and triple mutants of β‐lactamase TEM‐1 bearing the Q39K substitution showed lower thermal stability compared with the enzyme with Q39 intact. A sharp decrease in the stability was observed when Q39K was combined with E104K and M69V. The key R164S substitution demonstrated unusual ability to resist thermal inactivation. Computer analysis of the structure and molecular dynamics of β‐lactamase TEM‐1 revealed a network of hydrogen bonds from the residues Q39 and K32, related to the N‐terminal α‐helix, towards the residues R244 and G236, located in the vicinity of the enzyme's catalytic site. Replacement of Q39 by lysine in combination with the key drug resistance mutations may be responsible for loss of protein thermal stability and elevated mobility of its secondary structure elements. This effect on the activity of β‐lactamases can be used as a new potential target for inhibiting the enzyme.Highly mutable β‐lactamases are responsible for the ability of Gram‐negative bacteria to resist β‐lactam antibiotics. Using site‐directed mutagenesis technique, we have produced  ESBLextended spectrum β‐lactamasesIRinhibitor resistant β‐lactamasesMDmolecular dynamicsRMSDroot mean square deviationBacterial β‐lactamases determine the main mechanism of resistance to β‐lactam antibiotics, the most widely used class of antibacterial drugs http://www.lahey.org/Studies/temtable.asp/). These enzymes have a sequence of 286 amino acids. All TEM‐type β‐lactamases represent mutants of β‐lactamase TEM‐1 that differ from it by several single amino acid substitutions (one to seven). Currently, the mutations are found in 89 positions, five of them located in the signal sequence. Evolution of TEM‐type β‐lactamases and the role of individual mutations are actively studied TEM‐type β‐lactamases belong to the enzymes of molecular class A, most prevalent in Gram‐negative bacteria The variety of TEM‐type β‐lactamases promotes the study of structure–function relationships. Interaction networks were created for understanding the evolutionary patterns of resistance towards β‐lactams, involving all natural and artificial mutations described KM values towards the substrate CENTA and an increase in the thermal inactivation rate. Analysis of the tertiary structure of β‐lactamase TEM‐1 and its mutants performed by computer and MD simulations revealed an increase in the mobility of the site associated with the destruction of a hydrogen bond network.The aim of this work was to carry out experimental and structural studies to clarify the role of the mutation Q39K and its combinations with the key drug resistance mutations on the activity and stability of the enzyme. The Q39K mutation was the first natural mutation found in β‐lactamases E. coli strains DH5α and BL21(DE3) were purchased from Merck . The preparative work with DNA was performed using a Qiagen QIAprep Spin Miniprep Kit and a Qiagen QIAquick Gel Extraction Kit . Protein electrophoresis (SDS/PAGE) was performed according to Laemmli Reagents were purchased from Sigma‐Aldrich  and BD Biosciences  and used without further purification. Enzymes for DNA amplification, restriction and modification were purchased from Agilent Technologies , New England Biolabs  and Thermo Fisher Scientific . Oligonucleotides for sequencing and PCR were purchased from Syntol . The plasmid vectors pET‐20 and pET‐24a(+) and E. coli, ligations and restriction analysis were standard methods E. coli strain DH5α was used for cloning and BL21(DE3) for protein expression. The cells were cultivated in lysogeny broth (LB) medium  supplemented with 50 mg·L−1 of kanamycin.DNA techniques, such as plasmid isolation, transformation of E. coli DH5α cells. The previously developed pET‐bla expression vector PCR amplification was performed with Pfu DNA polymerase by site‐directed mutagenesis E. coli cells were grown in medium containing 1% Bacto Tryptone (BD Biosciences), 0.5% yeast extract, 1% NaCl, 0.1% glucose and 50 μg·mL−1 kanamycin. IPTG was used as an inducer. Cells were grown at 30 °C with stirring (180 r.p.m.) to a value of 0.8–1.2 absorbance units at 600 nm; then they were induced with 0.1 mm IPTG and the cultivation was continued for 5 h. The cells were centrifuged at 3000 g and 4 °C and stored at −20 °C.m EDTA and 10 mm Tris/HCL, pH 8.0, and incubated on ice for 15 min. The resulting spheroplasts were removed by centrifugation at 10 000 g at 4 °C for 15 min. The supernatant was dialyzed against 10 mm Tris/HCL (pH 8.0) and used for further purification. Ion exchange chromatography was performed on a SOURCE™ 15Q column , equilibrated with the same buffer; the recombinant enzyme preparation was eluted with a linear gradient . The estimated purity of all the recombinant proteins was of more than 95%. Fractions containing the active enzyme were stored at +4 °C or frozen at −20 °C.The periplasmic protein fraction was isolated using osmotic shock. In brief, the cells were thawed on ice, resuspended in a buffer containing 20% sucrose, 1 mm sodium phosphate buffer, pH 7.0 at 25 °C using a Shimadzu UV‐1602 spectrophotometer. CENTA stock solution of 4 mm was prepared in 50 mm sodium phosphate buffer, pH 7.0. The hydrolysis of CENTA was monitored by continuous recording of the absorbance at 405 nm (ε405 = 6400 m−1·cm−1). The concentration of β‐lactamase in the assay was 0.01 μm. The reaction was initiated by adding CENTA at concentrations of 20, 50, 100 and 200 μm to the solution containing the enzyme. The measurements were made in triplicate. Apparent Michaelis constants (KM) and kcat were determined using a weighted Lineweaver–Burk linearization. The weights were taken as V04/σ2(V0).Activity of β‐lactamases was measured against a chromogenic substrate, CENTA  m phosphate buffer, pH 7.0 with the enzyme concentration 0.1 mg·mL−1. The enzyme aliquots withdrawn at time intervals  were brought to room temperature, and their residual catalytic activity was measured.Study of thermal inactivation was carried out at 60 °C in 50 m1BTL) and TEM‐72 (PDB code 3P98) were taken as the enzymes’ initial structures sybyl 8.1 package . The protein molecule was positioned in a cell filled with TIP3P water molecules and periodic boundary conditions were used during simulations. The total charge of the system was neutralized by adding Na+ ions. MD simulations were carried out with the gromacs‐5.1 package (http://www.gromacs.org) using amber99‐SB forcefield. The potential energy of the full system was minimized (150 000 steps), followed by gradual heating up to 300 K (NVT ensemble) and raising pressure (NPT ensemble). Total length of MD trajectories was 25 ns (2 fs time step). Protein structures remained stable in the course of the MD simulation. The conformations saved each 200 ps were analyzed to calculate the number of established intramolecular hydrogen bonds using the gmx_hbond utility from the gromacs‐5.1 package.The spatial structures of β‐lactamases TEM‐1 , kcat and catalytic efficiency kcat/KM) for recombinant enzymes determined against the synthetic chromogenic substrate CENTA based on the antibiotic cephalothin Table KM obtained for the enzymes TEM‐1 and its mutants TEM‐2 and TEM‐135, bearing the single substitutions Q39K and M182T, respectively, were practically identical within the experimental error. These data are consistent with the results obtained earlier for β‐lactamase TEM‐1 and its natural mutant TEM‐2, having the single substitution Q39K KM values for the mutants having the substitutions at the key positions differ from those for wild‐type TEM‐1 enzyme: the M69V and R164S replacements led to an increase in KM by 15 and 1.5 times, respectively; replacement E104K led, on the contrary, to the value of KM decreasing by a factor of 2.The values of The catalytic constant was decreased by 4‐fold for the combination of Q39K and R164S and by 2‐fold for the combination of Q39K and E104K. In first case, catalytic efficiency was also reduced by 3‐fold.KM and catalytic constant values, which means improved binding of the substrate and less effective catalysis, compared with a single key replacement alone.The combination of the secondary mutation Q39K and one of the key mutations shows a decrease in both β‐Lactamases are quite stable enzymes, so to study their ability to show reverse renaturation, we used exposure at 60 °C as a stressful condition for the enzymes. All recombinant forms of TEM‐type β‐lactamases produced in this study were held at elevated temperature for a certain time interval, and then probes were cooled to room temperature and their residual catalytic activity towards the substrate CENTA was measured. The curves of the residual activities against the incubation time at 60 °C are shown in Fig. The magnitude of the residual enzymatic activity reflects the ability of enzymes to show reverse renaturation and proper protein refolding. To quantify the effect, we determined the residual activity of the enzymes after exposure at elevated temperature for 3 h , residue Q39 is located on the protein surface near the C end of the first α‐helix and is separated from the enzyme's catalytic site by a β‐sheet . Residue Q278 belongs to the C‐terminal α‐helix of the protein and is preceded by N276, whose side chain is directed towards the β‐sheet of the enzyme. The OD1 atom of N276 establishes a hydrogen bond with the NH2 group of R244 (the distance is 3.25 Å). This hydrogen bond enforces the interaction between the elements of the protein's secondary structure, enhancing its thermal stability. ND2 of N276 serves as a hydrogen donor with a bound crystallographic water molecule (the distance is 2.92 Å). Solvation of N276 also increases the thermal stability of the protein. A second nitrogen atom, NH1 of R244, forms a hydrogen bond with the main chain carbonyl oxygen of G236 (the distance is 3.03 Å), providing an extra bond between the strands of enzyme β‐sheet. Figure To explain the experimental data obtained, a computer analysis of β‐lactamase TEM‐1 and its mutant forms was carried out on the basis of available crystal structures. In the spatial structure of β‐lactamase TEM‐1  bearing a mutation Q39K in combination with the key mutations G238S and E240K and secondary mutation M182T To simulate changes in the β‐lactamase molecule upon replacement of glutamine at position 39 with lysine, we looked for available β‐lactamase structures having this substitution.There is only one solved structure of β‐lactamase TEM‐72 (PDB code The structures of β‐lactamases TEM‐1 and TEM‐72 look very similar. The root mean square deviation (RMSD) between the polypeptide chain heavy atoms of these structures is only 0.7 Å, which comes mainly from the difference in folding of the loop formed by residues 252–257. In the rest, the two proteins folds are very similar, with RMSD around 0.1 Å. Figure KM for β‐lactamase TEM‐1 and TEM‐2 with the Q39 substitution.The search for conformational changes that accompany the Q39K mutation did not reveal any substantial changes in neighboring residues. One can expect that this mutation will not affect the conformation of the residues forming the enzyme's catalytic site. So the binding affinity of the mutated enzyme for substrate should remain the same as for the wild‐type. This result is in agreement with approximately the same values of In order to estimate the mobility and degree of solvation of the residues involved in the hydrogen bond network we performed MD simulations for β‐lactamases TEM‐1 and TEM‐72. The MD trajectories obtained for each protein were processed to calculate the number of established hydrogen bonds in frames separated from each other by 200 ps. Dynamic stability of hydrogen bonds was analyzed for residue pairs Q(K)39–Q278, K32–Q278, N276–R244 and R244–G236. We used the criterion of maximal distance between the acceptor and the donor of the hydrogen bond of 3 Å to consider the bond as established. The results of the simulations are shown in Figs β‐Lactamases continue to be an attractive model for investigating the mutual influence of mutations due to their evolutionary variability. The term epistasis is increasingly used to explain polymorphism of β‐lactamases in the context of their molecular evolution and adaptation, although the molecular basis of the phenomenon still remains obscure KM values in all cases were decreased, the catalytic constants were decreased for ESBL phenotypes, and the thermal stability of mutant forms containing Q39K combinations was also reduced.Here we have attempted to shed light on the possible role of the secondary Q39K mutation in TEM‐type β‐lactamases. It attracted our attention as it is located on the surface of a protein globule and is quite frequently observed in different β‐lactamase phenotypes both as a single mutation and in combination with the key substitutions as well. We confirmed the data earlier obtained that a single replacement of Q39K in β‐lactamase TEM‐2 does not lead to any appreciable changes of the enzyme's properties, which is in agreement with the remoteness of this substitution from the enzyme's active site Tm) estimation and to analyze the influence of mutations on the fitness of a protein globule. It is important to study the dynamic stability of the structure, which is manifest in the ability of the enzyme to restore the catalytically active conformation after removal of the denaturing pressure. Considering this, the destabilizing effect of Q39K combinations with the key mutations means a reduction of the enzyme's ability to reverse thermal reactivation, which is especially pronounced in combination with the E104K and M69V substitutions. Our data demonstrate a non‐additive negative effect of these mutation combinations, which is partially in accordance with the unfavorable influence of the number of positively charged residues on stability of β‐lactamases Studying the thermal stability of different TEM‐1 β‐lactamase mutants, we suggested not limiting the melting temperature . Only the cis‐isomer demonstrates catalytic activity, providing proper orientation of the catalytically important glutamate in position 166. Based on the data obtained, we may suggest that all studied amino acid substitutions might affect the isomerization of this bond, which in turn leads to a different ratio of cis‐/trans‐isomers and therefore to active or inactive conformations of the enzyme. The antitropic effect of the Q39K and R164S substitutions may apparently be associated with an increase/decrease in the energy barrier of the cis–trans transition, which is about 20 kcal·mol−1Thermodynamic stability of different β‐lactamases was previously investigated and interpreted in terms of Currently, the spread of antibiotic resistance is certainly connected with anthropogenic factors. However, the phenomenon of bacterial resistance has deeper roots and is much older than the era of the use of antibiotics by humans. Broad polymorphism in the superfamily of β‐lactamases and the opposite effects of certain mutations on kinetic properties and stability appear to play a significant role in the adaptation and survival of microorganisms in changing external conditions. The literature data indicate that both mutation types, stabilizing protein structure and contrary ones, arise in the course of evolutionary adaptation of bacteria. Our study underlines the relevance of a systematic comprehensive study of the effect of so‐called secondary or ‘silent’ mutations in combination with the key ones on the properties of β‐lactamases, which helps in understanding the evolution of β‐lactamases. The study of the surface spots on the protein globule, where the residues affecting protein susceptibility to external influence are located, facilitates the search for new targets for directed β‐lactamase inhibition and overcoming resistance.AE supervised the project; MR, VG and AE designed the research; VG, IA, OS, MU and II performed the research; IU, AV and DS performed modeling and molecular dynamics; VG, MR, IU analyzed data; MR, VG, IU, AV and AE wrote the paper with contributions from the other authors.


This study aims to increase the precision of single‐compartment DESPOT relaxometry by two means: (i) a joint system relaxometry (JSR) approach that estimates parameters in a single step using all available data; and (ii) optimizing acquisition parameters by deploying a robust design tool based on the Crámer‐Rao lower bound (CRLB).Following the development of the analysis and design capabilities, phantom and four in vivo subject experiments were performed to compare directly the precision achieved with DESPOT and JSR estimation using published protocols and protocols designed using a proposed CRLB framework.Experimental data demonstrate JSR's ability to decrease relaxometry estimation variance. Phantom results show 72 to 77% improvement using the same data as conventional DESPOT. This is further improved to 81 to 87% using optimal parameters. Both experiments show systematic bias depending on the acquisition parameters used, which are shown to be highly reproducible and to vary with different magnetization transfer conditions.ρ,T1,T2, and B0 in an 11‐min examination, making the approach appealing for potential clinical use. Magn Reson Med 79:234–245, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Compared with DESPOT, JSR produces reproducible relaxation maps with improved precision. Further improvement was achieved using CRLB as a protocol design tool. With this combined approach, it is possible to achieve submillimeter maps of  T1 and T2 has proven to be useful—particularly in detecting and understanding neurological diseases T1 and T2 are 2D acquisitions based on spin‐echo (SE) methods, which require long acquisition times and have been shown to produce variable results across different studies T1 and T2 (DESPOT1/2) 3 3D maps of the human brain in clinically feasible times Despite the continued dominance of qualitative assessment in clinical applications of MRI, the ability to probe tissue relaxation times T1 and T2 of each measured voxel. The standard fitting procedure T1 from a linearized Ernst relationship using SPGR data alone, and then feeds the T1 value into a linearized bSSFP model to estimate T2  A DESPOT2 approach that avoids linearization of the equations (DESPOT‐FM) was also proposed, to estimate the underlying field map The DESPOT1/2 approach makes use of spoiled gradient echo (SPGR) and balanced steady‐state free precession (bSSFP) sequences over a range of varying flip angles (FAs) at fixed repetition times (TRs), to estimate T2 information after T1 had been assessed was suggested T2 or T2* dependency of the SPGR signals as well as T1 information present in the bSSFP curves. Both effects can be taken into consideration by carefully stating the signal evolution for different acquisition parameters. The expected SPGR signal equation, SPGRS, adopted in this work, is given by bSSFPS, is described by Originally called variable nutation angle TRSPGR, TESPGR and TRbSSFP, and TEbSSFP are the repetition and echo times of the SPGR and bSSFP sequences; T2′ is defined by 1/T2* = 1/T2 + 1/T2′; α is the FA modulated by a (dimensionless) transmit field inhomogeneity κ; E1=exp; and β=2πΔΩTRbSSFP+ϕRF is the off‐resonance dephasing angle in radians, resulting from frequency offset ΔΩz(Hz) plus the rotation induced by incrementing the phase, ϕRF, of the radiofrequency (RF) pulse at each TRbSSFPTRF) was considered by following the correction proposed in E2=exp(−(TR−ζβTRFTRbSSFP)/T2) with ζβ=ζcos2[β2(1−(1−ζ)TRFTRbSSFP)] and ζ≈0.68−0.125(1+TRFTRbSSFP)T2T1  Note that the two sequence types need not have the same repetition and echo times as each other. MxSS and MySS correspond, respectively, to bSSFP real and imaginary steady‐state components. In Equations [M0 as all measured signals have a scanner‐dependent scaling factor that must be explicitly accommodated during fitting (see “Methods” section). Both SPGR (Eq. [Equations  to 5] d d5] descPGR (Eq. ) signal e−TE/T2′ term is present in the SPGR and not in the bSSFP sequence model, which is a consequence of the latter's spin‐echo behavior as described in The proposed JSR approach requires careful balance between SPGR and bSSFP signal intensities during readout periods. Although possible to compute the echo‐time decay from the magnetization before the RF pulse, using the magnetization immediately after the excitation pulse allows a simple exponential decay to be added to the steady‐state solution. This is contrary to the approach described by Deoni in 2009 (9), in which the steady‐state magnetization immediately before each RF pulse was considered. The TR≫T1 however, RF and gradient spoiling methods need to be applied when TR≪T1. A good discussion of this issue is presented in T1 obtained is applied. However, the obtained steady state is still TR,T1, T2 and FA dependent. To ensure that imperfect spoiling was kept under control, the SPGR sequence was modeled using the extended phase graph (EPG) algorithm 1 and T2 ranges.Equation  is only x,θ), where x=T is a vector containing N independent measurements  and θ=T is a vector containing M model parameters  then the variances of the elements of the unbiased estimate vector of the model parameters θ^ based on noisy measurements y=T are described by the M × M covariance matrix Σθ^2 as follows:F is the Fisher information matrix. The CRLB, which is the lowest limit of Σθ^2 is obtained by taking the equality in Equation  and the maximum value obtained is minimized, resulting in the following cost function, CFgrid, which is to be minimized as follows:grid is conservative in that it always selects the worst‐case relative precision detected on the defined search space grid, and therefore minimizes the estimation variance for the worst case in all of the θG considered.To achieve equal relative precision 2+(ρi)2 resulting in the final considered JSR model of Equation  for each measured voxel are obtained through fitting Equation  a minimum of five measurements are required for a fully determined solution. In addition, given that equal echo times are used for all acquisitions, a minimum of two bSSFP measurements is required to estimate the field map ΔΩ.With five unknowns to be estimated TRSPGR, ϕRF and FAs were therefore determined for different combinations of NbSSFP and NSPGR, as expressed in Table The optimal In vivo scans were acquired in four healthy volunteers ), who gave written informed consent according to local ethics requirements.Each subject was imaged using the complete baseline protocol and an optimized set of parameters were obtained by making use of the proposed CRLB optimization framework. The optimized parameters are listed in the “Results” section.www.fsl.fmrib.ox.ac.uk) 1‐weighted SPGR image using the FSL fast tool (38‐40).Before relaxation‐map estimation, all images were skull‐stripped and aligned using standard FSL bet and flirt tools /pT1,2CRLB is below 1%, as can be observed in the right column of Figure Figure 1 = 2058 ± 27 ms and T2 = 185 ± 1 ms, as determined using the reference spin‐echo sequences and MTR = 5.8% ± 0.7%. Phantom data were used to validate the hypothesis that JSR precision outperforms the standard DESPOT1/2 approach, and allowed the proposed CRLB framework to be tested. To achieve this, an extensive set of phantom‐specific optimized acquisition protocols , respectively. Similar improvement can be observed in T2 estimation, in which the JSR‐1:4 and JSR‐2:4 interquartile ranges are 14 and 15 ms (46 and 43% reductions), respectively.The conventional two‐step fitting approach on the baseline protocol results in interquartile range estimations of 1162 ms for DESPOT1 TT1 spin‐echo measurement DESPOT1, JSR baseline, JSR 1:4, and JSR 2:4 demonstrate 15.1, 11.0, 3.3, and 4.4% bias, respectively. On the same note, the T2 percentage deviation is reported as 20.7% for DESPOT2‐FM, 10.9% for JSR baseline, −11.4% for JSR 1:4, and −9.2% for JSR 2:4.The percentage deviation of the median value relative to the reference ϵ=(Sspgr−SspgrEPG)/SspgrEPG for both WM (ϵWM) and GM (ϵGM) as a function of different TRSPGR and FA αSPGR To avoid solutions with a WM error larger than ±5%, the maximum allowed FA was set to 15o  using DESPOT1/2 (blue), JSR with baseline acquisition (red), and JSR with optimized acquisition (yellow) parameters. As with the phantom data, JSR produces narrower estimation distributions compared with the conventional DESPOT1/2 approach. However, the estimation mean relaxation‐time values show significant shifts toward lower relaxation time values.Figures 1 and WM/GM T2 histograms obtained using the optimized JSR acquisition for all of the healthy volunteers, demonstrating the reproducibility of the proposed method.Figures 1/T2 estimation between conventional DESPOT and JSR estimation. This is further corroborated by the in vivo data, in which visual assessment . For this phantom, the bias between spin echo and VFA measurements was between 5 and 43% for T1, and −18% and 22% for T2. This is a larger variation than for the 0.5% agarose experiment summarized in Figure 1 and − 11.4 to 20.7% for T2. In Figures This work aimed to improve the precision of DESPOT1/2 based on the JSR approach, in which all of the key parameters are estimated in a single calculation. Figure ent Fig.  shows thle Figs. e–6h. Num2 values show a significant underestimation when compared with other studies RF correction (8) for the phantom data resulted in an overestimation of the obtained T2 values by 15 to 20% compared with the ones displayed in Figure The in vivo Ttotal = 29.2 ms, which matches the baseline protocol taken from the literature o. This cut‐off guarantees that the expected WM spoiling inconsistencies are below 5%, whereas GM are kept bellow 8% for all optimization protocols explored. Although some solutions might benefit from allowing higher SPGR FA, this would imply either exploring optimal ways to weight Equation [1; we note that performing JSR allows the acquisition to require only one SPGR measurement. This is interchangeable with increasing the number of SPGR measurements, provided that the same time is spent sampling SPGR information . Although we restricted our acquisition time to a baseline protocol for comparison, it would be possible to trade off the JSR gain in precision for further reduction of the acquisition time.The JSR framework presented can help achieve optimal acquisition efficiency. In this study, full 3D 0.8‐mmT2' by allowing different echo times between acquisitions. In addition, it is feasible to include B1 field estimation directly from the VFA data by incorporating an inversion recovery sequence  into the joint system model. Investigation of such protocols showed that the CRLB design framework can be applied, but initial in vivo testing with JSR processing resulted in T1 values with higher systematic deviations for different acquisition parameters , compared with what is presented in this work. We attribute this to the very different RF conditions engendered by such sequences, causing a model bias as a result of MT effects that are not considered (data not shown). This is currently the subject of further investigation; in particular, the option to explicitly include MT in the JSR approach is an interesting future possibility. The JSR approach has a lot in common with the recently introduced fingerprinting concept (MRF) The work presented here is a proof of concept and demonstrates the benefit of simultaneously using images with different signal responses to minimize variance in relaxometry estimation. Although neglected throughout this work, the JSR framework can be expanded to estimate ρ,T1,T2, and B0 in an 11‐min examination, making the approach appealing for potential clinical use. For examinations in which the clinical protocol already includes SPGR, the additional time needed to achieve quantitative relaxometry maps is even smaller . As with all variable FA approaches, the absolute values of the relaxation times found tend to deviate systematically from spin echo–based measurements. However, high reproducibility combined with efficiency endow these methods with significant advantages for larger‐scale studies. The proposed framework enhances both precision and efficiency, further adding to their potential utility.This work shows an immediate benefit of the proposed JSR analysis approach, compared with conventional DESPOT1/2, in producing relaxation maps with improved precision. It also shows that further improvement can be achieved by making use of the CRLB as a protocol design tool. With this approach, it is possible to achieve submillimeter maps of


We were able to measure the magnetic structure of this Cd containing and highly neutron absorbing material by optimizing the sample geometry and by blending the CdMn7O12 with Aluminum powder. Below its Néel temperature TN1 all magnetic reflections can be indexed by a single commensurate propagation vector k = . This is different to the case of CaMn7O12 where the propagation vector is incommensurate and where an in-plane helical magnetic structure has been found. We observe a commensurate non-collinear magnetic structure in CdMn7O12 with in-plane aligned magnetic moments resembling the ones in CaMn7O12. However, the commensurate propagation vector prevents the appearance of a helical magnetic structure in CdMn7O12. Finally, we also observe a third structural phase transition below ~60 K that can be attributed to phase separation.We report on the magnetic structure of CdMn The A site is 12-fold coordinated and A′ site is square-coordinated, while the B site is octahedrally coordinated. The A′ site is occupied by Mn3+ ions and the B site by mixed valent Mn3.25+ ions. Below ~440 K, CaMn7O12 undergoes a structural transition from cubic TN2 becomes more complicated due to the appearance of multiple propagation vectorsMultiferroic CaMn7O12, the other quadruple perovskite manganites have to be synthesized under high pressure and high temperature conditions rendering the sample availability more difficult. A recent study on its analogue, CdMn7O12, has shown similar physical properties7O12 also exhibits a structural transition at Ts1 ~ 493 K and two successive magnetic transitions at TN1 = 88 K and TN2 = 33 K. Moreover, the magnetic transition at TN1 is robust against external magnetic fields, while TN2 can be gradually suppressed with applied magnetic field. The crystal structure of CdMn7O12 at room temperature is trigonal . We focus on the temperature range TN2 < T < TN1, where the magnetic peaks can be indexed by a single propagation vector like in CaMn7O12. However, for CdMn7O12 the propagation vector amounts to k =  which is in contrast to CaMn7O12 where an incommensurate propagation vector along the c-direction has been observed. The magnetic structure of CdMn7O12 is non-collinear.In this paper, we report the magnetic structure of CdMn7O12 could be obtained. The structural parameters are listed in 7O12 amount to 69.7(5)% and 30.3(2)% respectively.Ts2 ~ 254 K have been reported recentlyNote, that indications for a structural transition Ts3 ~ 60 K. Note, that there is a small anomaly at ~60 K in specific heat measurements at high magnetic fields of 9 T, whereas no obvious anomaly could be observed in zero field measurementsTs3 similar as observed in other transition metal oxides like SrCrO3Ts3, the intensity of the main peaks related to the Ts3 and where only a certain volume fraction of the compound undergoes the structural transition (that might be enhanced by applying magnetic fields of 9 T). The origin of this structural transition requires further studies. Here, we focus mainly on the magnetic structure determination of CdMn7O12 slightly above or around Ts3.We performed temperature dependant powder X-ray diffraction experiments down to 12 K and were able to observe an additional structural transition at a lattice constant decreases continuously with decreasing temperature until TN2. A small kink can be observed at Ts2 while no obvious anomaly can be found at TN1. Then, below TN2, a starts to increase with decreasing temperature. On the other hand, the c lattice constant exhibits distinct anomalies at Ts2 and also at 100 K and 50 K. This behaviour is similar to the one in CaMn7O12TN1 and TN2, suggesting that the magnetic transitions follow the structural ones.We determined the lattice parameters of the 7O12 at 40 K by means of powder neutron diffraction at the D2B diffractometer. The peak-splitting that has been observed in our high-resolution XRD measurements could not be resolved in our neutron measurements with less resolution. In We studied the crystal structure of CdMn7O12 between 2 K and 120 K at the D1B diffractometer using an incident neutron wavelength of 2.52 Å. Only two peaks from the Aluminum powder appear in the entire diffraction pattern. Thus, the dilution with fine Aluminum powder does not cause any interference with magnetic reflections that appear below the Néel temperature of CdMn7O12. As can be seen in 7O12. Hence, these reflections are magnetic in origin and can not be connected to the structural transition that takes place at 254 K. For TN2 < T < TN1 all magnetic peaks can be indexed with a single propagation vector k = , which is different from the incommensurate case in CaMn7O12. Below TN2 the magnetic structure is more complicated and multiple propagation vectors are necessary for describing the magnetic structure. Here, we will focus on the magnetic structure for TN2 < T < TN1 only. Due to the structural transition from We next focus on the magnetic structure refinement. We have measured the magnetic structure of CdMne and 9d is decomposed into three irreducible representations (IRs) with dimension one, asThe magnetic reducible representation Γ of site 9b, it is also decomposed into three IR with dimension one, asFor site 3ψi) of the IRs are listed in b and for the 9e(9d) site respectively.The basis vectors  reflection, which is not in agreement with our experimental observations  and rotate by 2π/3 and 4π/3 when going from one layer to the next according to the centering translations  and  respectively. The net moment in the unit cell is zero, which is consistent with the magnetization measurements between TN1 and TN2, showing linear curve of M vs. H. The magnetic structure resembles the one of CaMn7O1287O12 is incommensurate whereas the propagation vector is commensurate in the case of CdMn7O12.Thus, the IR Γrvations . For theTN1, the ordered moment size starts to increase. Then, at Ts3, the moments at the Mn1 site exhibit an anomalous decrease whereas the Mn2 and Mn3 sites continue their increase. Note, that the size of the magnetic moments of Mn3+ ions at the 9e (Mn1) and 9d (Mn2) sites  is very similar for CaMn7O12 which is in contrast to the case of CdMn7O12.The temperature dependence of the ordered magnetic moments for the distinct Mn sites is shown in 7O12 below its Néel temperature has been determined by PND. We observe a non-collinear magnetic structure with Mn spins that are aligned within the ab plane. The magnetic propagation vector is commensurate with k =  which prevents the occurrence of a helical structure. This is different to the case of CaMn7O12.In summary, the magnetic structure of CdMn7O12 were synthesized by high pressure method as described elsewherePolycrystalline samples of CdMnλ of 2.52 Å and 1.59 Å respectively. In order to reduce the absorption of neutrons by Cd nuclei, the CdMn7O12 powder was mixed with larger amount of Al powder and filled into the outer space of a hollow vanadium cylinder. This technique that we initially developed for SmFeO3Powder neutron diffraction (PND) measurements were performed at the D1B and D2B diffractometers at the Institut Laue-Langevin (ILL), France with a neutron wave length Phenix of Oxford Cryosystems) was used for cooling the flat plate powder sample.In addition, temperature dependent x-ray diffraction (XRD) measurements were also performed on a Bruker D8 Discover A25 diffractometer using Cu How to cite this article: Guo, H. et al. Non-collinear magnetic structure of manganese quadruple perovskite CdMn7O12. Sci. Rep.7, 45939; doi: 10.1038/srep45939 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In this study exposure behavior between CO2 and tight rock of the Ordos Basin has been studied experimentally by using nuclear magnetic resonance transverse relaxation time (NMR T2) spectrum and magnetic resonance imaging (MRI) under the reservoir pressure and temperature. Quantitative analysis of recovery at the pore scale and visualization of oil mobilization are achieved. Effects of CO2 injection, exposure times and pressure on recovery performance have been investigated. The experimental results indicate that oil in all pores can be gradually mobilized to the surface of rock by CO2 injection. Oil mobilization in tight rock is time-consuming while oil on the surface of tight rock can be mobilized easily. CO2 injection can effectively mobilize oil in all pores of tight rock, especially big size pores. This understanding of process of matrix exposed to CO2 could support the CO2 EOR in tight reservoirs.Understanding mechanisms of oil mobilization of tight matrix during CO CO2 EOR is a process in which CO2 under supercritical conditions acts as a powerful solvent and is routinely used for extracting more oil out of aging reservoirs2 EOR also considerably reduces greenhouse gas emissions2 EOR mechanisms include the oil viscosity reduction, oil swelling effect, interfacial tension (IFT) reduction, light-hydrocarbons extraction in immiscible and miscible conditions782 will flow along fractures and cannot permeate into rock matrix due to low matrix permeability in tight oil reservoir. So it is of significant importance to study the mechanisms of oil mobilization in CO2 injection process in tight reservoir.Tight oil is a new and unconventional resource. According to the U.S. Energy Information Administration (EIA) data, as of 2013 tight oil is more than 42% of U.S. domestic total crude oil production and it is expected to reach 59% in 2020. In China, tight oil has been explored in five main basins12 EOR in tight reservoir have been conducted. Hawthorne et al.2 Bakken tight rock and proposed conceptual mechanisms of CO2 EOR in a tight fractured system: (1) CO2 flows into and through the fractures, (2) rock matrix is exposed to CO2, (3) CO2 permeates the rock under pressure, then swelling of crude oil extrudes some oil out of the pores, (4) oil migrates to the bulk CO2 via swelling and reduced viscosity, and (5) oil production is slowly driven by concentration gradient diffusion from pores into the bulk CO2 in the fractures. Zekri et al.2 was capable of mobilizing oil in the rock with very low permeability (0.16 mD) and providing reasonable displacement efficiency. Furthermore, immiscible CO2 recovery was probably related to ability of oil extraction by supercritical CO22 EOR were low interfacial tension (IFT) displacement and extraction of oil components, and the latter dominated after CO2 breakthrough. Vega, et al.2 into siliceous shale. More than 54% of oil can be recovered by CO2 injection. CO2 diffusion was a major recovery mechanism, which results in a decrease in oil viscosity and an increase in CO2 molar fraction in the oil. Tovar et al.2 EOR in unconventional liquid reservoirs (ULR) with nanodarcy matrix permeability. CO2 was able to penetrate the cores, resulting in an estimated oil recovery in the range of 18–55%. Oil vaporization into the carbon dioxide was the main mechanism for oil production. In conclusion, CO2 diffusion into matrix to mobilize oil is the main mechanism of CO2 EOR in the tight matrix.Recently, a large numbers of studies about CO2 spectrum and MRI, is a quantitative and visual analytical tool2 distribution calibrated by mercury injection capillary pressure (MICP). Recently, NMR T2 distribution was conducted to characterize the tight reservoir. Saidian and Prasad2 distributions of Middle Bakken formation. Wang et al.2 distributions. Marschall et al.2 test of rock samples covering a wide range of permeability (0.078–4450 mD).NMR technology, including NMR Tet al.2 being injected into the bead-pack core (grain size distribution ranging from 0.177–0.250 mm) at high pressure and high temperature by using MRI. The piston-like miscible regions and CO2 front, onset of CO2 channeling or fingering, and the distribution of oil in porous media can be accurately detected. Suekane et al.212 injected into a packed bed of glass beads (with 70 um average diameter) and Berea sandstone (permeability of 19.6 mD and porosity of 18.5%.) containing water. Water saturation distributions can be successfully observed. Brautaset et al.in situ by MRI during water flooding followed by CO2 in four Portland Chalk core samples with different wettability.There have been many studies on the visualization of flow and transport in porous media using MRI techniques. Zhao 2 injection is crucial for CO2 EOR engineering design in fractured tight oil reservoir. Oil mobilization characteristic of tight matrix in pore scale during CO2 injection is the key scientific question for effective production of fractured tight oil reservoir. In this study, we focused on the process of tight matrix exposed to CO2 under the temperature of 40 °C and pressure of 12 MPa and 22 MPa. NMR T2 and MRI were used to detect the tight matrix exposure process in situ to study mechanisms of oil mobilization during CO2 injection. After tight matrix was exposed to CO2, NMR T2 test and MRI were continuously performed until the obtained T2 spectrum remained unchanged to investigate the effect of exposure time on oil mobilization. At the end of the first exposure experiment, interaction between CO2 and tight matrix reached equilibrium state. Then the second exposure experiment started with CO2 injection under a constant pressure of 12 MPa and at a constant rate to displace the CO2 used in the first exposure experiment in order to remain concentration of CO2 to be 100% in the gas phase. CO2 injection process also was detected by NMR to study oil mobilization in the CO2 displacement process. Finally, 2nd, 3rd and 4th exposure experiments were conducted to investigate the effect of exposure times sequentially. Effect of immiscible and miscible pressure on oil mobilization was also investigated.Understanding mechanisms of oil mobilization of tight matrix during CO2 and matrix with permeability of 0.2180 mD at 40 °C and 12 MPa. Tight matrix was exposed to CO2 and T2 test of tight matrix was continuously performed with NMR system. NMR T2 spectra of CO2 exposure experiment under different exposure time are shown in 2 amplitude reduction of pores with radius larger than 1 μm is greater than that of pores with radius smaller than 1 μm. Therefore, the recovery of oil in the former is larger. Initially, oil exists in pores with maximum radius of 21 μm in the original saturated tight matrix. After CO2 injection, oil flows to pores with radius greater than 21 μm, suggesting that oil in the tight matrix “diffuses” to the surface of matrix. And NMR T2 amplitude of pore with radius greater than 21 μm increases as exposure time increases, which indicates that more oil gradually “diffuses” to the surface of tight matrix. From NMR T2 amplitude reduction, the recovery of oil in pores with radius larger than 1 μm increases more sharply in initial exposure stage. For example, NMR T2 amplitude reduction of pores with radius larger than 1 μm from 0 to 27 hours is greater than that from 27 hours to 50 hours. Similar phenomenon was also observed in pores with radius smaller than 1 μm, but the amplitude of NMR T2 reduction is relatively small.Exposure experiment was conducted between CO2 has diffused into the whole core. During the slow-growing stage (27–120 h) oil saturation of core decreases from the inside outward from MRI images  and in the second stage pressure decreased from 7.5 MPa to atmospheric pressure. NMR Tariation . A smallariation . After 4st, 2nd, 3rd and 4th exposure experiments are 23.7%, 7.2%, 2.6% and 1.5%, respectively and decrease as exposure times increases. Recoveries of two depressurization experiments are 0.9% and 4.5%, respectively.2 and matrix under immiscible pressure of 12 MPa and miscible pressure of 22 MPa and temperature of 40 °C with core permeability of 0.5997 mD and 0.6038 mD, respectively. NMR T2 spectra of CO2 exposure experiment under different pressure are shown in 2 amplitude reduction of pores with radius larger than 1 μm is greater than that of pores with radius smaller than 1 μm. It can also be observed that oil in the tight matrix “diffuses” to the surface of matrix and more oil “diffuses” to the surface of tight matrix as exposure time increases. Recoveries of oil under different pressure versus exposure time are shown in Exposure experiments were conducted between CO2 diffuses into oil in the tight matrix, then oil swells and leaves from pores to surface. Oil signal can be observed on the surface of tight matrix , COt matrix  and incrt matrix  demonstr2 has diffused into the whole tight matrix and nearly contacted all the oils, at the same time oil in all pores swells. Oil diffuses gradually from the inside outward and oil saturation of core decreases from MRI images from the inside outward (At the beginning of slow-growing stage of the first exposure experiment (27–120 h), CO outward . Main mend, 3rd and 4th exposure experiments, interaction between CO2 and tight matrix has reached equilibrium and oil swelling no longer exists. MRI results  of exposure experiment with permeability of 0.2180 mD under immiscible pressure of 12 MPa, exposure experiment with permeability of 0.5997 mD under immiscible pressure of 12 MPa and exposure experiment with permeability of 0.6038 mD under miscible pressure of 22 MPa is 64.3, 49.7 and 32.8, respectively. These CO2 utilizations are much less than that mentioned by Trivedi and Babadagli2. Meanwhile, it can be observed that CO2 utilization increases from 64.3 to 49.7 as permeability of core increases from 0.2180 mD to 0.5997 mD under immiscible pressure of 12 MPa and even CO2 utilization can increase up to 32.8 after pressure raises to miscible pressure of 22 MPa.At the end of first exposure experiment CO2 EOR process is the most important for fractured tight oil reservoirs. The main oil recovery mechanism is diffusion in fractured reservoirs during injection of CO2 for EOR. Darvish et al.et al.2 injection using NMR T2 spectrum and MRI. Injected CO2 diffused into oil in the tight matrix and then oil swelled and oil composition diffused. These effects could mobilize oil from tight matrix to fracture. Meanwhile, increasing pressure could quickly recover more oil from tight matrix. Minimum miscibility pressure (MMP) between oil and CO2 was 17.8 MPa determined by slim tube test at 40 °C. However, miscibility was not observed in this study under the miscible pressure of 22 MPa. This results indicated that MMP determined by traditional slim tube is not suitable and more influences should be considered for CO2 EOR design of fractured tight oil reservoirs.Oil mobilization of tight matrix during CO2 and matrix was design for NMR measurement. The key units are NMR measurement system (PKU University) and exposure experiment device compatible for NMR test with a maximum temperature of 80 °C and a maximum pressure of 35 MPa. The NMR measurement system has a constant magnetic field strength of 2350 gauss (permanent magnets) and a resonance frequency of 10 MHz. The parameters for NMR T2 measurement were set as follows: echo time (TE), 0.23 ms; repetition time (TR), 2 s; echo numbers, 4096; numbers of scans, 64. After the measurements, transverse relaxation time (T2) was calculated by multi-exponential inversion of the echo data with 64 preset decay times logarithmically spaced from 0.1 ms to 10 s. The parameters for MRI were set as follows: echo spacing (TE), 3.2 ms; repetition time (TR), 500 ms; image data matrix, 256 × 256; field of view (FOV), 150 mm × 150 mm with a thickness of 20 mm.Exposure experiment between CO3 (at 40 °C) and 5.50 mPa.s (at 40 °C), respectively, at atmospheric pressure. Minimum miscibility pressure (MMP) between oil and CO2 was 17.8 MPa determined by slim tube test at 40 °C.Oil and tight matrix were collected from CHANG 8 layer of the Ordos Basin in Honghe Oilfield. Density and viscosity of oil were determined to be 0.8050 g/cm2 test of cleaned core clearly showed that there were no any signals of fluid inside the cleaned core. 2. Saturating and aging core procedure. The core was vacuumed up to 10−5 mbar for 48 h in the core-holding device under overburden pressure using molecular pump. Then the core was saturated with oil and aged at 80 °C and 30 MPa for 14 days. In this study effect of water saturation wasn’t investigated and so there was no procedure to establish the initial water saturation.Horizontal cylindrical core plugs were cut and then were prepared for exposure experiment according to following procedures. 1. Cleaning core procedure. The core plug was held in a suitable core-holding device under overburden pressure that will permit the flow of solvent through the matrix. Toluene was injected from the inlet of core-holding device to rinse the oil for two weeks and then methanol was injected to rinse the brine for one week in the core. Then the rinsed core was dried for two days in the oven at 116 °C. An NMR T2. The first exposure experiment started. Meanwhile, NMR T2 and MRI test of the core were continuously performed with the NMR system to investigate the effects of exposure time on EOR. At the end of the first exposure experiment, interaction between CO2 and core matrix reached equilibrium state with unchanged T2 spectrum. Then the second exposure experiment started with CO2 injection under a constant pressure of 12 MPa and at a constant rate to remain fresh CO2 in system. The procedure of NMR test was unchanged. The third and fourth exposures were conducted sequentially.The experimental procedure used in this study is briefly described as follows. The saturated core was enclosed into exposure experiment device. After the temperature of the whole system achieved setting experimental temperature and became stable, the core was exposed to CO2, of a fluid in a pore is given by the following As for the NMR transverse relaxation time, T2,bulk is the bulk relaxation time of the pore-filling fluid (ms), T2,surface is the surface relaxation time (ms), and T2,diffusion is the relaxation time as induced by diffusion (ms). As for fluid flow in porous media, T2,bulk is usually neglected because T2,bulk is much larger than T2,surface. T2,diffusion is also neglected when the magnetic field used is deemed to be uniform with a quite small field gradient. Then T2 is mainly dependent on T2,surface, which is associated with specific surface area of a pore. T2,surface can be expressed as the following where T2), and V is the pore volume (μm3). S/V can be rewritten as a function of the dimensionless shape factor of a pore, Fs, and pore throat radius, r (μm) by the following where ρ is the surface relaxivity (μm/ms), S is the pore surface area  of a given core can be assumed to be constant. Thus, T2 can be rewritten as Surface relaxivity (ρ) and shape factor (Fs), and C is a constant conversion coefficient (ms/μm).where C = 1/(ρFs) is introduced to account for the fact that NMR responds to pore body size whereas MICP is controlled by the size of pore throats. According to 2 can be converted into pore throat radius r using a constant conversion coefficient C. Thus conversion coefficient C that scales relaxation time T2 into MICP average pore throat radius r is determined by a method mentioned by Saidian and Prasad2LM)  is calculate using Relative average deviation (RAD) and relative standard deviation (RSD) in percentages are calculated using RAD and RSD of permeability test is less than 2% and RAD and RSD of porosity test is less than 1%. RAD and RSD of density test is less than 0.05% and RAD and RSD of viscosity test is less than 0.5%. RAD and RSD of NMR test is less than 0.6%.How to cite this article: Wang, H. et al. Measurement and Visualization of Tight Rock Exposed to CO2 Using NMR Relaxometry and MRI. Sci. Rep.7, 44354; doi: 10.1038/srep44354 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The surface was determined to be terminated by Al-O species, and was significantly different from the idealised bulk structure. Termination reversal was not observed at higher temperature (600 °C) and chamber pressure of 10−10 Torr, but rather an increased Al-O occupancy occurred, which was accompanied by a larger outwards relaxation of Al from the bulk positions. Changing the oxygen pressure to 10−6 Torr enriched the Al site occupancy fraction at the outermost surface from 0.245(10) to 0.325(9). In contrast the LaO, which is located at the next sub-surface atomic layer, showed no chemical enrichment and the structural relaxation was lower than for the top AlO2 layer. Knowledge of the surface structure will aid the understanding of how and which type of interface will be formed when LaAlO3 is used as a substrate as a function of temperature and pressure, and so lead to improved design of device structures.The surface crystallography and chemistry of a LaAlO One example is with SrTiO3 resulting in superconducting32Cu3Ox7− superconducting films3 undergoes a structural phase transition from rhombohedral R−3c to cubic Pm-3m at ~430–530 °C673 has been documented at different temperatures and pressures, the surface crystallography and chemistry are less well understood due to the lack of characterisation techniques with appropriate sensitivity. However, in order to prepare an excellent interface, crucial for device performance, this knowledge is vital: for example it can directly affect the electronic density of states, valence edge shift and chemical activity. The termination of the LaAlO3 (001) surface has been reported to vary with temperature and oxygen partial pressure and there are inconclusive results reported in the literature910et al.et al.−10 Torr a mixed Al-O/La-O (RT) and La-O (727 °C) termination. In addition, a mixed Al-O/La-O termination at 400 °C under 10−10 Torr was proposed using data from scanning tunnelling microscopy and ion scattering spectroscopyet al.−2) was energetically favoured over AlO2 (1.79 J m−2).Lanthanum aluminate (LaAlOon gases DEGs whenℓ), for integer values of the in-plane indices (h and k). The total structure factor20Surface X-ray diffraction (SXRD) is now recognised as a very high resolution structural probe of two dimensional crystals in the form of a surface or interface and complementary to other techniques such as scanning tunnelling microscopy and low energy electron diffraction13141618wherefj is the atomic scattering factor of atom j, B the Debye-Waller parameter,  the atomic coordinate within the unit cell, α an attenuation factor and θ the surface occupancy factor.where et al.3 described an Al-O termination on the (001) surface at both RT and 400 °C under UHV (1 × 10−10 Torr) conditions. At both temperatures, the top surface oxygen position was found to move vertically away from the bulk accompanied by an inward relaxation of the aluminium atom. Their results focused only on measurements of the h0ℓ rods and specular reflectivity meaning that they were insensitive to the full in-plane relaxation. Instead the lateral movements were constrained using symmetry arguments  to prevent chemically unrealistic solutions.The only report, by Francis in-situ on a single crystal LaAlO3 (001) substrate, over a large range of reciprocal space . The effect of temperature on the surface structure was probed ) whilst the role of oxygen pressure (10−10 and 10−6 Torr) was also investigated. This is key information to enable the deposition of high quality LaO3-δ and YBa2Cu3O7-δ thin films for intermediate temperature solid oxide fuel cell cathodes and high temperature superconductors, respectively.In this report, we collected a comprehensive set of CTRs, −10 Torr, (ii) 600 °C, 10−10 Torr, (iii) 600 °C, 10−6 Torr of oxygen and (iv) RT, 10−6 Torr of oxygen , clearly observed for the −20ℓ, 11ℓ and 12ℓ rods . However varying the oxygen pressure did not affect the profiles significantly (details of the fits to these data at different conditions are presented in the 2 – LaO – AlO2) were included in the surface model  LaAlO3 (001) single crystal substrate measured at RT and 10−10 Torr showed a very poor fit (χ2 = 10.6) for a simple bulk termination model, which was only marginally improved (χ2 = 10.4) when roughness was included as shown in 2 and LaO) was the initial fitting parameter refined during the analysis. An occupancy value of 1 corresponds to a complete layer of the bulk terminated structure. The best fits showed the outermost surface to be well oriented and terminated with AlO2 but with a reduced occupancy of 0.121(6), whilst the occupancy of the second layer (LaO) was 0.321(2). The next two atomic layers were fully occupied. The atomic positions in the out-of-plane direction  were subsequently refined, leading to an optimised χ2 value of 2.4. Subsequent relaxation of the lateral (in-plane) atomic positions resulted in an improved fit (χ2 = 1.9) especially visible for 01 L and −1–2L CTRs  and Al(3)) were outwards from the bulk positions by 0.10(6) and 0.136(10) Å respectively, accompanied by a smaller inward relaxation of the heavier La(2) and La(4) cations (within the second layer LaO) of −0.090(5) and −0.019(2) Å respectively.Crystal truncation rod (CTR) experiments on the as-received samples demonstrated a very high quality to the surface of the crystal as revealed by the presence of a CTR signal at all positions along the rod see , Fig. S1f oxygen . A compashown in  consiste–2L CTRs . The fit2 to 0.245(10), whilst that of the second layer LaO reduced slightly to 0.306(2). The data is not consistent with termination reversal to a La-O upper layer at this high temperature, in contrast to reports by Yao et al.et al.ℓ in the higher temperature data). Specifically we find that ΔzLa(2) = −0.090(5) Å at RT and −0.032(5) Å at 600 °C leading to detailed information not only about the chemical segregation but also about surface relaxation.Fits to the data recorded at a higher sample temperature of 600 °C in ultrahigh vacuum indicated an increase in the occupancy of the first atomic layer of AlO to 0.245, whilst 2 layer. In addition, the scattering cross-section of La is higher than Alet al.et al.2NiO4 predicted that the B site of A2BO4 Ruddlesden-Popper type oxides  would be the preferential surface termination, in agreement with our data. Experimentally, however, the B-O rich surface has not been found in other doped ABO3 perovskites: La0.6Sr0.4Co0.2Fe0.8O3−δ and its derivatives such as the GdBaCo2O5+δ double perovskites, and La2−xSrxNiO4+δ single crystals characterised by LEIS, CTR and angle-resolved X-ray photoelectron spectroscopy (AR-XPS), were reported to show surface segregation of the A cations283, the absence of transition metals and alkaline earth elements that tend to segregate to the surface30Low energy ion scattering (LEIS) spectroscopy is a highly sensitive technique used to measure the atomic composition of the outermost atomic surface233 has been used. For example Liu et al.3 films onto LaAlO3 substrates at 800 °C in which n-type behaviour was expected due to a LaO-TiO2 interface, from the presumed La-O termination on the LaAlO3 substrate. This n-type interface should have shown metallicity characteristic of a two dimensional electron gas (2DEG)3 substrate (caused by the surface reconstruction), interfacing with nonpolar SrTiO3, which supresses the existence of the polar discontinuity, was proposed to be the main reason. However, based on the current study, an AlO2 terminated surface would be expected to form a p-type interface with SrTiO3 which typically requires an extra half a hole or removal of half an electron per surface unit to maintain charge neutrality, by creating an oxygen vacancy (Sr2+ O0.751.5−)p-type of interface is reported to be insulatingKnowledge of the detailed structure of the interface aids in understanding the behaviour of device structures reported in the literature where LaAlO2 occupancy was determined to be even higher (0.325(9)) when oxygen was admitted into the chamber to a pressure of 10−6 Torr at 600 °C. This increase in occupancy is only apparent for the outermost species in the first atomic layer whilst the second layer LaO occupancy is relatively stable at 0.3120(17). The top layer Al atoms, Al(1) and Al(3), move further outwards, away from the bulk terminated position compared to the measurements under ultrahigh vacuum. Cooling the single crystal back to RT under the same oxygen pressure (10−6 Torr) resulted in a further change to the occupancy of the AlO2 outermost surface layer. The occupancy of this layer was refined to be 0.174(9), significantly reduced from that of the surface at 600 °C, but still slightly higher than that determined at the same temperature but under ultrahigh vacuum. The optimised fits under different experimental conditions are shown in The surface AlO3 perovskite where Ti was found at the surface with the displacement going away from the bulk for (1 × 1) RT UHV, (2 × 1) RT UHV and (1 × 1) 750 °C, 10−3 Pa, except for (2 × 2) RT UHV35The overall displacements of the cations along the surface normal and laterally are presented in 3 single crystal substrate was found to significantly deviate from the ideal bulk structure. The outermost surface is mainly terminated by Al-O species whose occupancy increases with elevated temperature and under higher oxygen pressures, as determined by crystal truncation rod analysis and low energy ion spectroscopy. This is not ideal for in 2DEG applications where an insulating p-type interface with SrTiO3 is likely to form. The La-O in the second atomic layer is not fully occupied but was relatively unmodified over the range of temperatures and pressures studied. Under all conditions there was an outwards relaxation of the AlO2 terminating layer, which increased at higher temperatures and pressures, whilst the second layer La cations showed a smaller inward relaxation (≤0.090(5) Å) that was independent of the measurement conditions.In summary, the surface of a LaAlO3 (001) single-side polished substrates (5 × 5 mm2) were obtained from CrysTec Kristalltechnologie. The CTR measurements were carried out at the ultrahigh vacuum endstation of beamline I07ℓ) was recorded using a θ–2θ geometry (incidence angle = exit angle) with the surface normal horizontal while the CTRs were recorded with a fixed incidence angle of 0.5°. The structure factor was calculated from each image by taking the background subtracted integrated intensity of the CTR and applying correction factors to account for the polarisation correction and rod interception, prior to taking the square root. Data fitting was performed using the ROD and WinRod programsSingle crystal LaAlO3 (001) samples was also analysed using low energy ion scattering . The primary beam was a 3 keV 4He+, positioned normal to the surface with a collection angle of 145°. The sampled raster area of the LaAlO3 substrate used was 750 × 750 μm2. The spectra were measured in-situ at different temperatures and a chamber pressure of 10−9 Torr using an ion current of ~5 nA. The spectra were fitted using SurfaceLab 6 software  with Gaussian peaks of different elemental components with the background described by error functions38One of the as-received LaAlOHow to cite this article: Pramana, S. S. et al. Understanding surface structure and chemistry of single crystal lanthanum aluminate. Sci. Rep.7, 43721; doi: 10.1038/srep43721 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.