Although EAE is predominantly considered a T helper 1-driven autoimmune disease, mounting evidence suggests that activated dendritic cells (DC), which are the bridge between innate and adaptive immunity, also contribute to its pathogenesis. Sirtuin 6 (SIRT6), a NADEAE was induced in female C57bl/6 mice by MOG35-55 injection. The effect of treatment with a small compound SIRT6 inhibitor, administered according to therapeutic and preventive protocols, was assessed by evaluating the clinical EAE score. SIRT6 inhibition was confirmed by Western blot analysis by assessing the acetylation of histone 3 lysine 9, a known SIRT6 substrate. The expression of DC activation and migration markers was evaluated by FACS in mouse lymph nodes. In addition, the expression of inflammatory and anti-inflammatory cytokines in the spinal cord were assessed by qPCR. T cell infiltration in spinal cords was evaluated by immunofluorescence imaging. The effect of Sirt6 inhibition on the migration of resting and activated bone marrow-derived dendritic cells was investigated in in vitro chemotaxis assays.Preventive pharmacological Sirt6 inhibition effectively delayed EAE disease onset through a novel regulatory mechanism, i.e., by reducing the representation of CXCR4-positive and of CXCR4/CCR7-double-positive DC in lymph nodes. The delay in EAE onset correlated with the early downregulation in the expression of CD40 on activated lymph node DC, with increased level of the anti-inflammatory cytokine IL-10, and with a reduced encephalitogenic T cell infiltration in the central nervous system. Consistent with the in vivo data, in vitro pharmacological Sirt6 inhibition in LPS-stimulated, bone marrow-derived DC reduced CCL19/CCL21- and SDF-1-induced DC migration.Our findings indicate the ability of Sirt6 inhibition to impair DC migration, to downregulate pathogenic T cell inflammatory responses and to delay EAE onset. Therefore, Sirt6 might represent a valuable target for developing novel therapeutic agents for the treatment of early stages of MS, or of other autoimmune disorders. DC were then challenged to migrate towards a CCL19-CCL21 cocktail (which stimulates CCR7) or towards SDF-1 (which activates CXCR4). As shown in Fig. 1, demonstrating that pharmacological Sirt6 inhibition blocks DC migration. These results are in line with our previous findings in DC from Sirt6 KO mice [As mentioned above, a role for SIRT6 in mature DC migration was previously reported based on findings with DC isolated from wild-type and from Sirt6KO mice . Here, wing Fig. a. Next, 1, both at 7 dpi and at 15–17 dpi, i.e., at disease onset in vehicle-treated mice  SIRT6 promotes the release of TNFα from different cells (including DC) through different mechanisms –4, 9; (b1, in the EAE model of autoimmunity according to both a “preventive” and a “therapeutic” protocol. The most interesting result from this study was the delay in EAE onset, which was obtained by inhibiting Sirt6 according to our “preventive” approach. By this approach, we aimed at investigating the effect of Sirt6 inhibition during the early phase of the inflammatory process preceding the overt disease. In line with the studies indicating SIRT6 as a key player in regulating pro-inflammatory responses, our results indicate that SIRT6 inhibition delayed the inflammatory cascade occurring upon immunization and before EAE onset. Specifically, SIRT6 inhibition with 1 reduced the number of splenocytes and of lymph node cells in mice (Fig. 1 reduced the percentage of mouse lymph node DC expressing the pro-migratory surface marker, CXCR4 (Fig. Thus, based on SIRT6 role in inflammation and in DC, we decided to test the SIRT6 inhibitor, ice Fig. b, c; bluice Fig. d; reduceice Fig. ; and incice Fig. . In addiCR4 Fig. .1 reflects the effect of Sirt6 inhibition on DC migration, as revealed by the reduced representation of CXCR4-positive and of CXCR4/CCR7-double-positive DC in lymph nodes [1-treated animals (Fig. 1-pre-treated DC in response to chemokines (Fig. in vivo might also reflect the fact that TNFα release (as measured by evaluating TNFα in plasma, Fig. Given the well-established role of peripheral and CNS-resident DC in the development of EAE , 32, we ph nodes  of 1-tre1 administration was started at disease onset; see Fig. Overall, our results provide a rationale for further exploring SIRT6 inhibition as a strategy to treat MS or other autoimmune disorders by virtue of its effects on the DC compartment. Given the fact that Sirt6 inhibition failed to show an impact on disease course in the therapeutic protocol (where The present study demonstrates that preventive pharmacological SIRT6 inhibition delayed EAE disease onset, reducing the representation of CXCR4-positive and of CXCR4/CCR7-double-positive DC in lymph nodes. The delay in EAE onset was associated with the early downregulation of CD40 expression on DC and with reduced encephalitogenic T cell infiltration in the CNS. Sirt6 inhibition reduced the production of the autoimmunity-promoting cytokines, IFNγ and IL12, and increased the production of the anti-inflammatory cytokine IL-10. Therefore, Sirt6 might represent a valuable target for developing novel therapeutic agents for the treatment of the early stages of MS or of other autoimmune disorders. The administration of SIRT6 inhibitors should be done with caution, limiting it to defined time windows, taking into account that this enzyme also has a role as a tumor suppressor in certain tissues and that complete SIRT6 depletion can be very detrimental . NeverthAdditional file 1:. Figure S1. Gating strategy for flow cytometry analysis of DCs


Summer upwelling has often been observed off Vietnam in the South China Sea (SCS). Occasional disappearance of the upwelling has attracted much attention because it modulates the regional climate and harms surrounding fisheries. Fluctuations of the East Asian monsoon associated with El Niño are considered responsible for the weakened or abolished upwelling. However, analyses of observations performed in the present study were equivocal in terms of the dominant influence of El Niño. Based on long-term sea surface temperature data, we demonstrated that weak upwelling off Vietnam occurs more frequently during periods of accelerated global warming compared to warming hiatus periods. Warming signals in the Indian Ocean vanished relatively quickly during the hiatus period. The accompanying easterly anomalies south of the anomalous anticyclone (AAC) in the northwestern Pacific were also weakened, reducing the impact of the El Niño teleconnection on the SCS summer monsoon and thus preserving the regular post-summer upwelling off Vietnam during warming hiatus periods. The SCS connects the Java Sea to the south via the Karimata Strait, the Sulu Sea to the southeast via the Mindoro and Balabac Straits, the Pacific to the northeast via the Luzon Strait, and the East China Sea to the north via the Taiwan Strait10. The warming signal extended across the entire SCS due to the weakened summer monsoon and deeper thermocline10. Earlier studies attributed the weak upwelling to the delayed 1997/98 El Niño teleconnection  occurring over the northwestern Pacific during the mature phase of El Niño and persists until the following summer13. Easterly anomalies in the southern part of the AAC diminish the southwesterly monsoon over the SCS, thereby decreasing the upwelling off Vietnam13. This constitutes a theoretical mechanism for the weak SCS upwelling off Vietnam caused by El Niño events. Alternatively, a recent study suggested that both the Pacific and Indian Oceans influence SCS upwelling off Vietnam, where their interaction results in a perturbation of wind direction that reduces the upwelling6.During the summer of 1998, SCS upwelling off Vietnam was severely weakened, resulting in the warmest basin conditions on record, which were in turn associated with massive flooding over the Yangtze River valleyHowever, El Niño events are not always associated with a weakened summer monsoon and SCS upwelling off Vietnam. For example, during the post-2006 El Niño summer, monsoonal winds were abnormally intense and SCS upwelling off Vietnam was obviously enhanced Fig. c, indica10. In this study, an ensemble empirical mode decomposition14 (EEMD) was performed to extract various intrinsic mode functions (IMFs). When applied to 68-year (1950–2018) SST data averaged over the upwelling region , the EEMD yielded six IMFs. The first three IMFs are short-term fluctuations below the interannual timescale. Eight weakened summer upwelling events were identified, including 1958, 1973, 1983, 1987, 1988, 1995, 1998, and 2015 , chlorophyll-a concentration, and sea surface height (SSH) are often used as proxies for SCS upwelling off Vietnamear 1950–018 SST dear 1950–018 SST d−1; mean ~ 6 m s−1) over the upwelling region 21 and/or Atlantic multidecadal oscillation (AMO)20 20 Fig. a. During)20 Fig. b20,23. T−1. Notably, high-pressure anomalies, such as the AAC . Grids of SSTs where data are incomplete in temporal are ignored. Daily 0.25° × 0.25° SSH and geostrophic velocity 25 are adopted from the Archiving, Validation, and Interpretation of Satellite Oceanographic data (AVISO) . Daily 1.875° × 1.875° 10 m wind velocity and 2.5° × 2.5° sea level pressure of the National Centers for Environmental Prediction reanalysis 1 (NCEPr1)26 are provided by the National Oceanic and Atmospheric Administration/National Centers for Environmental Prediction (NOAA/NCEP) (https://www.ncep.noaa.gov/). Daily chlorophyll-a concentration data of the SeaWiFS and the MODIS are given by National Aeronautics and Space Administration (NASA)/Ocean Color (https://oceancolor.gsfc.nasa.gov/).Monthly 1° × 1° SST data of the Hadley Centre Sea Ice and Sea Surface Temperature product (HadISST)18 and Paek et al.27. The PDO and AMO indices are adopted from the Joint Institute for the Study of the Atmosphere and Ocean (JISAO) and the NOAA/Earth System Research Laboratory (ESRL), respectively.The definition of El Niño diversity in Table


Amaranthus retroflexus pollens. The main gene of this allergen, Ama r 2, was cloned into the food grade plasmid pNZ7025 and then was electrotransformed into the food grade Lactococcus lactis NZ1330. No expression was observed in the primary structure due to the distance between the ribosome binding site and the start codon. Therefore, the vector structure was corrected using the site-directed mutagenesis (SDM) technique. The cell extract of this strain was used for assessing the expression of the recombinant allergen in western blot analysis, and the existence of this protein with a molecular weight of 14.2 kDa was confirmed. To evaluate the efficacy of this strain in the treatment of allergies as an oral vaccine, probiotic ice cream was prepared. After the sensitization of mice, the treatment was performed by oral immunotherapy for 4 weeks, 4 to 5 times per week. 20 μl of functional ice cream with 1012 CFU/ml of r-L. lactis NZ1330 significantly reduced the serum IgE level. The levels of IFN-γ and TGF-β cytokines increased in the 20 μl ice cream treatment group as well as 40 μg/ml pure allergen compared with the PBS-treated group, and IL-4 cytokine levels decreased compared with the PBS-treated group. Overall, 20 μl ice cream with 1012 CFU/ml of the recombinant bacteria resulted in the best performance in terms of improving allergies to Th1 and Treg responses.This study was conducted to evaluate the effects of recombinant probiotic bacteria as a candidate for oral vaccine with the potential of treating allergy to  Amaranthaceae mediated by immunoglobulin E (IgE) is prevalent in semidesert countries like Saudi Arabia, Iran, and Kuwait. In particular, among Iranian allergic patients, allergy to A. retroflexus pollen has been reported with a high positive rate of 68.8% [β-galactosidase through the recombinant L. lactis with the potential to alleviate lactose intolerance symptoms [L. lactis ameliorates nonalcoholic fatty liver and early atherogenesis in cholesterol-fed rabbits [Lactobacillus acidophilus (ATCC 4357D-5) and Bifidobacterium animalis subsp. lactis (ATCC 27536) in the ice cream after adding dietary fibre-rich. They said that these products could be used in ice cream with the improved survival of the probiotic bacteria [Allergy is a disorder created when the immune system reacts to harmless antigens. Allergic diseases comprise heterogeneous inflammatory pathologies such as respiratory, cutaneous, and food allergy , 2. 300 of 68.8% , 5. New of 68.8% . The rec rabbits . Kalicka rabbits . Ayar etbacteria .L. plantarum induces of Derp-1 which decreases the development of the Th2 allergic response by a reduction in specific IgE and the induction of allergen-specific IgG2a antibodies [L. lactis (LLDM) was engineered to deliver the Der p2 derivative to the intestinal mucosal surface. Oral immunotherapy of r-L. lactis with 2 × 109 cfu/ml dose was done for 5 days per week for 14 days. Oral administration of r-L. lactis alleviated the Der p2-induced airway inflammation, as shown by the reduced inflammatory infiltration and a reduction in Th2 cytokines in bronchoalveolar lavage [Lactic acid bacteria (LAB) are a series of Gram-positive bacteria such as Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, and Oenococcus species. LAB are employed in the production of fermented foods from agricultural raw materials, including milk, cereals, meat, and vegetables. Additionally, LAB strains are the most prevalent microorganisms applied as probiotics. Probiotics defines as alive microorganisms which confer the health effects when administered in adequate amounts , 13. Thetibodies . Ai et ar lavage .L.lactis in reducing allergic response against Amaranthus retroflexus pollens. To investigate the efficacy of the recombinant strain in the treatment of the allergy, the functional ice cream was prepared, and the performance was evaluated through animal model.The objectives of the present study are to investigate the effect of recombinant L. lactis NZ1330  was considered the host for cloning and expression. pNZ7025  was applied as the cloning and expression vector for the production of protein. L. lactis NZ1330 which was grown in M17 medium at 30°C  supplemented with 1.5% glucose and 200 μg/ml D-alanine without aeration. Escherichia coli was cultured on LB agar and broth  and incubated at 37°C with a shaking of 180 rpm.β, and interferon gamma (IFN-γ) enzyme-linked immunosorbent assay (ELISA) kits were obtained from Karmania Parsgene (Iran), and a IgE antibody ELISA kit was obtained from Parstous (Iran), respectively. Phytohaemagglutinin (PHA), culture medium RPMI 1640, fetal bovine serum (FBS), and penicillin/streptomycin were obtained by Gibco (UK).Taq DNA polymerase, PFU, Phusion High-Fidelity DNA Polymerase, dNTPs, DNA marker, restriction enzymes, T4 DNA ligase, Plasmid Miniprep Kit, gel extraction kit, and low molecular weight protein marker were supplied from Thermo Fisher Scientific (The USA), NEB (The USA), Roche (Germany), and Parstous (Iran). Mouse interleukin (IL)-4, TGF-L. lactis NZ1330. The signal peptide Usp45 (81 bp in length) and the Ama r 2 gene, which carries the restriction sites of Sph I and Spe I at each terminal, start and stop codon and 6×His tag  with an accession number of FJ899746.1. The Ama r 2 encoding sequence was codon-optimized for the appropriate expression in ×His tag , were sySph I and Spe I restriction enzymes, the respective gene was purified and then cloned into a digested pNZ7025 vector. Ligation was carried out for 15 min at ambient temperature using a T4 quick ligation kit. The pNZ7025-Ama r 2 was cloned directly in L. lactis through electroporation. Positive colonies can grow on M17 agar without D-alanine, approved by direct colony PCR using pNZ7025-F (AGATCTGTCGACCTGCAGT) and pNZ7025-R (CTATCGAAAGCGAAATCAAACG) primers specifically designed for the pNZ70225 vector. The PCR conditions were as follows: initial denaturation at 94°C for 10 min, 32 cycles , followed by a final elongation at 72°C for 10 min. Moreover, the plasmid DNA resulting from the positive clones was purified and introduced to DNA sequencing [After the digestion of the pBHA vector by quencing .L. lactis was mixed with 10 μl of 4X loading. The mixture was boiled at 95°C for 5 min and then electrophoresed on 12% SDS gel electrophoresis. For western blot, after the electrophoresis, the refolded protein was blotted onto polyvinyl difluoride (PVDF) membrane in western blot transfer apparatus . The gel was covered with the transfer buffer comprised of Tris (25 mM), glycine (190 mM), and methanol (20%). The transfer was conducted at a constant current of 300 mA in a cold room for 15 min. After washing and blocking with 2% bovine serum albumin (BSA) for 16 h at 4°C, membranes were incubated with the monoclonal anti-polyhistidine peroxidase conjugate antibody  at 1 : 5000 dilutions at RT for 3 h. Finally, the blot washed with PBS and subjected to chemiluminescense substrate based on the recommendations of the manufacturer [SDS-PAGE and western blot analyses were done for the samples which cultured during 5 days. Thirty microliters of M17 broth and the supernatant of the sonicated Blocking was performed with 2% BSA in PBS at 4°C overnight. It was subsequently washed thrice with PBS and probed with.The purpose of SDM was to modify the construct of the vector containing Ama r 2, because the expression of this protein was not observed in SDS-PAGE and western blot. For this purpose, 5 primers were designed whose sequences are presented in μl of the PCR product with the first pair primers and 9 μl of that with the second pair primers were mixed with 2 μl of the hybridization buffer under the following temperature cycles: 95°C for 10 min, vortex for 2-3 sec, 80°C for 3 min, 70°C for 3 min, 60°C for 3 min, 45°C for 3 min, and finally, ambient temperature. After hybridization, the excessive salts were removed from the final product so that no perturbation would be created in electroporation. After that, electroporation was conducted based on the previously mentioned protocol. The colonies grown on M17 agar were subjected to direct colony PCR with the specific primers of the vector so as to confirm the presence of the insert-containing vector. However, since some colonies might have belonged to the recombinant bacteria with the primary insert-containing vector, we should have distinguished between the colonies with the primary and modified vectors. To that end, a primer was designed (SDM-F3) whose 3′ end exactly overlapped the 8 nucleotides whose removal was the final goal. Consequently, direct colony PCR was performed on the picked up bacteria using SDM-F3 and SDM-R2 pair primers. In all the cases, in order to observe the bands, agarose gel 1% was prepared in TBE buffer and run at 100 V for 30 min [The correct design of primer, the application of the polymerase with the lowest error rate, and proper hybridization are the key factors in the appropriate conduct of this technique. PCR was carried out for each of the pair primers of SDM-F1 and SDM-R1 and SDM-F2 and SDM-R2 under the following conditions: initial denaturation at 95°C for 5 min, then 15 cycles , 30 sec; and 72°C, 2 min) followed by a final elongation at 72°C for 10 min. In the next stage, the hybridization of the replicated strands was performed so that the complementary strands paired with each other. For this purpose, the 10X hybridization buffer was prepared . Nine r 30 min .L. lactis NZ1330 with 1012 CFU/ml added, and incubation was done for 6 h. They kept the mixture in the refrigerator for 24 h to allow the ripening process. It was then added to the ice cream maker and processed for 20 min. To evaluate the viability of the r-L lactis NZ1330 strain in the product, before freezing and during storage, every 10 days sampling was performed [For the probiotic ice cream formulation, 57.92% skimmed milk, 20.87% heavy cream (40% fat), 2.71% dry milk, 17% sugar, 0.5% stabilizer and emulsifier (Pulsgard), and 1% vanillin flavor were used. At first, the milk and cream were poured into the formulation tank and heated slowly. When the temperature reached 50°C, the skim milk powder, dry milk, pulsgard, sucrose, and vanillin were added. The ice cream mixture was then pasteurized at 60°C for 30 minutes. Afterwards, the ice cream mixture was cooled to 30-40°C, r-erformed .E. coli containing pET-21b-Ama r 2 constructs was used [To produce a large-scale Ama r 2 protein, recombinant was used . The prowas used .35 Balb/c mice  were purchased from the Pasteur Institute of Iran. Animal keeping procedures were conformed according to the standard guidelines of animal care and were approved by the Animal Ethics Committee of Ferdowsi University of Mashhad (IR.UM.REC.1398.066). After one week of habituation, they were randomly divided into 7 groups .μg r-Ama r 2 plus alum adjuvant (2 mg) on days 0 and 14, and then subsequently, they were sensitized by the inhalation using the r-Ama r 2 solution at a concentration of 0.1 mg/ml and using a nebulizer since day 21, each day for 20 minutes over four consecutive days [Mice sensitization was performed according to a standard protocol with two intraperitoneal injections containing 10 μl of cell suspension was merged with 20 μl of trypan blue dye, and cells were counted with a neobar slide under the microscope. Cells were cultured in 24-well flat-bottomed culture plates in duplicate at a density of 2 × 106 cells/ml and stimulated with Ama r 2 (10 μg/ml), PHA (1 μg/ml), or media at 37°C. The cell supernatant was collected after 72 h to measure TGF-β, IFN-γ, and IL-4 cytokines [Treatment was performed orally with gavage needles, 4-5 times a week for a month at specified amount and concentrations. The mice were again exposed to the rAma r 2 allergen solution on days 60 and 61 using a nebulizer. At day 62, mice were euthanized, and serum and spleen of different groups were separated for the evaluation of the humoral and cellular immune responses. Serum from different groups was collected to evaluate changes in the IgE antibody levels and analyzed using the ELISA technique. The spleen cells were removed by sterile forceps and scissors and transferred to a 1.5 ml microtube from an incomplete cold RPMI medium. The cells were then ejected into petri dishes by injecting 2-3 ml of incomplete RPMI with a syringe needle from one side of the spleen. Next, the cells were collected and poured into a sterile 15 ml falcon . The tubes were then centrifuged at 1500 rpm for 4 min at 4°C, and the supernatant was removed. After that, the cells attached to the falcon body were removed and suspended. For the RBC lysis, 5 ml of cold lysis buffer or ammonium chloride buffer was added to the suspension and gently mixed by inverting the tube. The tubes were incubated on ice for 5 min and washed twice with 5 ml of sterile PBS to remove ammonium chloride. Next, 2 ml of complete RPMI was added to the cell suspension. Then, 20 ytokines .p values <0.05 were considered significant.Descriptive statistics, mean, standard deviation, tables, and graphs were used to describe the data. The statistical analyses were performed using GraphPad Prism 8.0.2. The data have been expressed as mean ± standard deviation. Means were compared using one-way analysis of variance (ANOVA) followed by Tukey–Kramer test. All L. lactis was examined with direct-colony PCR. Plasmid primers amplified a 1000 bp fragment which contained the Ama r 2 sequence. While PCR was being performed on self-plasmid colony, the segment which should have been observed on the agarose gel was 488 bp in length  was designed whose 3′ end exactly matched the 8 nucleotides which were going to be removed from the vector construct. If the vector were not modified, the primer would match the segment, be replicated, and its band would be observed on the agarose gel. On the other hand, if the 8 nucleotides were removed, the primer could not attach with the segment and consequently, no band would be observed on the gel.The spacing between the RBS and the translation initiation codon remarkably differs in naturally occurring mRNAs with a mean length of 7–8 nt. Extremely long or short spacing between the RBS and the start codon could be harmful to the effective translation initiation . ConsequAs a result, the grown colonies on the medium were analyzed using two pair primers: firstly, with the specific pair primers of the vector to ensure the presence of the pNZ7025 vector and secondly, the bacteria with positive responses were analyzed using SDM-F3 and SDM-R2. As can be seen in μg/ml after the band analysis in comparison to the standard sample.Western blotting is a widely used analytical technique in cell and molecular biology to detect specific proteins in a mixture of proteins . As the L. lactis which selected marker was erythromycin, and the expression induction was done with 2% lactose . Glenting et al. expressed Ara h 2, the main peanut allergen, in L. lactis. The Ara h 2 gene was cloned into pAMJ399 plasmid containing the P170 promoter and the SP310mut2 signal sequence. Also, the recombinant bacteria were selected based on the resistance to erythromycin . In the studies carried out on the production of recombinant allergens in prokaryotic and even probiotic hosts, in order to select the recombinant strain in the culture medium, the resistance marker to antibiotics such as erythromycin and chloramphenicol is used as a selector so that the vector-receiving strain can survive in the medium containing antibiotic (Rosano and Ceccarelli 2014). Based on our current knowledge, the utilization of food grade vectors and the hosts fulfilling food industry standards for allergen expression either have not been performed. FDA suggests not employing antibiotics for the selection of bacteria. In particular, beta-lactam antibiotics, like penicillin, should not be applied during the production of a therapeutic product for human consumption; this may allow the transfer of the resistance gene to the intestine microflora, which can cause problems in curing infectious diseases . As a result, in a previous research, Vasiee et al. investigated the resistance of the r-L. lactis NZ1330 containing the pNZ7025-Ama r 2 plasmid against 9 antibiotics, namely, vancomycin (30 μg), fosfomycin (200 μg), kanamycin (30 μg), gentamycin (10 μg), neomycin (30 μg), cefixime (5 μg), ciprofloxacin (5 μg), ampicillin (10 μg), and erythromycin (15 μg). Their results revealed that this recombinant strain did not resist against any of these antibiotics .The application of genetically modified and/or recombinant organisms causes many concerns. In order to lower these concerns, expression systems—also known as food grade—have been introduced . The distinguishing characteristic of such vectors is the lack of antibiotic resistance markers (Tagliavia and Nicosia 2019). Numerous studies have been conducted concerning the application of LAB hosts in the expression of allergens. Nasiraie et al. cloned Che a 2 allergen, as a candidate for oral immunotherapy, into L. lactis NZ1330 was prepared for oral use for treating sensitized mice to Amaranthus retroflexus pollen. In this study, probiotic bacteria were counted within 90 days of production (every 10 days) with the serial dilution method on M17 agar medium  on the reduction of IgE was significantly different compared to the control group. The treatment group with a daily consumption of 20 and 50 μl of probiotic ice cream containing r-L. lactis NZ1330 showed a significant difference (p < 0.001) compared to the PBS-treated group. There was also a significant difference between the treatment group with 100 μl probiotic ice cream (p < 0.01) with the control group. There was no significant difference between the treatment groups with a daily consumption of 20 and 50 μl of probiotic ice cream, although there was a significant difference at the p < 0.05 level with the 3 different ice cream formulations. One of the long standing immunotherapy methods is using low doses of pure allergen for treating allergy which in this study, recombinant allergen produced, purified, and dialyzed at a concentration of 40 μg/ml and was used to evaluate the efficacy of this treatment. As shown in μl daily functional ice cream intake group, although the difference was significant at the p < 0.01 level compared to the PBS-treated group. To ensure the efficacy of the recombinant probiotic strain for the treatment of allergic rhinitis, the recombinant bacterium with the Ama r 2 gene as well as the recombinant bacterium with the pNZ7025 vector without the Ama r 2 gene that were diluted to the 1012 CFU/ml in PBS buffer was also included in treating groups. Therapeutic effect of the r-L.lactis NZ1330 group (Ama r 2 gene) was not significantly decreased IgE compared to the PBS treatment group. However, the group treated with recombinant probiotic bacteria (without Ama r 2 gene) had a significant difference at the p < 0.01 level with the group treated with PBS. r-L. lactis NZ1330 (Ama r 2 gene) treatment groups and r-L. lactis NZ1330 had a significant difference at the p < 0.05 level.Although there are various methods for measuring this immunoglobulin, the most important and precise method is ELISA . Howeverμl of treatment and between the pure r-L. lactis NZ1330 group (Ama r 2 gene) and pure r-L. lactis NZ1330 showed no significant difference. There was a significant difference between the effect of probiotic ice cream groups compare to the PBS group at p < 0.0001. Difference between r-L. lactis NZ1330 groups (Ama r 2 gene) and pure r-L. lactis NZ1330 and PBS-treated group was also significant at the p < 0.001 level.IL-4 is the major cytokine in stimulating B lymphocytes to produce IgE. This cytokine also prevents antibody class switching to IgG2a and IgG3. IL-4 inhibits macrophage activity and many of the positive effects of interferon-gamma to increase the production of cytokines such as IL-1 . The IL-γ is a cytokine that is usually produced by Th0 and Th1 lymphocytes as well as most CD8+cells. The transfection of the IFN-γ gene begins immediately after the stimulation of the cell with the antigen, and IL-2 and IL-12 exert a resonant effect on it. This cytokine plays a major role in the development of cellular immune-mediated diseases. This cytokine is the most important activator of macrophages and unlike IL-4, IFN-γ decreases IgE levels. It can also modulate the antibody class to IgG2a and IgG3 subunits by affecting B cells and prevent the IgG1 and IgE production [γ levels after oral immunotherapy is shown in γ production than the PBS treatment group. This increase was significant in the treatment group with 40 μg/ml allergen compared to the PBS group at p < 0.0001. The difference between the 40 μg/ml allergen and 20 μl functional ice cream was significant at p < 0.05. Also, the difference between the three probiotic ice cream groups were significant at p < 0.0001. There was no significant difference between the treatment group of 50 and 100 μl of probiotic ice cream. There was a significant difference between the consumption of bacteria with the Ama r 2 gene and no Ama r 2 gene at the p < 0.05 level. Also, there was a significant difference between ice cream groups with 50 and 100 μl daily consumption and pure bacteria with and without the Ama r 2 gene at the p < 0.0001 level.IFN-oduction . The resβ is an anti-inflammatory cytokine that is produced by a large number of immune cells, including regulatory T lymphocytes and macrophages. This cytokine has many anti-inflammatory properties and is present on a large number of immune cells containing the receptor and can alter the type of antibody to IgA, which is of particular importance in mucosal immune responses [β cytokine production was significantly increased in the 6 treatment groups compared to the normal group .TGF-esponses . Based oal group . There wL. lactis NZ1330 against the development of aspiration allergy manifestations. The r-L. lactis contains the Ama r 2 gene as a means of producing and transmitting this allergen (simultaneously) to intestinal epithelial cells. Because of the patient's preference for the drug to be used until the injection, the efficacy of oral immunotherapy was chosen to treat the sensitive mice. To investigate the effect of bacterial counts on allergy treatment, three ice cream volumes were used . The results showed that by increasing the volume of ice cream gavage, the evaluated parameters became weaker. The results of using 20 μl of ice cream daily had a better response than 50 and 100 μl groups. Regarding the evaluation of cytokines, after ice cream with a volume of 20 μl, the pure allergen group provided the best response, but for IgE, the ice cream group with a volume of 50 μl was second in the reduction of this antibody. In the case of IL-4, the 50 μl ice cream group showed a better response to ELISA compared to the 100 μl group, whereas the IFN-γ treatment group received 100 μl probiotic ice cream. In addition, analysis TGF-β showed that the treatment group containing 20 μl of probiotic ice cream showed a better response to pure allergen. Hajavi et al. reported that sublingual immunotherapy by 12.5 μg/dose of rChe a 3 encapsulated in PLGA was effective than other doses (25 and 50 μg) in reducing the eosinophilic infiltration and Th2 response [L. plantarum induces of Derp-1 which decreases the development of the Th2 allergic response by a reduction in specific IgE and the induction of allergen specific IgG2a antibodies [L. lactis (LLDM) was engineered to deliver the Der p2 derivative to the intestinal mucosal surface. Oral immunotherapy of r-L. lactis with 2 × 109 cfu/ml dose was done for 5 days per week for 14 days. Oral administration of r-L. lactis alleviated the Der p2-induced airway inflammation, as shown by the reduced inflammatory infiltration and a reduction in Th2 cytokines in bronchoalveolar lavage [The current study explored the potential efficacy and mechanism of action of r-response . Rigaux tibodies . Ai et ar lavage .Lactococcus lactis, is one of great interest in the areas of recombinant enzyme production, industrial food fermentation, gene, and metabolic engineering, as well as antigen delivery for oral vaccination. In this study, the expression of the recombinant allergen, Ama r 2, was evaluated in the probiotic host, L. lactis NZ1330. The recombinant bacteria were fed to mice, and their effects on allergy treatment were evaluated. IgE, IL-4, TGF-β, and IFN-γ were evaluated using ELISA. The best response was seen in the treatment group with 20 μl of ice cream daily. Generally, the food grade L. lactis NZ1330 can be considered as a good vehicle to deliver therapeutic proteins to target sites in the body.The food-grade production of recombinant proteins in Gram-positive bacteria, especially in


Dermatophagoides farinae (DF) and D. pteronyssinus (DP) as biomarkers by using in-house ELISAs in healthy (n = 33) and atopic dogs (AD) (n = 44) that were either positive or negative by IDT to HDM.In dogs with atopic dermatitis, intradermal testing (IDT) or allergen specific IgE serological testing are routinely employed to identify causative allergens. These allergens can then be used for allergen-specific immunotherapy and allergy management. The clinical relevance of this testing is affected by the source of allergen, and other biomarkers that are more related to specific allergens still need to be identified. The aim of this study was to investigate levels of specific IgE, total IgG, and IgG1 and IgG2 subclasses against the local house dust mites (HDM) p = 0.0049), but there was no relation to IDT outcomes . High levels of all antibody isotypes  against HDM were found in aged healthy dogs (> 3 years old). In AD, HDM-IgE and IgG1 levels were higher in dogs that were IDT positive to HDM than in IDT negative animals. Levels of IgE and IgG1 could be used to distinguish the specific allergens, whereas total IgG and IgG2 levels were not different between IDT-positive and IDT-negative AD. By the receiver operating characteristic curve at a false-positive rate = 0.10, both IgE and IgG1 showed better sensitivity than IgG and IgG2. Similar to IgE, serum IgG1 concentration was also relevant to IDT outcomes.Being over 3 years of age was a risk factor for AD (Odds Ratio (OD) = 4.10, 95% Confidence interval (CI) 1.57–10.75, Our in-house ELISAs coated with local HDM were useful for evaluating antibody levels, and we propose use of the HDM-specific IgG1 subclass as a biomarker to detect HDM specific allergens in AD, potentially together with an IgE based platform.The online version contains supplementary material available at 10.1186/s12917-021-02768-2. Antigens extracted from locally collected house dust mites (HDM) improved the sensitivity and specificity of ELISAs used to detect evidence of exposure to HDM in atopic dogs (AD)HDM-specific IgG1 was identified as a useful biomarker to detect HDM allergens in AD, potentially used together with an IgE based platformThe serological approach to detection of HDM allergy gave less consensual results in dogs over 3 years old.Canine atopic dermatitis (CAD) is a major chronic immune-mediated inflammatory and pruritic skin disease, with a genetic predisposition . UnderlyDermatophagoides farinae (DF) and D. pteronyssinus (DP) extracts in healthy dogs and in atopic dogs which had positive or negative IDT reactions to the HDM.The aim of the study was to identify an improved biomarker for use in CAD. This involved investigating levels of allergen-specific IgE, total IgG, IgG1 and IgG2 antibody against the local house dust mites (HDM) p = 0.3359) (Table p = 0.0049), but there was no relation to IDT outcomes   of the dogs were positive to HDM (DF and DP) by IDT Table . The num9) Table . Being oHDM extracts contained at least 32 mg of crude protein per gram of purify lived HDM. The concentration of group 1 allergens in DF and DP extracts were 40 and 36.75 μg/ml, respectively. As shown in the cropped electrophoretic gels in Fig. The IgE ELISA for HDM presented an acceptable reproducibility value, and %CV of intra-and inter-assay were 5.4 and 7.1%, respectively. For HDM specific IgG and its subclasses, the % CV of intra-and inter-assays were about 3.0 and 5.8%, respectively.Aged healthy dogs had significantly higher DF specific-IgE levels than both younger healthy dogs and AD Fig. a. ConsidFor DF-specific IgG1 levels, the aged healthy dogs showed the highest median levels compared to the other groups Fig. c. In theSimilarly, the pattern of antibody levels specific to the DP antigen Fig. e-h reassThe ratios of DF-IgG1/IgG and IgG2/IgG were calculated to reduce the individual confounder of their total specific IgG difference. A low DF-IgG1/IgG ratio and high value for the DF-IgG2/IgG ratio were observed in the young healthy dogs whereas the patterns were switched in the aged healthy dogs Fig. a. The raThe DP-IgG1/IgG ratio level was higher in the aged healthy group than in the young group Fig. b, as witBy using our in-house ELISAs, the sensitivity and specificity values achieved for the different antibody classes and antigens varied considerably as shown by the receiver operating characteristic curve (ROC) Fig. . For conIn this study we explored levels of specific IgE, IgG, IgG1 and IgG2 antibody against local sources of DF and DP extracts in healthy dogs and in AD by using in-house ELISAs. IgE levels measured by ELISA were not satisfactory as a screening test without clinical diagnosis, because healthy dogs still presented high levels of DF- and DP- IgE . The HDM extracts especially standard groups 1 allergen extracts (Der f 1 and Der p 1)  are routIn addition to the IgE level, the finding of high levels of HDM–specific IgG, IgG1 and IgG2 among the aged healthy group confirmed the need for caution about background antibody levels: although the antibodies were specific to HDM the latter still consist of a number of proteins . ConversD. farinae- and D. pteronyssinus-specific IgG1/total IgG reflecting true positives. In contrast, the IgG2/ total IgG levels clearly distinguished the healthy and HDM-IDT negative group from the HDM-IDT positive group, indicating an increase in specificity. A bias towards IgG1 is believed to occur as a response in TH2-mediated disease in mice and dogs, especially in the case of canine leishmaniasis [Leishmania chagasi-specific IgG2 was present in vaccinated dogs or asymptomatic dogs exposed to this protozoan [Regarding the IgG subclass volume in dogs, the proportions of IgG1 and IgG2 are approximately equal and deviate during episode of inflammation; for instance, high IgG2 levels occur in furunculosis, otitis externa and in autoimmune haemolytic anaemia . Varyingmaniasis , 20. On rotozoan . Our finIn this study, IDT was used as the gold standard, but associated technical problems were not fully evaluated in clinical samples. Regarding the agreement between IDT and ELISA, serological detection might serve as a tool for specific allergen identification as confirmed by using HDM allergenic model, but this was not recommended for investigation in healthy dogs. In Thailand, IDT is widely used because the commercial ELISA is costly and time-consuming. Identification of DF allergy through our IgE ELISA had a similar sensitivity to a commercial IgE ELISA using high affinity Fc-epsilon receptor alpha chain protein (FcεRIα) , this coInterestingly, the DP-IgE ELISA in this study showed better sensitivity than that commercial test. Different sources of DP might influence the results of sensitivity , and locThere has been a lack of information on the role of IgG1, which may act as another allergic marker, or possibly function as a blocking antibody after an IgE response. In humans with allergic rhinitis, IgG1 production depends on the frequency of protein exposure, and its response becomes dominant after ASIT . The lacLevels of IgG1 and IgE had similar patterns during episodes of HDM allergy in CAD. An in-house ELISA using IgG1 could be used to differentiate between DF or DP allergy, and showed the similar trend as IgE detection. Unfortunately, this serological approach gave less consensual results in dogs over 3 years old. The detection platform for HDM allergy identification using IgG1 and ratio of IgG1/IgG was affordable, valid, and only required a very small serum sample.A total of 33 healthy dogs were enrolled from the Chulalongkorn University blood bank, with the following inclusion criteria: normal at physical examination; not suffering from any diseases; no previous history of skin problems; and normal skin appearance. Serum samples were collected and stored at − 20 °C until used.Our AD subjects were enrolled from the Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Kasetsart University. CAD was defined in 44 dogs by a combination of their clinical histories, clinical signs, match to Favrot’s criteria , ruling g, 4 °C for 5 min . Composition analysis of extracts were performed for quality control. All the processes of validation and production of DF and DP extracts were executed following good manufacturing practice (GMP) regulations, under the supervision of an SDMC specialist. Protein content was measured by Bradford’s assay , and the supernatant was kept in aliquots at − 20 °C until used. Antigenic bands of HDM extracts were checked by SDS-PAGE, as previous reported [Purified DF (batch no. DF-SDMC 080158) and DP (batch no. DPT-BKK 060158), both with > 99% purity, were obtained from SDMC. Two grams of each mite had been individually resuspended in 8 mL of 0.01 M phosphate buffered saline (PBS), pH 7.4 and then were homogenized using an Omni Sonic Ruptor 4000  at 35% amplitude with 0.5 cycles . The supernatant was collected after centrifugation at 12,000 x reported . To confreported  In additPooled serum samples from five AD dogs and five young healthy dogs were used in a checker-board titration. AD dogs were selected for their strongly positive IDT (+ 4) to DF/DP from commercial (ALK Abello and Greer Labs Inc) and SDMC allergens and also their positive results to both DF and DP in a commercial ASIS . Sera from young healthy dogs (6-8 months old) negative by commercial ASIS were used as negative controls. DF and DP extracts were separately prepared in 0.2 M sodium carbonate-bicarbonate buffer, pH 9.4 . One hundred microliters of HDM solution was added into the wells of 96-well plates  and incubated overnight at 37 °C. After washing with washing buffer consisting of PBS containing 0.05% Tween 20  (PBST), 200 μl of blocking buffer ) was added and incubated at 37 °C for 1 h. Plates were rinsed before adding 100 μl of diluted pooled serum samples in duplicate, consisting of two-fold serial dilutions in blocking buffer starting at 1/5. Plates were incubated at 37 °C for 2 h before washing. Monoclonal anti-dog IgE  diluted in blocking buffer (1/2000) were added and incubated at 37 °C for 1 h. After washing, 100 μl of a phosphatase conjugate (1/2000 in blocking buffer)  was added, incubated at 37 °C for 1 h, and washed as before. Alkaline phosphatase substrate  was added at 25 °C for 20 min, and plates were immediately read at an optical density (OD) of 450 nm using an ELx808 ultra microplate reader and KC4 3.3 Rev. Ten software (Bio-Tek instruments Inc.). Results were subtracted with the OD of the blank control before generating titration curves. The middle point of the near-linear part was selected as an optimal point. The optimal HDM concentration for coating was 20 μg/mL with a serum dilution of 1/5. The reaction was quantified in ELISA units to act as a reference, as s previously described . ResultsChecker-board titrations for the other antibody classes were performed as described for the IgE ELISA, with some modifications. Three different peroxidase conjugates were used to detect allergen-specific IgG, IgG1 and IgG2, included polyclonal anti-dog IgG (IgG antibody (AAI32P), Bio-Rad.), polyclonal anti-dog IgG1 (IgG1 antibody (AHP947P), Bio-Rad), and polyclonal anti-dog IgG2 (IgG2 antibody (AHP948P), Bio-Rad), respectively. These conjugates were individually diluted in blocking buffer at 1/4000 before use. Peroxidase substrate  was added for colour development. The optimal serum dilution was 1/500.p values were less than 0.05. The validity of each antibody isotypes to indicate HDM allergy in AD dogs was analyzed by ROC curve, and AUC considered their significant if p values were less than 0.05.GraphPad Prism (GraphPad Software Incorporated) was used for statistical analyses. Risk factors were analyzed in the sample population. Levels of DF- and DP- specific immunoglobulin between healthy and AD dogs were determined for normality by the D’Agostino & Pearson omnibus, and medians were compared by the Kruskal-Wallis. The Dunn’s multiple comparisons test was applied for post doc analysis. Differences were considered significant if Additional file 1: Supplementary Table 1. Sensitivity and specificity (%) of in-house ELISAs through different type of specific immunoglobulins.


Clustering and visualization are essential parts of single-cell gene expression data analysis. The Euclidean distance used in most distance-based methods is not optimal. Batch effect, i.e., the variability among samples gathered from different times, tissues, and patients, introduces large between-group distance and obscures the true identities of cells. To solve this problem, we introduce Batch-Corrected Distance (BCD), a metric using temporal/spatial locality of the batch effect to control for such factors. We validate BCD on a simulated data as well as applied it to a mouse retina development dataset and a lung dataset. We also found the utility of our approach in understanding the progression of the Coronavirus Disease 2019 (COVID-19). BCD achieves more accurate clusters and better visualizations than state-of-the-art batch correction methods on longitudinal datasets. BCD can be directly integrated with most clustering and visualization methods to enable more scientific findings. Single-cell RNA sequencing (scRNA-seq) technologies profile thousands of cells simultaneously , enablinAs a practical example, researchers have collected data from the developing retina of 13 mice at different ages . IdeallyTo date, multiple methods have been published to correct batch effects . For exaIn an effort to address this issue, we noticed the “locality” among the single-cell samples. Specifically, samples gathered in proximal time points are expected to be more alike than from distant times. Similarly, the later two cell types are differentiated in development, the more alike they will be. That means the difference between two samples is more likely to be nuisance if those samples are collected from the same time point or adjacent time points. Here, we define a new distance function, termed Batch-Corrected Distance (BCD) which exploits the locality to precisely remove the batch effect  but keep22.1G =  which reflects the hop-by-hop gradual change (E) of cells  by optimizingxi is the profile of cell i, which can be the whole gene expression, or the first few principal components (PCs). Eulidean distance is the most widely used metric, while the Mahalanobis distanceCj = {i | cell i is in batch j} is the batch information and mj is the centroid of each batch. If weight Wij ≡ 1, it degrades to the metric defined by Qi and Davidson  to label each sample, where week is the number of weeks mentioned above, and location is set to be 0, 2, and 4 for trachea, small airways, and distal lung, respectively. The result is shown in We explored the dataset using BCD. Because both temporal and spatial information of the samples is available, we use a vector [4In order to build a reliable trajectory of cell type development from a longitudinal dataset, the batch effect should be corrected for. This is a new problem as state-of-the-art batch effect correction methods cannot utilize the time/spacial information, and thus result in over-correction. Our method, BCD, utilizes such information to accurately identify and remove the batch effect, while preserving the correct structure of the data. The results based on the BCD clearly show the evolution of the cell types through time. The discovered trajectory helps researchers make hypotheses of the physiological changes during development or pathological changes as a result of disease infection. The changes can be ascertained by differential gene expression analysis.BCD assumes that the batch effect is shared by all the samples. This is a reasonable assumption because many kinds of batch effects have a biological basis. For example, Cellular stress response stimulated by the sample preparation affects certain genes. In the case the set of genes do change, the local linear approximation may be used. Because the sums and products of distances are guaranteed to be valid distances, multiple BCDs each correcting for the batch effect in a specific set of samples can be combined as a consensus distance.BCD is intrinsically a linear transformation, which may be insufficient for more complex batch effects including interactions of genes. Nevertheless, it is a proof of concept that the time/spatial locality should be considered in batch correction for longitudinal datasets, which are on the rise. Nonlinear methods may be invented based on the same concept. For example, kernelization can be a direct extension to BCD. Besides, BCD is very fast to run and can be used in the exploration of the dataset with negligible cost.Although all the experiments are from a biology background, the scope of BCD is not confined to it. It can be applied to any longitudinal/spatial dataset affected by batch effects where the temporal/spatial locality holds. BCD also illustrates that batch the effect correction problem is related to the alternative clustering problems. Over the past few years, many advanced alternative clustering models have been introduced, and translate them to this context may result in better performance.5We defined a novel pairwise distance of the cells, namely Batch-Corrected Distance (BCD), where the effect of the unwanted clustering is controlled. Results show our method achieves more accurate clusters and better visualizations than state-of-the-art methods on longitudinal datasets. The BCD can be directly integrated into most clustering and visualization methods to enable more scientific findings.


Mutations in LRRK2 cause familial Parkinson’s disease and common variants increase disease risk. LRRK2 kinase activity and cellular localization are tightly regulated by phosphorylation of key residues, primarily Ser1292 and Ser935, which impacts downstream phosphorylation of its substrates, among which Rab10. A comprehensive characterization of LRRK2 activity and phosphorylation in brain as a function of age and mutations is missing. Here, we monitored Ser935 and Ser1292 phosphorylation in midbrain, striatum, and cortex of 1, 6, and 12 months-old mice carrying G2019S and R1441C mutations or murine bacterial artificial chromosome (BAC)-Lrrk2-G2019S. We observed that G2019S and, at a greater extent, R1441C brains display decreased phospho-Ser935, while Ser1292 autophosphorylation increased in G2019S but not in R1441C brain, lung, and kidney compared to wild-type. Further, Rab10 phosphorylation, is elevated in R1441C carrying mice, indicating that the effect of LRRK2 mutations on substrate phosphorylation is not generalizable. In BAC-Lrrk2-G2019S striatum and midbrain, Rab10 phosphorylation, but not Ser1292 autophosphorylation, decreases at 12-months, pointing to autophosphorylation and substrate phosphorylation as uncoupled events. Taken together, our study provides novel evidence that LRRK2 phosphorylation in mouse brain is differentially impacted by mutations, brain area, and age, with important implications as diagnostic markers of disease progression and stratification. Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson’s disease (PD), while common variants in the LRRK2 locus increase the lifetime risk of disease . LRRK2 iAutophosphorylation of Ser1292 is a robust readout of LRRK2 kinase activity in cells , which wTo gain insights into the effects of LRRK2 mutations on protein activity and phosphorylation ex-vivo, we performed a comprehensive survey of LRRK2 and Rab10 phosphorylation in midbrain, striatum, and cortex, as well as in peripheral tissues of three LRRK2 mutant mice, namely knockin (KI) Lrrk2-G2019S, KI Lrrk2-R1441C, and BAC overexpressing murine Lrrk2-G2019S. We observed that KI G2019S animals exhibit elevated pSer1292 in brain and lung/kidney but no increased pThr73-Rab10 in lung/kidney as compared to wild-type (WT), whilst KI mice carrying the ROC R1441C mutation display pSer1292 levels surprisingly similar to WT brain/lung/kidney but increased pT73-Rab10. Phospho-Ser935 is barely detectable in KI R1441C brains, and halved in KI G2019S compared to WT. In mice overexpressing the murine Lrrk2 locus (BAC-Lrrk2-G2019S), total and phospho-LRRK2 levels increase in the aging striatum, while phospho-Thr73 Rab10 is decreased at 12 months, indicating that LRRK2 and Rab10 phosphorylations are uncoupled, at least in this model. Finally, histological evaluation of BAC-G2019S brains confirmed that Lrrk2 expression is considerably higher in the striatum compared to the cortex and further revealed that aged mice display elevated GFAP levels, suggestive of enhanced gliosis.Taken together, our comprehensive ex-vivo study provides evidence that LRRK2 phosphorylation in murine brain is differentially impacted by mutations and that pSer1292/pSer935-LRRK2 and pThr73-Rab10 may be diagnostic of G2019S and R1441C-linked diseases, respectively.C57BL/6 LRRK2 wild-type and mouse LRRK2 G2019S BAC (GS BAC) mice were obtained from Jackson Laboratory [B6.Cg-Tg(Lrrk2∗G2019S)2Yue/J]. Non-transgenic wild-type (WT) mice were littermates obtained from the heterozygous breeding. Housing and handling of mice were done in compliance with national guidelines. All animal procedures were approved by the Ethical Committee of the University of Padova and the Italian Ministry of Health (#200/2019-PR and 1041/2016-PR).Homozygous LRRK2 G2019S knock-in (GSKI), R1441C knock-in (RCKI), knockout (KO) and parental WT mice were housed at the National Institute on Aging, NIH, ,13 accorDissections of cortex, midbrain, and striatal regions were performed in 1, 6, and 12 months-old mice of all genotypes. 4–5 mice/genotype/age were used in all experiments.For western blotting the following antibodies were used: total LRRK2 [MJFF2 (c41-2)] , phospho-S935 LRRK2 , phospho-S1292 LRRK2 , total Rab10 , phospho-T73 Rab10 , tyrosine hydroxylase , DARPP32  and β-actin . For immunofluorescence: β-tubulin-III ; total LRRK2 [MJFF2 (c41-2)] , phospho-S935 LRRK2 [UDD2 10(12)] , GLT1 , GFAP  CD11b [M1/70] .v/v) NP40, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 2 mM EGTA, 20 mM sodium fluoride, 50 mM beta glycerophosphate, 50 mM sodium pyrophosphate, 20 mM sodium orthovanadate. 70 μg of protein samples were resolved on pre-casted 4–20% Tris-glycine polyacrylamide gels .Brain regions were mechanically lysed in 25 mM pH = 7.5 Tris-HCl, 150 mM NaCl, 1% (Resolved proteins were transferred to polyvinylidenedifluoride (PVDF) membranes using semidry Biorad transfer machine . After 1h of saturation in 0.1% Tween-20 (TBS-T) plus 5% non-fat dry milk, PVDF membranes were incubated overnight at 4 °C with specific primary antibodies. The PVDF sheets were washed in TBS-T (3 × 10 min) at room temperature (RT) followed by incubation for 1 h at RT with horseradish peroxidase-conjugated IgG. Immunoreactive proteins were visualized using chemiluminescence . Densitometric analysis was carried out using Image J software . Levels of phospho-LRRK2/total LRRK2, phospho-RAB10/total RAB10 ratios were compared between control and mutant mice at different ages.®) and ketamine (Zoletil®) and transcardially perfused with 0.9% saline followed by ice cold 4% paraformaldehyde (PFA). Brains were dissected and post-fixed in 4% PFA at 4 °C overnight, then transferred to a sucrose gradient in phosphate-buffered saline (PBS) (20% and 30%) at 4 °C for cryopreservation. Once saturated in sucrose, 40 μm thick coronal slices were obtained by sectioning the brains with a vibratome. Sections were rinsed three times with PBS and then the sample autofluorescence was quenched in 50 nM NH4Cl in PBS. After three more washings, tissue sections were immersed in a solution made of 0.1% Sudan Black B (SBB) and 70% ethanol for 15 min at RT. To remove the excess of SBB the slices were rinsed three times with PBS and then they were permeabilized and saturated for 2 h in blocking solution  containing 0.5% Triton X-100. Incubation with the primary antibodies was carried out overnight at 4 °C in blocking solution. Samples were washed three times with PBS and then sections were incubated with appropriate secondary antibodies diluted 1:200 in blocking solution. Images were acquired with Zeiss LSM700 confocal microscope, using 20×/0.8 M27 objective.Animals were terminally anesthetized with xylazine (RompunAll quantitative data are expressed as mean ± SEM (standard error of the mean) from at least 4 different mice/genotype/age. Significance of differences between two groups was verified by Student t-test while comparisons between 3 or more groups were performed by one-way analysis of variance (ANOVA) with Dunnett’s Multiple comparison test/Bonferroni’s post-hoc test.To explore LRRK2 phosphorylation in mouse brain, we dissected three brain regions, namely striatum, cortex, and midbrain a. These Phosphorylation levels of Ser935 and Ser1292 are a readout of LRRK2 activity, and pSer935 is widely used to assess on-target LRRK2 inhibitor engagement . While pFirst, the levels of phospho-Ser935 in RCKI brains are ~5 times lower than in the WT in all brain areas, in 1 and 6 month-old animals b–d. InstLast, we observed some differences in Lrrk2 steady state levels across genotypes, but without a consistent trend in one specific region or at a specific age, with the exception of 6 months mice were total Lrrk2 levels are lower in GSKI cortex and midbrain a–d.Based on the unexpected finding that R1441C KI brains display undetectable Ser1292 autophosphorylation, we next asked whether this phenotype is restricted to the brain or whether it also applies to other tissues. Because lungs and kidneys have been previously shown to express high levels of LRRK2 ,20,21 anTo understand whether the lack of phospho-Ser1292 activation in RCKI mice reflects a general inability of this mutation to manifest a similar gain of kinase function observed in cell models , we evalWe next examined the effect of the G2019S mutation in a different mouse model, namely the hemizygous BAC murine Lrrk2 G2019S (mBAC G2019S) mouse. This model, which was developed by Zhenyu Yue and collaborators , displays ~six-fold higher Lrrk2 expression compared to the endogenous locus  and it wIn this dataset, we observed that: (i) LRRK2 expression increases upon aging in striatum and cortex; (ii) pSer935 and pSer1292 increase with aging in the striatum; (iii) Rab10 phosphorylation decreases at 12 months when Lrrk2 expression peaks, further supporting the notion that autophosphorylation and Rab10 phosphorylation are uncoupled events, at least in this context a–c. The β-III-tubulin, a neuronal marker (β-III-tubulin but no (or very faint) co-localization with GLT1 could be observed  partially colocalizes with l marker a,b and tl marker c–d. Instl marker e,f. Quitobserved , suggestobserved , we wondobserved i,j.Understanding the effects of pathogenic mutations on LRRK2 phosphorylation and cellular activity is critical to clarify their impact on LRRK2 signaling and to identify mutation-specific effects important for disease diagnosis. There is general consensus that both kinase and non-kinase mutations increase LRRK2 activity towards its major substrates, namely LRRK2 itself and a subset of RAB GTPases ,7. HowevHigh levels of LRRK2 in the striatum were previously reported ,16, as wBy comparing LRRK2 and Rab10 phosphorylation in KI G2019S and R1441C brains and lung/kidney, we made two major observations. First, the G2019S mutation increases autophosphorylation at Ser1292 while the R1441C mutation does not b. SecondTaking these and our findings together, we can postulate that GTP-locked LRRK2 R1441C does not need to undergo autophosphorylation in vivo being “constitutively” localized at RAB10-membranes (or other compartments), bypassing RAB29-mediated recruitment and activation d. The coper se the catalytic properties of the kinase, the G2019S mutation located in the activation loop of the kinase increases activity by doubling the Vmax [Our study further highlights that LRRK2 mutations located in different domains  likely operate through different pathogenic mechanisms. Knock-in mice carrying the G2019S mutation at the endogenous locus exhibit increased autophosphorylation at Ser1292 but Rab10 phosphorylation levels are similar to wild-type b–d. In cthe Vmax . Based othe Vmax ,7,47. Ththe Vmax  , strategies that can correct the lower GTP hydrolysis of RocCOR mutants may represent an alternative, and possibly more effective, therapeutic approach for LRRK2-R1441C carriers. Another implication of the different mode of action for these two mutations is that they may require distinct pharmacodynamic readouts to assess on-target kinase inhibition: dephosphorylation of Ser935/Ser1292 for G2019S carriers and Rab10 dephosphorylation for R1441C carriers.One intriguing observation from our analysis is that phosphorylation of Ser935 in GSKI brain lung and kidney is reduced by ~half. These results were not observed in cell lines ,49, overIn line with a previous study by Mercatelli and collaborators , we alsoFinally, by comparing the effect of aging on Lrrk2 and Rab10 phosphorylation in the context of BAC-Lrrk2-G2019S overexpressing mice, we collected further evidence that Ser1292 autophosphorylation and Rab10 phosphorylation are uncoupled phenomena. Specifically, while Lrrk2 levels and phosphorylation increase with aging (up to 12 months), phosphorylation of Rab10 decreases at 12 months, with the strongest effect observed in the striatum c. The rePD is a multisystemic disorder for which no cure is available. LRRK2 inhibitors are under clinical development and phase I clinical trials have already proved efficacy and safety . However


Inhibition of ABC transporters is considered the most effective way to circumvent multidrug resistance (MDR). In the present study, we evaluated the MDR modulatory potential of ERK5-IN-1, a potent extracelluar signal regulated kinase 5 (ERK5) inhibitor.125I]-iodoarylazidoprazosin (IAAP) photolabeling assay. Effect of ERK5-IN-1 on expression of ABCB1 and its downstream markers was measured by PCR and/or Western blot. Cell surface expression and subcellular localization of ABCB1 were tested by flow cytometry and immunofluorescence.The cytotoxicity and MDR reversal effect of ERK5-IN-1 were assessed by MTT assay. The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Doxorubicin efflux and accumulation were measured by flow cytometry. The modulation of ABCB1 activity was measured by colorimetric ATPase assay and [125I]-iodoarylazidoprazosin (IAAP) into ABCB1 in a concentration-dependent manner. In addition, ERK5-IN-1 treatment neither altered the expression level of ABCB1 nor blocked the phosphorylation of downstream Akt or Erk1/2. No significant reversal effect was observed on ABCG2-, ABCC1-, MRP7- and LRP-mediated drug resistance.Our results showed that ERK5-IN-1 significantly increased the sensitivity of vincristine, paclitaxel and doxorubicin in KBv200, MCF7/adr and HEK293/ABCB1 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Moreover, in vivo combination studies showed that ERK5-IN-1 effectively enhanced the antitumor activity of paclitaxel in KBv200 xenografts without causing addition toxicity. Mechanistically, ERK5-IN-1 increased intracellular accumulation of doxorubicin dose dependently by directly inhibiting the efflux function of ABCB1. ERK5-IN-1 stimulated the ABCB1 ATPase activity and inhibited the incorporation of [Collectively, these results indicated that ERK5-IN-1 efficiently reversed ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. The use of ERK5-IN-1 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients. Despite considerable advances in chemotherapies against cancer, multi-drug resistance (MDR) remains a major obstacle to positive therapeutic outcomes , 2. One The most direct way to restore drug sensitivity in MDR cancer cells caused by ABC transporters is to block or modulate their activity . So far,Small molecule tyrosine kinase inhibitors (TKIs) are developed to block the uncontrolled activity of intracellular signaling pathways in tumor cells. Our studies have previously revealed that some TKIs are either inhibitors or substrates to alter efflux mechanisms mediated by ABC transporters. TKIs such as EGFR inhibitors , VEGFR inhibitors  and others can significantly inhibit the efflux function of ABC transporters, thereby sensitizing cancer cells to chemotherapeutic agents –26.ERK5-IN-1, benzo [e] pyrimido- diazepine-6 (11H)-one was discovered as a novel extracelluar-signal-regulated kinase 5 (ERK5) inhibitor . ERK5, aERK5-IN-1 was purchased from Selleck Chemicals . 1--3, 5-diphenylformazan (MTT), vincristine, paclitaxel, verapamil, cisplatin, doxorubicin (Dox), mitoxantrone, fumitremorgin C (FTC) and other chemicals were purchased from Sigma-Aldrich . DMEM and RPMI 1640 medium were obtained from Thermo Fisher Scientific Inc. . Antibodies against ABCB1/P-gp , Akt (sc-8312), p-Akt (sc-7985-R), ERK 1/2 , and p-ERK (sc-7383) were from Santa Cruz Biotechnology Inc. . ABCG2 antibody (MAB4146) was obtained from Chemicon International, Inc. . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Kangcheng Co. . SYBR Green qPCR Master Mix was from ExCell Bio . Flow cytometry antibodies against ABCB1 (#557002) and Mouse IgG2b/κ (#559532) were purchased from BD Biosciences .2 3. When the average tumor volume reached 2000 mm3 in the saline group, mice were euthanized and the xenografts were excised and weighed. The ratio of tumor growth inhibition (IR) was estimated according to the following formula -IAAP  was then added and incubated for another 5 min under subdued light. The samples were cross-linked by UV illumination (365 nm) on ice and labeled ABCB1 was immunoprecipitated using the C219 antibody. Finally, samples were subjected to SDS-PAGE using a 7% Tris-acetate NuPAGE gel, dried and exposed to Bio-Max MR film  at -80 °C for 4 h. The radioactivity incorporated into the transporter protein was quantified using the Storm 860 PhosphorImager system .Crude membrane from High Five insect cells expressing ABCB1 (50 μg) was incubated with 0–5 μM ERK5-IN-1 for 5 min at room temperature in 50 mM Tris-HCl (pH 7.5). -IAAP binding to ABCB1 dose dependently. The 50% inhibitory concentration of [125I]-IAAP photoaffinity labeling of ABCB1 by ERK5-IN-1 was 3.7 μM  such as imatinib, gefitinib and erlotinib are small molecules developed to inhibit the uncontrolled activity of various tyrosine kinases involved in cancer , 44. ManERK5, also known as BMK1, is the least studied member of mitogen-activated protein kinases (MAPKs) family, but has been reported to play an important role in tumor neovascularization, tumor cell invasion and migration. Scientists have also identified MEK5/ERK5 pathway as a novel target implicated in chemoresistance by gene expression profiling analysis . ERK5-INThe in vitro modulating potency was first studied by the potentiation of cytotoxic drug activity using the MTT assay. The cytotoxic activity of ERK5-IN-1 were evaluated in vitro using a panel of cancer cell lines stabling expressing ABCB1 or ABCG2 and ERK5-IN-1 at non-toxic concentrations up to 0.4 μM were used for reversal assay in vitro Fig. .The promising activity of ERK5-IN-1 demonstrated in vitro was further confirmed in vivo, we examined the effect of ERK5-IN-1 on enhancing the antitumor activity of paclitaxel in KBv200 xenograft model. Consistent with the in vitro results, our results indicated that ERK5-IN-1 in combination with paclitaxel resulted in markedly enhanced antitumor activity of paclitaxel in mice  which could also reverse ABCB1-mediated MDR effectively [ABC transporters exert their drug efflux function by utilizing energy derived from the hydrolysis of ATP, and thus the change of ATPase activity of the transporters directly reflects their transport activity . We founCB1 Fig. . These dectively , 52. ThiThe reversal of MDR may be achieved by downregulation of the ABC transporter expression, thus the possible regulation of expression of ABCB1 by ERK5-IN-1 was also examined , 54. OurAs a kinase inhibitor, ERK5-IN-1 inhibited cell proliferation via blocking downstream signaling pathways such as phosphorylation of Akt and Erk1/2, which could also enhance the efficacy of chemotherapeutic agents. To elucidate whether the reversal effect in ABCB1-mediated MDR cells was related to the altered activation of downstream targets, cells were pretreated with ERK5-IN-1 and then subjected to Western blot analysis. Our data revealed no significant alteration of the phosphorylation of Akt and Erk1/2 in the tested cell lines, suggesting that the reversal activity of ERK5-IN-1 was not associated with the inhibition Akt and Erk pathways Fig. . A schemReversal of MDR by ERK5-IN-1 was through selective and potent inhibition of ABCB1 function. These findings provide strong support for further development of ERK5-IN-1 or its derivates as candidate inhibitors of MDR in cancer treatment.Additional file 1: Figure S1. Effect of ERK5-IN-1 on the subcellular distribution of ABCB1. KBv200 cells were treated with or without ERK5-IN-1 at 0.4 μM for 48 h. The subcellular localization pattern of ABCB1 was evaluated using confocal laser scanning microscopy. ABCB1 (red) and nuclei  were visualizedAdditional file 2: Table S1. Effect of ERK5-IN-1 on reversing ABCC1, MRP7 and LRP-mediated drug resistance


Combined X-ray and neutron diffraction refinements and electron probe micro-analysis were employed to locate Mo atoms in the crystal structure of Nd y6−WO12−δ (δ is the oxygen deficiency) provide excellent stability in harsh environments containing strongly reactive gases such as CO2, CO, H2, H2O or H2S. Due to this chemical stability, they are promising and cost-efficient candidate materials for gas separation, catalytic membrane reactors and protonic ceramic fuel cell technologies. As in Lay6−WO12−δ, the ionic/electronic transport mechanism in Ndy6−WO12−δ is expected to be largely controlled by the crystal structure, the conclusive determination of which is still lacking. This work presents a crystallographic study of Nd5.8WO12−δ and molybdenum-substituted Nd5.7W0.75Mo0.25O12−δ prepared by the citrate complexation route. High-resolution synchrotron and neutron powder diffraction data were used in combined Rietveld refinements to unravel the crystal structure of Nd5.8WO12−δ and Nd5.7W0.75Mo0.25O12−δ. Both investigated samples crystallize in a defect fluorite crystal structure with space group Fm3m and doubled unit-cell parameter due to cation ordering. Mo replaces W at both Wyckoff sites 4a and 48h and is evenly distributed, in contrast with Lay6−WO12−δ. X-ray absorption spectroscopy as a function of partial pressure pO2 in the near-edge regions excludes oxidation state changes of Nd (Nd3+) and W (W6+) in reducing conditions: the enhanced hydrogen permeation, i.e. ambipolar conduction, observed in Mo-substituted Ndy6−WO12−δ is therefore explained by the higher Mo reducibility and the creation of additional – disordered – oxygen vacancies.Mixed ionic electronic conducting ceramics Nd Hydrogen production from water electrolysis has also been studied thoroughly , allowing simultaneous chemical reaction and product separation and improving thermodynamically limited reaction yields, are promising devices for process intensification owing to their compactness, simplicity and energy efficiency. In the past few years electrochemically driven proton-conducting membranes have been employed in CMR for methane dehydroaromatization  are presented as promising candidates due to their superior mixed conductivity and their ability to operate at high temperatures and pressures  ceramics are selective to Hy6−WO12−δ (NWO) and substituted NWO compounds have proved to be stable mechanically   and substituted LWO crystallize in the fluorite-type crystal structure with a doubled cubic lattice due to the ordering of La and W . It was assumed that the stoichiometric differences in Nd content between the literature and specimen compositions were not relevant enough to induce any substantial deviation from the cubic crystal structure when z = 0.2, which was proved to be the case. The presented results will further support a comprehensive understanding of H2 transport within the NWO structure.Little is known about the crystal structure of NWO and substituted NWO. Trunov 1968 and McCa al. 1972 proposed al. 2015 reported2.2.1.et al., 2013et al., 2009−1 to obtain single-phase materials with high crystallinity suitable for diffraction investigations. The sintered discs were polished to remove secondary phases  on the surface formed during sintering. Two different samples were produced: an undoped neodymium tungstate with a nominal composition of Nd5.7WO12−δ (labelled NWO) and an Mo-substituted sample with a nominal composition of Nd5.4W0.8Mo0.2O12−δ (labelled NWM). In order to remove adsorbed and/or absorbed water, the samples were dried under constant Ar or synthetic air flow at 1173 K for 4 h in a tubular furnace prior to the structural analysis. Deuteration was achieved by connecting two bubble bottles filled with D2O to the synthetic air stream at room temperature. The furnace was kept at 623 K for 4 h. Following heat treatments, the samples were transferred to the experimental stations in air-tight boxes filled with argon to prevent changes to the pre-treated specimens.Specimens were produced following a wet chemical route, the citrate complexation route  using a JEOL JXA 8200 device. Wavelength-dispersive X-ray spectroscopy was carried out on 10 to 20 points measured on the surface of each grain and on cross sections of pieces of the sintered pellets to ensure homogeneous sampling of the powders. Only grains without visible pores and only obtained measurements with a calculated sum in mass percent of at least 95% were considered in the analysis. Elemental standards for tungsten, neodymium oxide and molybdenum oxide were used for the calibration, in order to increase the accuracy of the local composition measurements.−1 and Ar atmosphere.The mass loss of the pre-treated samples was determined during heating with a Netzsch TG209F1 Iris microbalance equipped with a mass spectrometer QMS 403 Aëolos with a heating ramp of 5 K min2.3.Kα X-ray diffraction (XRD) using a Bruker D8 diffractometer in Bragg–Brentano geometry. Phase identification was aided by the International Centre for Diffraction Data  PDF2 database within the EVA14 software  and high-resolution synchrotron X-ray powder diffraction (HRSXRD). Neutron diffraction was carried out on the fine-resolution powder diffractometer (FIREPOD) at the neutron reactor BERII at HZB  with wavelengths of 1.3084 (2), 1.7982 (1) and 2.8172 (2) Å  which was used as internal standard and filled into polyimide capillaries of diameter 0.7 mm inside a glove bag. Further HRSXRD measurements for samples dried and deuterated under synthetic air were conducted on the material science (MS) beamline  Å], reducing the effective number of electrons (6+ from 36 e− to 31.35 HRSXRD measurements were performed on beamline ID22 (formerly ID31) .(i) The instrumental profile, wavelength and zero correction were refined using reference materials (HRSXRD: LaB(ii) The background was fitted with a Chebychev polynomial function using 15 (BERII data), 21 (ESRF data) and/or 36 (SLS data) parameters and kept fixed until the last stage of the refinement, when it was refined together with the other structural parameters.2O3. The internal standard was kept fixed to the tabulated values during refinement.(iii) Unit-cell parameters and sample displacement parallel and perpendicular to the beam were refined together with the internal standard Alx, y, z) were refined (see Results and discussion(iv) Finally, structural parameters  were performed on the LISA beamline (BM-08)  were used for harmonic rejection. Measurements were performed on pellets (13 mm diameter) in transmission mode at room temperature; W and Cr standard foils were measured in transmission mode for energy calibration.XANES measurements at the W and Nd Epe (E0 − 200 eV ≤ ΔEpe ≤ E0 − 25 eV), a step of 0.025 eV in the XANES region (E0 − 25 eV < ΔEXANES ≤ E0 + 25 eV) and a fixed k step of 0.03 Å−1 above E0 + 25 eV, up to maximum values of kmax(Nd) = 11.6 Å−1 and kmax(W) = 12 Å−1, for proper post-edge spectrum normalization.The spectra were acquired with a step of 5 eV in the pre-edge region Δet al., 1981ATHENA , which eluded both laboratory XRD and EPMA. The existence of this secondary phase can be explained by segregation of W-rich phases at the surface of the sintered discs, which also clarifies the difference between nominal and measured Nd, W and Mo content. The Nd10W2O21 phase was indexed and refined for the HRSXRD pattern with unit-cell parameters a = 16.416 (1) Å, b = 10.903 (1) Å and c = 10.929 (1) Å. However, due to the high number of reflections (6407) within the refined 2θ range of the HRSXRD data, Rietveld refinement with the main NWO phase and secondary Nd10W2O21 phase becomes excessively slow. Since the refinement of the main NWO phase is not influenced by the secondary Nd10W2O21 phase, the latter was not considered further for the combined X-ray and neutron refinements. Isosurface plots of the observed electron and nuclear scattering densities extracted from the HRSXRD and ND powder patterns  in the [110] direction, away from one of the three mirror planes onto Wyckoff site 48h (0yy).High-resolution XRD showed the absence of any tetragonal distortion of the fluorite structure, contrary to previous reports . The oxygen site O1 is split from a regular arrangement on a cube on Wyckoff site 32f (xxx) onto 96k (xxy) by leaving the ternary axis along the mirror plane, observed already for LWO  was located on the 48h site  with chemical restraints did not improve the refinement. In this case only the neutron patterns were used because of the better contrast between Nd and W. A subsequent combined refinement with the anti-site disorder fixed to the result from ND leads to even larger ADPs for the Nd1 site and smaller ADPs for the W1 site. Therefore, anti-site defects occupying the 4a and 4b Wyckoff sites can be ruled out. Closer inspection of the electron-density maps at the 4b site shows a remarkable disorder in the direction towards the 4a site. Although splitting the 4b (½½½) site into 1/6 occupied 24e (00x), where x is close to 0.5, reduced the ADPs for the Nd1 site, it did not improve the fit any further. Since the SOFs for the Nd1 and W1 sites refined to 1 (within the uncertainties), both SOFs were kept at 100% occupation for the rest of the analysis. Only the SOFs of the 48h and of the two oxygen Wyckoff sites were refined. Even this did not improve the fit or change the SOFs significantly from the starting values obtained from EPMA. Therefore, the SOFs for all cation sites were kept fixed during the final refinement and only the SOFs for O1 and O2 together with the ADPs and fractional coordinates were refined. The resulting atomic coordinates, displacement parameters, SOFs and residuals from the refinement are summarized in Table 2At first the ADPs were refined as isotropic while keeping the SOFs fixed. Then the fractional coordinates were refined, keeping SOFs and ADPs fixed. Then the ADPs were refined together with the coordinates and fixed SOFs. In the final cycles of the refinement, atomic anisotropic displacement parameters were used and refined together with the coordinates. This resulted in abnormally high ADPs for Nd1 on site 4a), surrounded by the split O1 site, which is displaced from site 32f to 96k with 24 possible positions. This results in an average oxygen coordination of 5.6 (1). Nd occupies site 4b (edge centres) and is coordinated by O2 on site 32f, equivalent to 7.2 (1) oxygen neighbours arranged regularly on a cube. The higher ADPs for Nd on Wyckoff site 4b can be rationalized by local static disorder. In the disordered average picture the mixed occupied and split site 48h with an average oxygen coordination of 6.4 (1) is highly disordered, which can be better understood if the local arrangement is considered. On this site 0.7 cations out of 24 are occupied by W, which acts as a donor dopant ], occupying one of the possible three positions of the disordered average model, i.e. the one next to the oxygen vacant site. The orientation of one W octahedron propagates ordering to the next octahedron via the split and half-occupied Nd2/W2 site [Fig. 6b)]. In the local description, the Nd2/W2 site moves away from the defect and is closest and bonds to two oxygen ions of the WO6 octahedron.The highly disordered average structure is described through local rearrangements and relaxation of both anions and cations. By analogy with LWO, in NWO an octahedral coordination for W can be assumed if two oxygen vacancies are placed on a diagonal of the cube and the remaining six oxygen ions relax in a direction towards the vacancies :Comparing the ionic radii of six-coordinated WM0: refinement performed with W on 4a and Nd on 4b and 48h sites and SOFs free (no Mo in the structure).(i) M1: SOFs for sites 4a and 48h calculated directly from the single-atom model.(ii) M2: statistical distribution of Mo on both Wyckoff sites 4a and 48h.(iii) M3: Mo only on 4a site (no Mo on 48h site).(iv) M4: W only on 4a site (no W on 48h site).(v) M1–M4 were refined keeping all M0-refined global parameters  constant. The SOFs for the different distribution models, as listed in Table 3a and 48h are plotted for the models M1–M4 in Fig. 7b).Models a, 4b and 48h.Cation order is mainly reflected by the superstructure reflections already indexed as 111, 200, 220 and 311 of the double fluorite cell in the laboratory XRD patterns Fig. 2. The weahkl) shown in Figs. 7c)–7f) and the corresponding fitted reflections Fig. 7g)–7j) reveals that the best fits occur for models M1 and M4. This is also reflected in the listed R values in Table 3M2 and M3 show a lack of electrons on site 4a, which can be excluded on the basis of the larger R values reported in Table 3M3, the effective numbers of electrons are equal for both sites 4a and 4b, differing only slightly from site 48h (56.6 to 57.0). Therefore, as no detectable cation order is observed, the superstructure lines in M3 are only weakly fitted. In M4 the superstructure reflections are over-fitted, related to the higher scattering power for the 4a site having the smallest amount of Mo and the highest amount of W. Taking the quality factors and careful inspection of the refinement results into account, M1 is proposed to fit the average crystal structure of NWM best. The substituted amount of Mo for W is more or less equally distributed over Wyckoff sites 4a and 48h (∼0.6 atoms per site) and not distributed proportionally to the W amount on each site proposed for Mo-substituted LWO. Fantin and co-workers found for Re-  Mo ions on site 4a and 0.57 (5) on site 48h. The details and results of the combined refinement are summarized in Table 4M1, which showed the best fit to the HRSXRD data collected close to the Mo K edge with increased scattering contrast for Mo ions. This leads to an almost even distribution of Mo on both W-containing Wyckoff sites. The crystal structure for NWM, except for small differences in the Nd/(W+Mo) ratio and in the SOFs for the oxygen sites, can be compared directly with the NWO structure depicted in Fig. 5In order to validate the Mo distribution model 3.5.p(O2) and p(D2O) by annealing specimens under dry Ar, synthetic air and deuterated synthetic air. The neutron diffraction patterns showed negligible differences for deuterated and dry samples, due to the small amount of incorporated O or OD upon humidification. More interesting is the role of the cations, whose contrast is more visible with the HRSXRD measurements, presented and discussed in the following. The substitution of W by Mo leads to a decrease in the unit-cell parameter for NWM, rationalized by the smaller ionic radius of Mo6+ (0.59 Å) compared with that of W6+ (0.60 Å). However, Nd is the largest element in the compound. The Nd content, and in this specific case the Nd/(W + Mo) ratio, plays an important role and must be considered for the unit-cell parameter discussion. Indeed, the cationic W/Mo anti-site defects on Nd2 site 48h are directly determined by the x values in Ndx28−(Wz1−Moz)x4+Ox/254+3νx/22−3. Having a larger ratio for NWO  than for NWM  corresponds to more Nd3+ (rion = 1.109 Å) ions in the crystal structure and thus to a smaller number of p(O2) and p(D2O) partial pressures as well, due to filling of the oxygen vacancies with O and OD, respectively. This is described in Fig. 9m obtained from thermogravimetric analysis (TG) is shown as a function of the corresponding unit-cell parameter, hinting at direct proportionality between p(O2)/p(D2O) and unit-cell size.Unit-cell parameters and bond lengths in NWO and NWM were investigated in relation to the partial pressures p(O2) and p(D2O) can be explained by the higher Nd/W ratio and hence higher number of oxygen vacancies that can be filled. From Fig. 9i.e. oxygen vacancy filling, is more relevant for unit-cell expansion than the partial substitution of W by Mo. On the other hand, the small unit-cell parameter difference between the two NWM and NWO reduced specimens (dry Ar) is given by the lower Nd content in NWM combined with the partial reduction of Mo6+ to Mo5+ and/or Mo4+, i.e. by the larger ionic radius of reduced Mo ions  ratio of NWO compared with NWM, where a higher Nd/(W + Mo) ratio equals more Nd atoms (largest ionic radii) on Wyckoff site 48h. From the anion side, the filling of oxygen vacancies, i.e. an increase in p(O2)/p(D2O), leads to an increase in coordination number and a larger cation ionic radius (see Table 5ca 23 atoms). The large difference in the Nd1—O2 bond distance could also be correlated to the space group used for the refinements, as the oxygen site O2 on 32f (xxx) can only move in the direction of the Nd1—O2 bonds and cannot relax in the dry Ar state. This interpretation would also explain the large ADPs found for the O2 and Nd1 sites, though it is likely that the decrease in the Nd1—O2 bond length with increasing p(O2)/p(D2O) is a function of all the above-mentioned factors, and a final statement cannot be provided. The partial reduction of Nd3+ to Nd2+ under dry Ar is, however, excluded by the X-ray absorption spectroscopy studies reported in the following section, corroborated by the findings of Escolástico et al. (20152).The cation–oxygen bond distances for the different treatments, obtained from Rietveld refinements, are listed in Table 6 al. 2015 using X- al. 2015 under ev3.6.et al., 2014L3 edges of Nd and W in reduced and oxidized NWO and NWM, the transitions from 2p3/2 to vacant s and d orbitals are probed. As the p→d transition contribution is about 50 times larger than that of the p→s transition ] and Nd [Fig. 10b)] in dry Ar/H2 (reducing atmosphere) and dry synthetic air (oxidizing atmosphere) NWO and NWM specimens are presented.Through XANES measurements one investigates the oxidation state, bonding environment and local geometry (Henderson cf. inset) at the W edges, corresponding to the main absorption edge at 10209.90 (5) eV marked in Fig. 10a), lie well below 0.05 eV, which is taken as a strict reasonable limit above which meaningful differences may occur. It is recalled that a change in oxidation state would produce shifts of the order of a few electronvolts. Similarly, all the peak maxima of the first derivatives at the Nd L3 edge are found to lie at 6210.90 (5) eV. The oxidation states of both Nd and W are, therefore, independent of the pre-treatment used and of Mo concentration, i.e. Nd and W preserve their +3 and +6 oxidation states, respectively, even in highly reducing atmospheres such as dry Ar/H2.From Fig. 10L3 edge and XANES is not easy to interpret, as the ligand field is the sum of a sevenfold oxygen coordination  and an eightfold coordination . Asakura et al. , a larger FWHM for NWO dry Ar/H2 [8.3 (3) eV] compared with that for NWO dry synthetic air [7.4 (3) eV] hints at a larger oxygen loss in NWO dry Ar/H2, which in turn decreases the average Nd—O coordination. A slight change in the FWHM is seen in NWM as well , which agrees with the fact that water uptake in NWM is about a factor of two less than that of NWO  and a minority of six- or sevenfold symmetry . In Fig. 10a), a clear split of the W L3-edge white line of about ΔEcf = 3.7 eV  is observed. The split is the consequence of the crystal field which acts on W, splitting the 5d orbital into the tg2 and eg components, with ΔEcf = 3.7 eV their energy difference. ΔEcf is commonly referred to as 10Dq and it is a measure of how strong the ligand crystal field is. Different information on the point-group symmetry and orbital overlapping of W in NWO can be inferred from the 10Dq value when compared with the literature. It is reported that the higher the 10Dq value, the less distorted the hO symmetry of a six-coordinate environment  and nearly octahedral symmetry , respectively . The 10Dq value for LWO is slightly higher than the corresponding value for NWO, hinting at a more regular hO-like symmetry in the former. A similar 10Dq value to NWO is found for WO3 (10Dq = 4.0 eV) which presents a more distorted octahedral symmetry.The average W—O coordination number is the result of a majority of octahedral symmetry , which shows a fluorite structure like NWO, it is reported that the displacive cubic to tetragonal transition is driven by the 4.A site of Lax6−WO12−δ and the partial substitution of the B site of Ndx6−WO12−δ by Mo were studied by combined neutron and high-resolution synchrotron powder diffraction. The crystal structure is characterized by the following: (i) a defect fluorite structure  a highly disordered anion sublattice, which enables excellent transport properties, as reported in the literature  no ordering of oxygen vacancies is observed, crucially, since proton mobility strongly inversely correlates with oxygen vacancy ordering. Unlike Mo-  NWO_dry_Ar_X+N_final_3_publ, NWO_dry_Ar_X+N_final_3_overall, NWO_dry_Ar_X+N_final_3_phase_1, NWO_dry_Ar_X+N_final_3_phase_0, NWO_dry_Ar_X+N_final_3_pwd_0, NWO_dry_Ar_X+N_final_3_pwd_1, NWO_dry_Ar_X+N_final_3_pwd_2. DOI: