Rhodococcus sp. VLD-10 using the conventional and genomic (16S rRNA) approaches. Bioactive compounds responsible for the antimicrobial activity of the strain were elucidated by cultivating the strain VLD-10 in a modified yeast extract-malt extract-lactose broth followed by subsequent chromatographic and spectroscopic analyses. Extraction, purification, and structural confirmation of five compounds, viz., benzoic acid, 2-nitrobenzaldehyde, 4-chlorobenzaldehyde, nonadeconoic acid, and 3-isopropylhexahydro-1H-pyrido pyrazine-1,4(6H)-dione, from Rhodococcus sp. VLD-10 were fruitfully described. The bioactivity of the compounds isolated from the strain VLD-10 against Gram-positive as well as Gram-negative bacteria, yeast, and molds was tested and their minimum inhibition concentration was reported. Antibacterial activity of 3-isopropylhexahydro-1H-pyrido pyrazine-1,4(6H)-dione is more prominent against Bacillus subtilis, B. cereus, B. megaterium, Corynebacterium diphtheriae, and Escherichia coli, whereas its antifungal spectrum showed less potency against yeast and fungi. This is the first report on the natural occurrence and bioactivity of 3-isopropylhexahydro-1H-pyrido pyrazine-1,4(6H)-dione from Rhodococcus sp. VLD-10.A potent actinobacterial strain isolated from the marine samples of Bheemunipatnam beach, Visakhapatnam, India, was identified as The online version of this article (doi:10.1007/s13205-016-0576-6) contains supplementary material, which is available to authorized users. Actinomycetales  are distributed ubiquitous in nature and account for more than 50% of the compounds reported in the Dictionary of Natural Products.Microorganisms are capable of carrying out a tremendous variety of reactions and can adapt to a range of environments allowing them to be transplanted from nature to the laboratory where they can be grown on inexpensive carbon and nitrogen sources to produce valuable compounds  followed by plating on YMD agar medium using soil dilution technique  analysis of the strain were studied.Streptomyces Project (ISP) media, such as ISP-1 (Tryptone-yeast extract agar), ISP-2 , ISP-3 , ISP-4 , ISP-5 , ISP-6 (Peptone yeast extract iron agar medium), and ISP-7 (Tyrosine agar), as well as on five nonISP media, such as Nutrient agar (NA), Czapek–Dox (CD) agar, Bennett’s agar, Glucose–tryptone (GT) agar, and Starch casein (SC) agar, with the initial pH 7.2 maintained at 30 °C  as previously described (Bozzola and Russell 2 (25 mM), 2 μl of dNTP (0.4 mM), 1 μl of 16S rDNA forward primer—5′-CCCATG TTGGGTATTCCTCCAGGCGAAAACGGG 3′ (10 pmol/μl), 1 μl of 16S rDNA reverse primer—5′CCCGCATTATCCGTACTCCCCAGGCGGGGC-3′ (10 pmol/μl), Taq polymerase (2 U/μl), and 2 μl template DNA. PCR amplification was carried out as follows: the initial denaturation step at 94 °C for 3 min followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 65 °C for 1 min, and extension at 72 °C for 1 min, with a further 5 min extension at 72 °C. The PCR product was purified with agarose gel DNA purification kit  followed by sequencing of 750 bp. The deduced partial 16S rDNA gene sequence was compared with the accessible sequences in GenBank (http://www.ncbi.nlm.nih.gov/) using Basic Local Alignment Search Tool (BLAST) in NCBI GenBank databases. Phylogenetic and molecular evolutionary analyses were conducted using Molecular Evolutionary Genetic Analysis (MEGA) version 4.0  for the enhanced secondary metabolite production. The fermentation was carried out in 1 L Roux bottles for 120 h at 30 °C.The bioactive compounds from the fermented broth were harvested by filtration of biomass through Whatman filter paper no. 42 . The culture filtrate (25 L) was extracted twice with an equal volume of ethyl acetate and pooled, and the organic layer was concentrated in a Rotovac. The deep brown semi-solid compound (3.8 g) obtained was applied to a silica gel G column .Rf) were combined and assayed for antimicrobial activities. The crude eluent was recuperated in 5–10 ml of ethyl acetate and was further purified.The separation of the crude extract was conducted via gradient elution with hexane: ethyl acetate. The eluent was run over the column and small volumes of eluent collected in test tubes were analyzed via thin-layer chromatography (TLC) using silica gel plates  with hexane: ethyl acetate solvent system. Compounds with identical retention factors (1H NMR and 13C NMR); and model: Varian Gemini 200, and samples were made in CDCl3 with trimethyl silane as standard.Among the 11 main fractions eluted, 10 fractions were found polar and 1 was nonpolar residue. Antimicrobial activity was tested for all the fractions obtained. Among the 10 polar fractions eluted from the crude extract, three fractions along with the nonpolar fraction exhibited high antimicrobial activity. D1 (70–30 v/v), D2 (30–70 v/v), D3 (20–80 v/v), and D4 (100–0 v/v) were the fractions collected at different hexane: ethyl acetate solvent system. All the fractions were rechromatographed using different gradient eluent systems for final elucidation of compounds. The fraction D1 on further purification yielded two compounds each in pure form (D1Ba and D1Bb). The second fraction D2 also yielded two subfractions in pure form namely D2Ba and D2Bb. The fraction D3 was single and obtained in pure form. The structure of these active fractions was analyzed on the basis of Fourier Transform Infrared (FTIR); model: Thermo Nicolet Nexus 670 spectrophotometer with NaCl optics, Electron Ionization Mass/Electron Spray Ionization Mass Spectrophotometry (EIMS/ESIMS); model: Micromass VG-7070H, 70 eV spectrophotometer and Nuclear Magnetic Resonance (B. cereus (MTCC 430), B. megaterium (NCIM 2187), B. subtilis (MTCC 441), Corynebacterium diphtheriae (MTCC 116), E. coli (MTCC 40), Pseudomonas aeruginosa (MTCC 424), Salmonella typhi (ATCC 14028), Serratia marcescens (MTCC 118), Shigella flexneri (MTCC 1457), Staphylococcus aureus (MTCC 96), Xanthomonas campestris (NCIM 2310), and fungi, including Aspergillus niger (ATCC 1015), Alternaria alternata (MTCC 6572), Botrytis cinerea, C. albicans (MTCC 183), Fusarium oxysporum (MTCC 218), F. solani (MTCC 4634), and Verticillium alboatrum. The inoculated plates were examined after 24–48 h of incubation at 37 °C for bacteria and 48–72 h at 28 °C for fungi. The lowest concentration of the bioactive metabolite exhibiting significant antimicrobial activity against the test microbes was taken as the MIC of the compound.The antimicrobial spectra of the bioactive compounds produced by the strain were determined in terms of minimum inhibitory concentration (MIC) against a wide variety of Gram-positive, Gram-negative bacteria, and fungi using agar plate diffusion assay  NaCl, while growth was moderate between 1 and 6% (w/v). No growth was found in the medium without NaCl indicating its halophilic nature. It could produce a broad range of commercially important enzymes like amylase, cellulase, chitinase, in Table .Rhodococcus sp. VLD-10 belonging to the family Corynebacteriaceae.Using 16S rRNA analysis, the gene sequence of the strain was compared and aligned with those sequences retrieved from NCBI GenBank database using the BLAST analysis. The phylogenetic tree was constructed by neighbour-joining method using the MEGA software Fig.  and depoRf) on thin-layer chromatography (TLC) silica gel plates were pooled together. Out of 11 eluted main fractions, 10 are polar residues and 1 is nonpolar residue. Among the ten polar fractions, three fractions and the single nonpolar fraction exhibited high antimicrobial activity. These four fractions, namely D1, D2, D3, and D4, were collected at different eluent systems of hexane: ethyl acetate solvent systems, viz., 70–30 v/v, 30–70 v/v, 20–80 v/v, and 100–0 v/v, respectively. All the fractions were rechromatographed using different gradient eluent systems to obtain the fractions in pure form for structural elucidation. The flowchart showing the isolation and purification of bioactive fractions is illustrated in Fig S1.For the isolation and purification of bioactive compounds, crude extract was applied on a silica gel G column  and their separation was conducted via gradient elution with hexane: ethyl acetate. Elutions were collected sequentially in small test tubes and those fractions having similar retention factors . In ESIMS, the compound showed molecular ions at m/z = 123 [M+] inferring the molecular weight of C7H6O2 [M]+ . The proton NMR of the compound displayed proton signals at signals at δ 8.12–8.15 , 7.59–7.62 , and 7.44–7.46  due to five aromatic protons . 13C NMR depicted peak and showed signal at δ 172.7 for carbonyl group . Based on these spectral data, the first active fraction (D1Ba) was identified as benzoic acid.D1 fraction was rechromatographed into two pure fractions, (D1Ba) and (D1Bb). The fraction D1Ba appeared as yellow solid which was soluble in CHClVmax at 1705 cm−1 suggested the presence of functional aldehyde . In ESIMS, the compound showed molecular ions at m/z = 301[2M-1] inferring the molecular weight of 2(C7H5O3N)-1 [2M-1] . The proton NMR of the compound displayed proton signals at δ 10.43 , and one proton for aldehyde, at δ 8.13 , 7.96 , and 7.75–7.83  for four aromatic protons . 13C NMR depicted peaks at δ (188.1) for aldehyde carbon . Based on the spectral data, the fraction D1Bb was identified as 2-nitrobenzaldehyde.D1Bb fraction soluble in MeOH, DCM, and DMSO appeared as white crystalline powder. The IR absorption maxima 3, MeOH, and DCM. The IR absorption maxima Vmax at 1683 cm−1 suggested the presence of aldehyde . In ESIMS, the compound showed molecular ions at m/z = 141 [M+1]+ inferring the molecular weight of C7H6OCl [M+1]+ . The proton NMR of the compound displayed proton signals at δ 9.99  for one aldehyde proton, and at δ 7.83  and 7.53  for four aromatic protons . 13C NMR depicted peak at δ 190.7 for aldehyde . D2Ba was identified as 4-chlorobenzaldehyde based on the spectral data.D2 fraction was rechromatographed into two pure fractions, (D2Ba) and (D2Bb). D2Ba fraction appeared as white crystalline powder and was soluble in CHCl3, MeOH, DCM, and DMSO. The IR absorption maxima Vmax at 1709 cm−1 suggested the presence of carboxylic group . In ESIMS, the compound showed molecular ions at m/z = 299 [M+1]+ inferring the molecular weight of C19H38O2 [M+1]+ . The proton NMR of the compound displayed at δ 2.35  for alpha methylene protons; at δ 1.65–1.55  and 1.25–1.99  for aliphatic methylene protons; at δ 1.25–1.99  for methylene protons; and at δ 0.82  for methyl protons . 13C NMR depicted peak at δ 180.8 for carboxylic group . Based on spectral data, the D2Bb fraction was identified as nonadeconoic acid.The second fraction D2Bb in pure form appeared as light green liquid soluble in CHCl3, MeOH, DCM, and DMSO. The IR absorption maxima Vmax at 1687 cm−1 suggested the presence of functional groups like carbonyl group . In ESIMS, the compound showed molecular ions at m/z = 211.1474 [M+1] inferring the molecular weight of C11H1902N2[M+1]+ . The proton NMR of the compound displayed proton signals at δ 5.91  for amide protons; 4.12  for methylene protons; 4.02  for methylene protons; 3.67–3.48  for methylene protons; 2.45–2.24  and 2.23–1.97  for methylene proton; 1.96–1.83 , 1.82–1.69 , and 1.60–1.45  for methylene protons; and 1.05  and 0.96  for methyl protons. . 13C NMR depicted peaks at δ 170.2 (1C), 166.1 (1C), 76.5 (1C), 58.9 (1C), 53.3 (1C), 45.4 (1C), 38.5 (1C), 29.6 (1C), 28.0 (1C), 24.6 (1C), 23.2 (1C), 22.7 (1C), and at δ 21.1 (1C) . DEPT spectrum exhibiting methyl groups , methylene groups , and methylene groups  . 1H–1H COSY NMR spectrum exhibits correlation between H12–H11, H10–H11, H10–H9, H6–H5, H5–H4, H4–H3 . HSQC spectrum exhibits correlation between 13 C NMR with 1H NMR: C3–H3, C4–H4, C5–H5, C6–H6, C9–H9, C10–H10, C11–H11, C12–H12, and C13–H13 . HMBC spectrum exhibits following correlation of 13 C NMR with 1H NMR 23.2–1.05; 21.1–0.96; 24.6–1.55; 38.5–1.55, 2.08; 53.3–4.03; 58.9–4.15; 28.0–2.18, 2.38; 22.7–1.97, 2.05; 45.4–3.60; 170–4.12; and 166.1–4.02. . Based on these spectral data, the active fraction D3 was identified as 3-isobutylhexahydropyrrolopyrazine-1,4-dione This is the first report from actinobacteria.Fraction D3 appeared as white solid, which was soluble in CHClB. cereus, S. aureus, and X. campestris as compared to the other bacteria tested, while Botrytis cinerea is sensitive among fungi. 2-nitrobenzal dehyde (D1Bb) is mostly active against bacteria like B. subtilis, Shigella flexneri, and Staphylococcus aureus. 4-chlorobenzaldehyde (D2Ba) is active against S. aureus, B. subtilis, and S. flexneri. Nonadeconoic acid (D2Bb) is active against S. flexneri, S. aureus, B. subtilis, and X. campestris. Among all the compounds, 3-isopropylhexahydro-1H-pyrido pyrazine-1,4(6H)-dione (D3) is more active against B. subtilis, B. cereus, B. megaterium, C. diphtheriae, and E. coli, but it showed less activity against the fungi when compared to that of standard control (nystatin). The partially purified fraction (D4) exhibited good activity against Bacillus spp. tested.MIC of the bioactive compounds, viz., benzoic acid, 2-nitrobenzaldehyde, 4-chlorobenzaldehyde, nonadeconoic acid, and 3-isopropylhexahydro-1H-pyrido pyrazine-1,4(6H)-dione  and reversed-phase HPLC (Lichrospher 60RP-select B) from the culture broths of Borisova  found thRhodococcus sp. VLD-10. Benzoic acid is a well-known food preservative that inhibits the growth of bacteria, yeasts, and molds -dione isolated from Rhodococcus sp. VLD-10.This study has revealed the production of five bioactive compounds, such as benzoic acid, 2-nitrobenzaldehyde, 4-chlorobenzaldehyde, nonadeconoic acid, and 3-isopropylhexahydro-1H-pyrido pyrazine-1,4(6H)-dione, from ds Warth , which iSupplementary material 1 (DOC 1321 kb)Below is the link to the electronic supplementary material.


Although there has been considerable work on immune regulation and autoimmunity, there is a relative paucity of work directed at the functional implications of the key peritoneal cavity (PC) B cell subset, coined B1a cells in PBC. We used flow cytometry and high-resolution microarrays to study the qualitative and quantitative characteristics of B cells, particularly B1a cells, in the PC of −/−IL-2Rα−/−p40 mice compared to controls. Importantly, B1a cell proliferation was markedly lower as the expression of Ki67 decreased. Meanwhile, the apoptosis level was much higher. These lead to a reduction of B1a cells in the PC of −/−IL-2Rα−/−p40 mice compared to controls. In contrast, there was a dramatic increase of CD4+ and CD8+ T cells accompanied by elevated production of IFN-γ. In addition, we found a negative correlation between the frequency of B1a cells and the presence of autoreactive CD8+ T cells in both liver and PC of −/−IL-2Rα−/−p40 mice. From a functional perspective, B cells from −/−IL-2Rα−/−p40 mice downregulated IL-10 production and CTLA-4 expression, leading to loss of B cell regulatory function. We suggest that the dysfunction of B1a cells in the PC in this murine model of autoimmune cholangitis results in defective regulatory function. This highlights a new potential therapeutic target in PBC.Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease with progressive cholestasis and liver fibrosis. Similar to human patients with PBC,  It is interesting that the liver, whose immunological function is to facilitate immune tolerance, itself becomes a victim of autoimmunity, including primary biliary cholangitis (PBC), a disease characterized by lymphocytic infiltrates in portal tracts and the presence of anti-mitochondrial Abs (AMAs) secreted by autoreactive B cells –7. A maj−/−IL-2Rα−/−p40 mice [An interesting feature of both humans and mice with autoimmune cholangitis is that although there are high titer AMAs, the role of B cells in the pathogenesis of disease has remained enigmatic –16. Prev−/− mice . This mo−/−IL-2Rα−/−p40 mice , CD4+ T cells (P = 0.0008) and CD8+ T cells (P = 0.0024) were much higher in PC of −/−IL-2Rα−/−p40 compared to −/−IL-2Rα+/−p40 mice, while B cell number (P < 0.0001) was dramatically lower   Figure . As note) Figure . Thence P = 0.0120, Figure −/−IL-2Rα−/−p40 mice, and the frequency of B1a in B cells was negatively correlated with PC and liver MNC numbers  (P < 0.0001) and B1b cells (CD11b+CD5−) (P < 0.0001) in B cells were much lower in −/−IL-2Rα−/−p40 mice compared with −/−IL-2Rα+/−p40 mice. B1a cells were almost undetectable, and the frequency of B2 (CD11b−CD5−) cells (P < 0.0001) in B cells were higher in PC from −/−IL-2Rα−/−p40 mice compared to −/−IL-2Rα+/−p40 mice  and B1b cells (P < 0.0001) were markedly reduced in −/−IL-2Rα−/−p40 mice compared with −/−IL-2Rα+/−p40 mice, whereas the number of B2 cells (P = 0.3752) was not altered , B1b (P = 0.3832) and B2 (P = 0.5434) cells in spleen B cells did not change  did not change, but the frequency of Annexin V+7-AAD+ late stage apoptotic B1a cells (P = 0.0284) was significantly increased in the PC of −/−IL-2Rα−/−p40 mice. These data demonstrated a positive correlation of the change in B1a cell frequency and disease progression in −/−IL-2Rα−/−p40 mice.The frequency of B1a  domain, was also up-regulated , Cd28 and Cd86, were also closely related  and −/−p40 (tm1JmB6.129S1-Il12b) mice on a C57BL/6J background were initially obtained from The Jackson Laboratory . +/−IL-2Rα mice were crossed with −/−p40 mice to generate +/−IL-2Rα+/−p40 mice, then backcrossed with −/−p40 mice to generate −/−IL-2Rα+/−p40 mice as breeding pairs. +/−IL-2Rα mice were used as breeding pairs to generate +/−IL-2Rα mice. We initially compared −/−IL-2Rα+/−p40 mice to +/−IL-2Rα and control C57BL/6 (WT) mice and demonstrated that these strains were similar and healthy. Hence, −/−IL-2Rα+/−p40 mice were used as controls throughout these studies. All mice were studied at 3-18 weeks and serially studied at 3, 6, 9, 12 and 18 weeks of age, with a minimum of 3 mice at each data point. Microarray and Real Time PCR assays were performed in 7-week old female mice. dnTGFβRII (B6.Cg-Tg(Cd4-TGFBR2)16Flv/J) mice, abbreviated as TG and used as another PBC mouse model [se model , were obLiver tissues were excised and immediately fixed with 10% buffered formalin solution for 2 days at room temperature, then embedded in paraffin and cut into 4 μm sections for hematoxylin-eosin (H&E) staining . Images As previously outlined , we isolA syringe containing 100ul ice cold PBS, with a 29G (0.33 mm) needle was carefully inserted into the peritoneum to avoid puncture of organs. After injection, the peritoneum was gently massaged for 2 minutes to dislodge any attached cells into the PBS solution. Thence an incision was made into the peritoneum and fluid collected with a Pasteur pipette; the fluid was then centrifuged at 5500g for 5 minutes and supernatant assayed using a cytometric bead assay (CBA) [6 cell preparations were incubated with purified anti-CD16/CD32 antibody  for 15 minutes at 4°C. Cells were stained for 20 minutes at 4°C with cocktails containing combinations of fluorochrome-conjugated monoclonal antibodies for cell surface markers including Pacific Blue-CD3, Pacific Blue-PDCA-1, FITC-B220, APC/Cy7-CD4, APC/Cy7-IgD, APC/Cy7-CD19, PE/Cy7-NK1.1, PE/Cy7-CD23, PerCP/Cy5.5-IgM, APC-CD21/35, Alexa Fluor 647-CD11b, PE-F4/80, PE-CD5 (BioLegend), V500-CD4 and V500-CD8a . In PC, B cells were defined as CD19+CD3−. Gated on B cells, B1a cells were defined as CD11b+CD5+ and B1b cells were defined as CD11b+CD5− whereas B2 cells were defined as CD11b−CD5− [+CD3−PDCA-1−. Gated on B cells, B1a cells were defined as CD23−CD5+IgMhiIgDlo and B1b cells were defined as CD23−CD5−IgMhiIgDloCD21/35−, whereas B2 cells, contained Follicular B cells (CD21/35loCD23+) and Marginal zone B cells (CD21/35hiCD23−) [1×1011b−CD5− . In splehiCD23−) . The numhiCD23−) .For intracellular cytokine staining, cells were suspended in RPMI-1640 with 10% fetal bovine serum (FBS) and stimulated with Cell Stimulation Cocktail (plus protein transport inhibitors) (eBioscience) at 37°C for 4 hours. Cells were incubated with purified anti-CD16/CD32 antibody and then stained with Pacific Blue-CD3, APC/Cy7-CD4, PerCP/Cy5.5-CD19, Alexa Fluor 647-CD11b, FITC-CD5, PE/Cy7-CD11b and PE/Cy7-NK1.1 (BioLegend), V500-CD8a (BD Biosciences) as above, fixed with fixation buffer, and permeabilized with permeabilization wash buffer (BioLegend); cells were thereafter stained for intracellular PE-IFN-γ, PE-IL-10, APC-IL-10, or PE-CTLA-4 (BioLegend). Normal IgG isotype controls (BioLegend) were used in parallel . Intrace6 cells were incubated with purified anti-CD16/CD32 antibody and then stained with FITC-CD4, PE-CD5, APC/Cy7-CD19 (BioLegend), and PE/Cy7-CD11b. The washed cells were resuspended in Annexin V binding buffer (BioLegend), and stained with Alexa Fluor 647-Annexin V (BioLegend) and 7-AAD (BioLegend) at room temperature for 15 min. Early stage apoptotic cells were defined as Annexin V+7-AAD− and late stage apoptotic cells were defined as Annexin V+7-AAD+. FACSVerse flow cytometry  was used to acquire data, which was analyzed by Flowjo software .Detection of apoptosis using a combined Annexin V and 7-AAD assay. 1×10−/−IL-2Rα−/−p40 and −/−IL-2Rα+/−p40 mice were isolated as above; the sorting purity was greater than 96%. Cells were suspended in TRIzol and total RNA extracted with an RNeasy Mini Kit  and hybridized to Agilent chips. Fluorescence was detected using the Agilent Scanner G2505C (Agilent Technologies) and images were analyzed using Feature Extraction software . A transcription profile chip service was provided by Shanghai OE Biotechnology Cooperation . Expression fluorescence values were log2-transformed, and subsequently analyzed using Genespring. Differentially expressed genes were defined as equal to or more than 2 fold differences between the two groups; signal values were confirmed beyond background signals, and the genes were classified based on the annotation of the Gene Ontology (GO) project. Volcano plot, heat map and red-green scale schemes were designed using Multiple Experiment Viewer 4.8 software. The microarray data was deposited on the National Center for Biotechnology Information GEO repository under accession number GSE79190.PC B1a cells from 7-week old female http://www.genemania.org), STRING (http://string-db.org) and Gene Network Central (http://www.sabiosciences.com) [http://www.cytoscape.org) was used to create the interaction networks.The target genes for all selected B cell functional genes were identified or predicted using three online bioinformatic databases, including GeneMANIA (ces.com) . The opeThe levels of IFN-γ, TNF-α, IL-6, MCP-1 and IL-10 from PC lavage fluid were measured with a BD cytometric bead assay (CBA) “Mouse inflammation” kit (BD Pharmingen), using a BD FACSVerse flow cytometer with CBA software.2 and P values were calculated.Correlation analysis was made using GraphPad Prism 5 with linear regression analysis. The linear correlation index r−/−IL-2Rα−/−p40 and −/−IL-2Rα+/−p40 mice; cell extraction was performed using Trizol Reagent , and cDNA synthesized with the PrimeScript® RT reagent Kit (Takara). Quantitative real-time PCR was performed by AB Stepone real-time PCR system  and using SYBR® Premix Ex TaqTM II (Takara). The primers used were noted in Table Gapdh (ΔCt). The results were calculated by the 2−ΔΔCt method.B1a peritoneal isolated cells were isolated from Statistical significance was analyzed using GraphPad Prism 5 . All experiments were replicated at least three times with similar results. All data are expressed as mean ± standard deviation (SD), and evaluated using a 2-tailed unpaired Student t test.


This elevation was suppressively controlled by targeting Ser129-phosphorylated α-synuclein to the proteasome pathway. Rotenone-induced insoluble α-synuclein was also targeted by Ser129-phosphoryation to the proteasome pathway. Experiments with epoxomicin and chloroquine showed that proteasomal targeting of insoluble Ser129-phosphorylated α-synuclein was enhanced under lysosome inhibition and it reduced accumulation of insoluble total α-synuclein. However, in a rat AAV-mediated α-synuclein overexpression model, there was no difference in the number of total α-synuclein aggregates between A53T mutant and A53T plus S129A double mutant α-synuclein, although Ser129-phosphorylated α-synuclein-positive aggregates were increased in rats expressing A53T α-synuclein. These findings suggest that Ser129-phosphorylation occurs against stress conditions, which increases influx of extracellular Ca2+, and it prevents accumulation of insoluble α-synuclein by evoking proteasomal clearance complementary to lysosomal one. However, Ser129-phosphorylation may provide an ineffective signal for degradation-resistant aggregates, causing extensive phosphorylation in aggregates.Parkinson’s disease (PD) is characterized neuropathologically by intracellular aggregates of fibrillar α-synuclein, termed Lewy bodies (LBs). Approximately 90% of α-synuclein deposited as LBs is phosphorylated at Ser129 in brains with PD. In contrast, only 4% of total α-synuclein is phosphorylated at Ser129 in brains with normal individuals. It is unclear why extensive phosphorylation occurs in the pathological process of PD. To address this issue, we investigated a mechanism and role of Ser129-phosphorylation in regulating accumulation of α-synuclein. In CHO cells, the levels of Ser129-phosphorylated soluble α-synuclein were maintained constantly to those of total α-synuclein in intracellular and extracellular spaces. In SH-SY5Y cells and rat primary cortical neurons, mitochondrial impairment by rotenone or MPPThe online version of this article (doi:10.1186/s40478-017-0452-6) contains supplementary material, which is available to authorized users. Drosophila model of PD, co-expression of α-syn and Drosophila G-protein-coupled receptor kinase 2 (Gprk2) was shown to generate Ser129-phosphorylated α-syn and enhance α-syn toxicity ), sonicated at 30 W for 1 s 5 times, and kept on ice for 30 min. After centrifugation at 12,000×g for 30 min, the resulting supernatant was collected and stored at −80 °C until use. Protein concentrations were measured by the BCA assay (Thermo Fisher Scientific).For preparation of cell lysates, cultured cells were suspended in buffer A , cells were plated onto 10 cm dishes. When cells were 90% confluent, we exchanged the growth media with 6 mL of Opti-MEM (Thermo Fisher Scientific). After further 24 h incubation, CM was collected and centrifuged at 6,000×g for 5 min to remove cell debris. Immediately, 6 mL of CM was added with 1/4 volume of 100% trichloroacetic acid (TCA), incubated for 30 min on ice, and centrifuged at 14,000×g for 5 min. The resultant pellet was washed three times with 300 μL of cold acetone, air dried, and dissolved in 100 μL of Laemmli’s sample buffer containing 2.5% β-mercaptoethanol.For analyzing the levels of insoluble α-syn, 1% Triton X-100 cell lysates were separated by centrifugation at 100,000×g for 30 min . Supernav/v) Tween 20 (TBS-T) for 10 min 3 times. The membrane was blocked by TBS-T containing 5% skim milk for 30 min, incubated in TBS-T containing 2.5% skim milk and primary antibody overnight in the cold room, and further incubated in the same buffer containing the corresponding secondary antibody overnight in the cold room. When we detected phosphorylated α-syn, 50 mM NaF was added to TBS-T containing skim milk. To visualize the signal, membranes were treated with ECL plus (Thermo Fisher Scientific) for detection of total α-syn, including non-phosphorylated and Ser129-phosphorylated forms, and phosphorylated α-syn. Other proteins were detected by supersignal West Pico chemiluminescent substrate (Thermo Fisher Scientific). Signals were recorded using a CCD camera, VersaDog 5000 (Bio-Rad). Levels of total α-syn and Ser129-phosphorylated α-syn were estimated by measuring band intensities with Quantity One software (Bio-Rad). For more quantitative estimation of the expression levels of total α-syn and Ser129-phosphorylated α-syn, we used purified recombinant α-syn proteins and Ser129-phosphorylated α-syn proteins as standards [Samples were denatured at 95 °C for 5 min in Laemmli’s sample buffer containing 2.5% β- mercaptoethanol. Equal protein amounts of denatured samples were subjected to SDS-PAGE on 13.5% polyacrylamide gels and then transferred to PVDF membranes . The transferred membrane was incubated in phosphate-buffered saline (PBS)  containing 4% paraformaldehyde (PFA) with 0.1% glutaraldehyde for detecting Ser129-phosphorylated α-syn for 30 to 60 min or without glutaraldehyde for detecting other proteins as described previously . After itandards , 12. A sThe experiments using rats had been approved by the Animal Subjects Committee of Yamagata University . We usedP values <0.05 were considered statistically significant.We performed each experiment at least three times. Data are expressed as mean ± standard deviation and n represents the number of samples. Homogeneity of variances was checked with Levene’s test. If the variances were homogenous, comparisons were performed by one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. In case of nonhomogeneity of variances, comparisons were performed by Welch-ANOVA with Games-Howell post hoc test . The authors declare that the main data supporting the findings of this study are available within the article and Additional files n = 12, r2 = 0.885) , although endogenous α-syn was undetectable  Fig. . The samble Fig. . These f2+ on Ser129-phosphorylation of α-syn, we incubated SH-SY5Y cell lines, which stably expressed wild-type α-syn (wt-aS/SH #4) [P = 0.033; 3.40 ± 0.45-fold increase at 8-h incubation, P = 0.001, n = 5, each group), compared with vehicle control cells     was significantly blocked by the addition of CaM inhibitor W-7 from 0.05 μM  and different CaM inhibitor calmidazolium chloride from 5.0 μM , compared with vehicle control cells  , for 8 hlls Fig. . Cells wup) Fig. . Howeverup) Fig. . The A23up) Fig. . Additioup) Fig. . EGTA anlls Fig. . These fP = 0.013, n = 5), compared with vehicle control cells , and A23187-mediated Ser129-phosphorylation of α-syn (3.30 ± 0.28-fold increase) was significantly inhibited by BAPTA-AM   , compared with vehicle control cells   or 5 μM of rotenone  in a dose-dependent manner  Fig. . After iner Fig. . Total αone Fig. .Fig. 3Ef+ in the presence or absence of Ca2+ chelators. MPP+-mediated Ser129-phosphorylation of α-syn  was significantly inhibited by BAPTA-AM   was significantly inhibited by EGTA    was significantly inhibited by EGTA   was inhibited by W-7    Fig. . To asseup) Fig. . Similarup) Fig. . Rotenonup) Fig. . Additioup) Fig. . Ser129-up) Fig. . These fP < 0.001; 1.68 ± 0.09-fold increase at 10 nM, P = 0.036, n = 4, each group), compared with vehicle control cells . The rotenone-mediated Ser129-phosphorylation of α-syn (1.54 ± 0.11-fold increase) was significantly inhibited by 0.5 mM EGTA   -chase experiments in wt-aS/SH cells. As previously reported, the levels of Ser129-phosphorylated α-syn rapidly decreased Fig. . When ceP = 0.011, n = 4) as compared with vehicle control cells   for 5 days. The cells were then separated into 1% Triton X-100-soluble and 1% Triton X-100-insoluble fractions by centrifugation at 100,000×g for 30 min. The 1% Triton X-100-insoluble fractions were resolved in a solution containing 8 M urea / 2% SDS. Western blots of 1% Triton X-100-soluble fractions showed that 10 and 50 nM rotenone elevated Ser129-phosphorylated α-syn levels without altering total α-syn levels. When we analyzed 1% Triton X-100-insoluble fractions, 50 nM rotenone generated insoluble total α-syn Fig. . The levlls Fig. . Althoug 4) Fig. . We then 4) Fig. . MG132 s 4) Fig. . However 4) Fig. . These fleft panels), epoxomicin did not affect the levels of 1% Triton X-100-insoluble Ser129-phosphorylated α-syn, but did increase 1% Triton X-100-soluble Ser129-phosphorylated α-syn levels. Additionally, epoxomicin did not affect 1% Triton X-100-insoluble total α-syn levels. Chloroquine treatment failed to induce expression of insoluble Ser129-phosphorylated α-syn, but insoluble total α-syn did accumulate . When cells were co-incubated in media containing epoxomicin and chloroquine, insoluble Ser129-phosphorylated α-syn proteins were generated in conjunction with the accumulation of insoluble total α-syn . These findings showed that proteasomal targeting of insoluble Ser129-phosphorylated α-syn was more activated under lysosome inhibition. To further test the effect of Ser129-phosphorylation on the metabolism of insoluble α-syn proteins, we compared insoluble total α-syn levels between cells expressing wild-type α-syn (wt-aS/SH cells) and Ser129-phosphorylation incompetent S129A mutant α-syn (S129A-aS/SH cells). In the wt-aS/SH cells, epoxomicin and chloroquine treatment yielded insoluble total α-syn  more abundantly than chloroquine single treatment    Fig. . Additio 5) Fig. . S129A-a 5) Fig. . These f3 at 2 weeks (n = 5) and 591.9 ± 34.6/mm3 at 4 weeks . Rats expressing A53T plus S129A double-mutant α-syn had 285.8 ± 103.9/mm3 α-syn aggregates at 2 weeks (n = 4) and 585.4 ± 103.3/mm3 at 4 weeks (n = 3). There was no significant difference in the number of total α-syn aggregates between rats expressing A53T single mutant and A53T plus S129A double-mutant α-syn (P = 1.000). These findings showed that Ser129-phosphorylation had no impact on the accumulation of α-syn aggregates in vivo.To determine whether Ser129-phosphorylation affects the formation of α-syn aggregates in vivo, we quantified the number of α-syn aggregates in the striatum of the rat AAV-mediated α-syn overexpression model. These samples were obtained from our previous study . In rats2+ concentration was a key factor in kinase-modulated Ser129-phosphorylation. In support of this, Ser129-phosphorylation required CaM function, which controls a variety of kinases in a Ca2+-dependent manner [+, Ser129-phosphorylation was enhanced through an increased influx of extracellular Ca2+. However, the CHX-chase experiments showed that rotenone-induced Ser129-phosphorylated α-syn was targeted to the proteasome pathway at the same rate as normally phosphorylated α-syn. It should be noted that in the present experimental condition, ATP-dependent proteasome activity was not lost by mitochondrial impairment. These findings suggested that proteasomal targeting played a role in suppressively controlling Ser129-phosphorylated α-syn levels. Then, we examined the role of Ser129-phosphorylation in insoluble α-syn accumulation. Chronic treatment with a low concentration of rotenone for 5 days induced small amounts of insoluble α-syn. CHX-chase experiments with MG132 showed that insoluble Ser129-phosphorylated α-syn was targeted to the proteasome pathway. This finding suggested that proteasomal targeting also played a role in suppressing accumulation of insoluble Ser129-phosphorylated α-syn.To determine the mechanisms and biological role of Ser129-phosphorylation in α-syn aggregate formation, we first examined the regulation of Ser129-phosphorylation in normal soluble α-syn. Our results showed that α-syn was phosphorylated at Ser129 in proportion to the levels of total α-syn. This phenomenon could be explained by two possibilities: 1) responsible kinases are constitutively active in a substrate-dose dependent manner; or 2) this regulation system is maintained by dephosphorylating or degrading excess amounts of Ser129-phosphorylated α-syn in reaction to total α-syn levels. To further explore these possibilities, we examined how the regulation mechanism of Ser129-phosphorylation was disrupted. Our results showed that the intracellular Cat manner , 22. In The present data raised a question of why increased levels of Ser129-phosphorylated α-syn were not accompanied by alteration in the levels of total α-syn in soluble and insoluble forms. To address the issue, we measured the change in levels of Ser129-phosphorylation and total α-syn under proteasome or lysosome inhibition, because α-syn is known to degrade through the autophagy-lysosome pathway . TreatmeThese findings were consistent with results from a previous yeast study showing that Ser129-phosphorylation and sumoylation push α-syn aggregates into the proteasome pathway and autophagy-lysosome pathway, respectively, and Ser129-phosphorylation rescues autophagy-lysosome clearance of α-syn by promoting proteasomal clearance when sumoylation is impaired . This prThe present data also raised a question as to relationship between effects of Ser129-phosphorylation on proteasomal targeting of soluble and insoluble α-syn proteins and extensive phosphorylation in α-syn aggregates. To address this, we assessed α-syn aggregates in a rat rAAV model expressing A53T α-syn with or without the S129A mutation. The present data showed that Ser129-phosphorylation had no impact on α-syn aggregate accumulation despite extensive phosphorylation. A previous study demonstrated that fibrillar α-syn proteins were phosphorylated by casein kinase (CK) I or CK II, and they were not good substrates for phosphatases in vitro . Because+ enhanced Ser129-phosphorylation through increased influx of extracellular Ca2+, this elevation was suppressively controlled by targeting Ser129-phosphorylated α-syn to the proteasome pathway. This targeting was seen in insoluble α-syn induced by rotenone. Additionally, proteasomal targeting of insoluble Ser129-phosphorylated α-syn was promoted under lysosome inhibition. This complementary action prevented accumulation of insoluble total α-syn. However, in a rat AAV-mediated α-syn overexpression model, Ser129-phosphorylation did not affect α-syn aggregate formation. Taken together, we propose a model that extensive phosphorylation in α-syn aggregates was consequently generated by an ineffective action of Ser129-phosphorylation for removing degradation-resistant α-syn aggregates. We report that a role of Ser129-phosphorylation in regulating α-syn expression levels is associated with extensive phosphorylation in α-syn aggregates. The levels of Ser129-phosphorylated α-syn were suppressively maintained to be constant to those of total α-syn in intracellular and extracellular spaces. Although mitochondrial impairment by rotenone or MPPAdditional file 1: Figure S1.2+ on Ser129-phosphorylation of α-syn in rat primary cortical neurons. Cell lysates (10 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or anti-β-actin (AC-15) antibody. a Effect of A23187 concentrations on Ser129-phosphorylation. Primary cortical neurons were treated with A23187 at the indicated concentrations for 8 h. b, c Effect of extracellular Ca2+ chelator EGTA (b) or intracellular Ca2+ chelator BAPTA-AM (B-AM) (c) on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing 0.25 μM A23187 with the indicated concentrations of EGTA or BAPTA-AM for 8 h. d Effect of CaM inhibitor W-7 on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing 0.25 μM A23187 with the indicated concentrations of W-7 for 8 h. Representative blots are shown. Relative band intensities of Ser129-phosphorylated α-syn and total α-syn were normalized to those of β-actin. Graphs show relative ratios to vehicle control cells. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction or Welch-ANOVA with Games-Howell post hoc test for unequal-variances . (TIFF 2872 kb)Effects of CaAdditional file 2: Figure S2.2+ chelator BAPTA-AM (b) or extracelluar Ca2+ chelator EGTA (c) on rotenone-induced Ser129-phosphorylation. Neurons were incubated in media containing 1 nM rotenone and the indicated concentrations of BAPTA-AM or EGTA for 1 h. d Effect of CaM inhibitor W-7 on rotenone-induced Ser129-phosphorylation. Neurons were incubated in media containing 1 nM rotenone with W-7 at the indicated concentrations for 1 h. Representative blots are shown. Relative band intensities of Ser129-phosphorylated α-syn and total α-syn were normalized to those of β-actin. Graphs show relative ratios to vehicle control cells. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction or Welch-ANOVA with Games-Howell post hoc test for unequal variances . (TIFF 4854 kb)Effects of mitochondria complex I inhibitor rotenone on Ser129-phosphorylation of α-syn in rat primary cortical neurons. Cell lysates (5 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a Effect of rotenone concentrations on Ser129-phosphorylation. Primary cortical neurons were incubated in media containing the indicated concentrations of rotenone for 1 h. Vehicle controls were treated with DMSO at the same final concentration. b, c Effect of intracellular Ca


The substituent planes at the C=C double bond of this fragment are twisted by 16.0 (3)° as a result of steric effects. The amine N(Et) N atom has a trigonal–pyramidal configuration (sum of the bond angles = 346.3°). The interplanar angle between the two carboxylate substituents is 60.39 (8)°. In the crystal, molecules form zigzag chains along [010] by intermolecular N—H⋯O hydrogen-bonding interactions, which are further packed in stacks toward [100]. The title azoninoindole might be considered as a candidate for the design of new Alzheimer drugs.The title compound, C Meanwhile, such medium-sized heterocycles, in particular azonine, frequently occur in natural products, such as alkaloids . 3-Methoxymethyl-substituted indole II was isolated as a by-product of this reaction.Azoninoindole re Fig. 1. The domet al., 1987et al., 2003et al., 2003et al., 2009et al., 2014et al., 2015et al., 2017The azonine systems, as a result of their specific structure, are known to act as ligands towards different receptors, thus demonstrating diverse types of biological activity . It should be noted that the analogous nine-membered azonine ring in the related compound methyl 4-ethyl-11-methyl-1,4,5,6,7,8-hexahydroazoninoindole-2-carboxylate adopts a twisted boat conformation  dissolved in methanol (10 ml). The reaction mixture was stirred for 2 h at room temperature with the TLC real-time control. Then the solvent was removed in vacuo and the residue was chromatographed over silica with ethylacetate:hexane as eluent to yield the target fluorinated azoninoindole I (22%) and 3-methoxymethylindole II. Light-yellow crystals of azoninoindole I suitable for X-ray crystallographic analysis were grown by slow evaporation of an ethylacetate:hexane (1:1) solution, m.p. 456–458 K.Dimethyl acetylenedicarboxylate  was added to 2-ethyl-9-fluoro-1,2,3,4,5,6-hexahydroazepino: 374 [M+] (100), 345 (20), 315 (100), 285 (30), 227 (10), 198 (20), 174 (30), 161 (30), 148 (10), 58 (40), 45 (10).SCALA program  = 1.2Ueq(N)]. The other hydrogen atoms were placed in calculated positions with C—H = 0.95–0.99 Å and refined in the riding model with fixed isotropic displacement parameters [Uiso(H) = 1.2Ueq(C)].The hydrogen atoms of the amino groups were localized in the difference-Fourier map and refined isotropically with fixed displacement parameters [10.1107/S2056989018001329/kq2018sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989018001329/kq2018Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018001329/kq2018Isup3.cmlSupporting information file. DOI: 1818381CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports6: Article number: 32403; 10.1038/srep32403 published online: 09132016; updated: 12132016T. absoluta development’,This Article contains typographical errors. In the Results section under subheading ‘“The average pupal weight of the individual feeding on plants treated with OW, OW S+, LW and LW S+ was 3.91 g, 3.58 g, 3.15 g and 3.27 g, respectively”.should read:“The average pupal weight of the individual feeding on plants treated with OW, OW S+, LW and LW S+ was 3.91 mg, 3.58 mg, 3.15 mg and 3.27 mg, respectively”.In Figure 4, the y-axis ‘Pupal weight (mg)’ is incorrectly given as ‘Pupal weight (g)’. The correct Figure 4 appears below as


For nearly a century, aluminum salts have been the most widely used vaccine adjuvant formulation, and have thus established a history of safety and efficacy. Nevertheless, for extremely challenging disease targets such as tuberculosis or HIV, the adjuvant activity of aluminum salts may not be potent enough to achieve protective efficacy. Adsorption of TLR ligands to aluminum salts facilitates enhanced adjuvant activity, such as in the human papilloma virus vaccine Cervarix®. However, some TLR ligands such as TLR7/8 agonist imidazoquinolines do not efficiently adsorb to aluminum salts. The present report describes a formulation approach to solving this challenge by developing a lipid-based nanosuspension of a synthetic TLR7/8 ligand (3M-052) that facilitates adsorption to aluminum oxyhydroxide via the structural properties of the helper lipid employed. In immunized mice, the aluminum oxyhydroxide-adsorbed formulation of 3M-052 enhanced antibody and TH1-type cellular immune responses to vaccine antigens for tuberculosis and HIV.  The ability of imidazoquinolines to target TLR7 and/or TLR8 to generate enhanced TH1-type innate immune responses, including IgG2 antibodies in mice, has been documented in the literature 22.1Synthetic 1,2-dilauroyl-sn-glycero-3-phosphcocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DLPG), 1,2-dimyrsitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), and glucopyranosyl lipid adjuvant  were purchased from Avanti Polar Lipids Inc. . Polysorbate 80 was purchased from J.T. Baker . Poloxamer 188 was purchased from Spectrum Chemical . Saline solution (0.9% w/v) was purchased from Teknova . CpG ODN was obtained from Avecia . Alhydrogel® ‘85’ and AdjuPhos® were purchased from E.M. Sergeant Pulp & Chemical Co. . 3M-052 (S-36862) was synthesized by 3M Drug Delivery Systems. The chemical structure has been previously disclosed 2.2Aqueous nanosuspensions were manufactured by dispersing 3M-052 or GLA with each lipid excipient see  at a 1:22.3Z-ave. Zeta potential was measured using the Zetasizer Nano-ZS using a disposable capillary cell, with nine consecutive measurements collected from each sample prepared at 1:10 dilution in water. In general, 3M-052 concentration was measured by UV absorbance at 322.5 nm after diluting formulations 1:20 into ethanol:HCl (98:2 v:v) and comparing to a standard curve. The dilution into organic solvent removes potential interference from light scattering of the nanosuspension particles. However, for the 3M-052 binding isotherm, where maximum sensitivity was desired, no dilution was performed for the samples or the standards. CpG concentration was measured by UV absorbance at 260 nm after 1:20 dilution into ethanol:HCl. GLA concentration was measured by reverse-phase HPLC with an C18 column (Atlantis T3 or Agilent XBridge) and charged aerosol detection (CAD) using a methanol:chloroform:water mobile gradient as described previously  g.Aqueous nanosuspensions were characterized for particle size by dynamic light scattering (DLS) using the Malvern Instruments  Zetasizer Nano-S or –ZS. Aqueous nanosuspensions were diluted 1:10 or 1:100 fold in water in a polystyrene cuvette prior to analysis, which consisted of three measurements resulting in a scattering intensity-biased average diameter value reported as the 2.42.Samples were preserved in vitrified ice supported by holey carbon films on 400-mesh copper grids. Samples were prepared by applying a 3 μl drop of sample suspension to a cleaned grid, blotting away with filter paper, and immediately proceeding with vitrification in liquid ethane. Grids were stored under liquid nitrogen until transferred to the electron microscope for imaging. Electron microscopy was performed using an FEI Tecnai T12 electron microscope, operating at 120 keV equipped with an FEI Eagle 4 k × 4 k CCD camera. Vitreous ice grids were transferred into the electron microscope using a cryostage that maintains the grids at a temperature below − 170 °C. Images of each grid were acquired at multiple scales to assess the overall distribution of the specimen. After identifying potentially suitable target areas for imaging at lower magnifications, high magnification images were acquired at nominal magnifications of 110,000 × (0.10 nm/pixel), 52,000 × (0.21 nm/pixel) and 21,000 × (0.50 nm/pixel). The images were acquired at a nominal underfocus of –2 μm , − 3 μm to –2 μm  and –5 μm  and electron doses of ~ 9–42 e/Å2.53COOH:H2O for overnight. The gels were then stained according to the directions provided by Sigma-Aldrich  ProteoSilver Plus Silver Stain Kit.Binding efficiency of 3M-052 and ID93 or FLSC to Alhydrogel® and AdjuPhos® was determined by UV–Vis Spectroscopy and SDS-PAGE with silver stain. 1-mL of formulation was prepared by mixing saline diluent, antigen, 3M-052-AF (aqueous nanosuspension of 3M-052 and DSPG), and/or aluminum salt. To determine FLSC adsorption, 30 μl of sample supernatant was mixed with 10 μl of 4 × reducing or non-reducing LDS Sample Buffer, following which 20–25 μl was loaded into a 10-lane SDS-PAGE gel with 15 μl of SeeBlue2 Prestained Standard. To determine ID93 adsorption, 45 μl of sample supernatant was mixed with 15 μl of 4 × reducing LDS Sample Buffer, following which 25 μl was loaded into a 10-lane SDS-PAGE gel with 15 μl of SeeBlue2 Prestained Standard. The gels were run for 55 min at 190 V and then placed into a fixing solution of 50:40:10 EtOH:CH2.6tm1Flv/J (TLR7-/-) were purchased from Jackson Laboratories . Mice were immunized by intramuscular injection with the recombinant TB vaccine antigen ID93 (0.5 μg/dose) or the HIV vaccine antigen FSLC1 (10 μg/dose) adjuvanted with AdjuPhos, Alhydrogel®, 3M-052 + Alhydrogel®, 3M-052 + AdjuPhos, or GLA + Alhydrogel®. The final adjuvant dose was 5 μg GLA or 0.1–10 μg 3M-052 with 200 μg of AdjuPhos or Alhydrogel® in 100 μl. Mice were boosted three weeks after the first immunization. All mice were maintained in specific pathogen-free conditions. All procedures were approved by the IDRI Institutional Animal Care and Use Committee.C57Bl/6 and B6.129S1-TLR72.7N = 5/group) were prepared 21 days after immunization by collection of retro-orbital blood into microtainer serum collection tubes , followed by centrifugation. Each serum sample was then analyzed by antibody capture ELISA. Briefly, ELISA plates  were coated with 2 μg/ml of the immunizing antigen in 0.1 M bicarbonate buffer and blocked with 1% BSA-PBS. Then, in consecutive order and following washes in PBS/Tween20, serially diluted serum samples, anti-mouse IgG, IgG1 or IgG2c-HRP  and ABTS-H2O2  were added to the plates. Plates were analyzed at 405 nm . Endpoint titers were calculated using Prism software V6 (GraphPad). Alternatively vaginal lavage fluid was collected three weeks after the third immunization with FLSC and analyzed for antibody titers using the same methods.Mouse sera (2.86 cells/well in 96-well plates and were stimulated for 2 h with the immunizing antigen (10 μg/ml), or unstimulated at 37 °C. GolgiPlug (BD Biosciences) was added and the cells were incubated for an additional 8 h at 37 °C. Cells were washed and surface stained with fluorochrome-labeled antibodies to CD4 (clone GK1.5), CD44 (clone IM7) and CD8 (clone 53-6.7) (BioLegend and eBioscience) in the presence of anti-CD16/32 (clone 2.4G2) for 20 min. Cells were washed and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 20 min. Cells were washed twice with Perm/Wash (BD Biosciences) and stained intracellularly with fluorochrome-labeled antibodies to CD154 (clone MR1), IFN-γ (clone XMG-1.2), IL-2 (clone JES6-5H4), TNF (clone MP6-XT22), GM-CSF (clone MP1-22E9), IL-5 (clone TRFK5), and IL-17A (clone TC11-18H10.1) (BioLegend and eBioscience) for 20 min at room temperature. Cells were washed and resuspended in PBS. Up to 106 events were collected on an LSRFortessa flow cytometer (BD Biosciences). Data were analyzed with FlowJo (TreeStar). Cells were gated as singlets > lymphocytes > CD4 + CD8– > cytokine positive or CD44hi > cytokine positive. In the case of ID93, antigen-specific response frequencies were determined by subtracting the frequency of response positives of unstimulated cells from antigen stimulated cells.One week after the final immunization splenocytes were isolated. Red blood cells were lysed using Red Blood Cell Lysis Buffer (eBioscience) and resuspended in RPMI 1640 and 10% fetal bovine serum (FBS). Cells were plated at 2 × 102.96 cells/mL. Cells were serially diluted 3-fold, added to plates, and incubated for 5 h at 37 °C. Secreted antibody was detected by addition of a 1:100 dilution of horse radish peroxidase (HRP) conjugated goat anti-mouse IgG antibody (Southern Biotech). Spots were visualized with AEC Peroxidase substrate kit (Vector Labs) according to manufacturer's instructions. Spots were quantitated on a CTL bioanalyzer.Antigen-specific antibody secreting cells present in the bone marrow were quantified using an ELISPOT assay. One day prior to assay initiation, Multiscreen ELISPOT plates (Millipore) were coated with 1μg of antigen/well, and incubated overnight. Blocked plates were washed three times with washing buffer (PBS + 0.5% Tween 20), blocked with collection medium for 2 h, and washed 3 times. Bone marrow was collected 21 days post-immunization in RPMI medium supplemented with 10% FBS, quantified using a Guava automated cell counter (Millipore) and resuspended to 1 × 102.106 events were collected on an LSRFortessa flow cytometer (BD Biosciences). Data were analyzed with FlowJo (TreeStar). Cells were gated as singlets > cells > CD19 + CD90.2– (B cells), CD8 + CD90.2 + (CD8 T cells), CD8– CD90.2 + (CD4 T cells), CD8– CD19– NK1.1 + (NK cells), CD8– CD19– CD11c + (DCs), CD8–CD19– CD11b + Ly6C + (inflammatory monocytes), or CD19–CD11b + Ly6G + (PMNs).18 h after intramuscular immunization into the gastrocnemius muscle the draining popliteal lymph node was collected and dissociated in PBS containing protease inhibitors (Thermo Fisher Scientific). Cells were surface stained for CD8, CD90.2 (clone 53-2.1), CD19 (clone 1D3), NK1.1 (clone PK136), CD11c (clone N418), CD11b (clone M1/70), Ly6G (clone 1A8), Ly6C (HK1.4), CD69 (clone H1·2F3), and CD86 (clone GL1) for 20 min on ice. Cells were washed and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 20 min. Cells were washed twice with Perm/Wash (BD Biosciences) and stained intracellularly with fluorochrome-labeled antibodies to IFN-γ and proIL-1β (clone NJTEN3) (BioLegend and eBioscience) for 20 min at room temperature. Cells were washed and resuspended in PBS. Up to 102.11p-values < 0.05 were considered significant. Only meaningful comparisons are reported in the figures .Antibody and T-cell responses were analyzed by two-way ANOVA with Tukey's multiple comparison correction using Prism version 5 or later (GraphPad). Comparisons resulting in 33.1Aqueous nanosuspensions of TLR ligands are formed by adding a suitable helper lipid or surfactant and inputting energy (e.g. sonication) to break down the particle size of the lipid complex Z-ave values were generally over 100 nm. However, the light scattering intensity-based Z-ave value reported by dynamic light scattering is heavily influenced by even a small proportion of large particles since they scatter more light than smaller particles  as evident in 3 . Moreover, the presence of other TLR ligands did not appear to interfere with the adsorption of 3M-052-AF to aluminum oxyhydroxide, indicating that an Alum-based formulation containing multiple adsorbed PRR ligands could be feasible (Table S2). In contrast to aluminum oxyhydroxide, we did not observe binding of 3M-052-AF to aluminum phosphate.To determine whether 3M-052-AF adsorbed to aluminum oxyhydroxide via a ligand exchange or electrostatic mechanism we evaluated the effect of ionic strength on adsorption, which neutralizes electrostatic mediated binding Recent seminal work by Wu et al. demonstrated that chemical synthesis of new TLR7 ligands with phosphonate groups facilitated adsorption to aluminum oxyhydroxide, resulting in improved transient localized adjuvant activity while reducing systemic activation 3.2To determine whether the binding of 3M-052-AF to aluminum oxyhydroxide alters the in vivo adjuvanticity of aluminum oxyhydroxide or 3M-052-AF, we immunized C57BL/6 mice with the tuberculosis vaccine antigen ID93 While the Th1-type response to 3M-052-AF-Alhydrogel® was generally consistent across multiple experiments, we note that there was more variability in biological response to 3M-052-AF (without Alhydrogel®), with some batches of 3M-052-AF inducing TH1 T cell adjuvant activity comparable to 3M-052-AF-Alhydrogel® (data not shown). Adsorption of 3M-052-AF to Alhydrogel® appeared to result in a more consistent biological activity between batches. Significant variation in biological response upon seemingly minor changes in formulation properties was also noted earlier for another aqueous nanosuspension, GLA-AF, from our lab 10 dose range. Three weeks after the first immunization with adjuvanted ID93 the Alum-adsorbed formulation of 3M-052-AF consistently elicited higher serum antibody titers across the entire dose range compared to either Alhydrogel® alone or the same dose range of 3M-052-AF  activates human TLR7 and TLR8 As noted above, we have previously employed the helper lipid approach to adsorb TLR4 ligands to aluminum oxyhydroxide. The otherwise insoluble TLR4 ligand GLA can be formulated as an aqueous suspension using a helper lipid, which can then be mixed with aluminum oxyhydroxide to allow for adsorption. However, in the case of GLA the agonist itself contains a phosphate group which promotes adsorption through ligand exchange. In the case of 3M-052, the agonist contains no phosphate group, thus adsorption due to ligand exchange must be entirely attributed to the helper lipid. Although in the present study ID93 + 3M-052-AF-Alhydrogel® appeared to induce a more potent TH1 response than ID93 + GLA-AF-Alhydrogel® , furtherTo test the in vivo adjuvant activity of 3M-052-AF with a different vaccine antigen as well as the effect of different aluminum salts, we immunized C57BL/6 mice with the HIV vaccine antigen FLSC + Ly6C+) in the draining lymph node 18 h after i.m. injection (Induction of a robust adaptive immune response to vaccine antigens requires appropriate activation of the innate immune system to provide the necessary costimulatory and cytokine milieu. Thus we analyzed the innate immune responses in the draining lymph node that are altered by immunization with 3M-052-AF ± aluminum phosphate or aluminum oxyhydroxide. Although not statistically significant in all readouts, overall 3M-052-AF synergized with aluminum oxyhydroxide to enhance various innate immune responses . Thus, 3njection A. Similanjection B and C. njection D, both m3M-052-AF and aluminum hydroxide synergized to increase expression of the co-stimulatory molecule CD86 on APCs in the draining LN and to transiently activate lymphocytes to remain in the draining lymph node in an antigen-independent fashion. By activating APCs and trapping lymphocytes in the same draining LN this synergy creates an optimal environment for lymphocyte priming and expansion. Interestingly, innate responses including early production of IFN-γ and IL-1β which we found to be important for the adjuvanticity of the GLA-SE 4In summary, we have developed a method to formulate lipid-based PRR ligands into aqueous nanosuspensions that can be made to adsorb to aluminum salts based on the properties of the helper lipid. The ability to develop Alum-compatible formulations of new PRR ligands may enable more rapid translation to the clinic since such formulations are analogous to the TLR4 ligand-Alum combination employed in Cervarix®, and aluminum salts are the most widely employed class of adjuvants in human vaccines, with a well-established safety and immunogenicity record.


N2,N3-bis(pyridin-4-ylmethyl)pyrazine-2,3-dicarboxamide with silver(I) nitrate led to the formation of a three-dimensional coordination polymer.The reaction of the ligand  18H16N6O2·2H2O (L1) , crystallized as a dihydrate. The molecule is U-shaped with the carboxamide groups being cis to one another, making a dihedral angle of 81.6 (5)°. The terminal pyridine rings are inclined to one another by 58.5 (4)°. There is an intramolecular N—H⋯Npyrazine hydrogen bond present, forming an S(5) ring motif. In the crystal, adjacent molecules are linked by N—H⋯Ocarboxamide hydrogen bonds, forming a chain along [001]. A chain of hydrogen-bonded water molecules is linked to the chain of (L1) molecules by O—H⋯N hydrogen bonds, forming columns propagating along the c axis. The columns are linked by C—H⋯O and C—H⋯N hydrogen bonds, forming a three-dimensional supramolecular structure. The reaction of ligand (L1) with silver(I) nitrate led to the formation of a new three-dimensional coordination polymer, {[Ag(C18H16N6O2)]NO3}n, polysilver(I)] nitrate] (I). The asymmetric unit is composed of half of one silver ion, located on a twofold rotation axis, half a ligand molecule and half a positionally disordered nitrate anion located about a twofold rotation axis. The full molecule of the ligand is generated by twofold rotational symmetry, with this twofold axis bisecting the Car—Car bonds of the pyrazine ring and the Ag—Ag bond. The carboxamide groups are now trans to one another, making a dihedral angle of 65.8 (4)°. The two terminal pyridine rings are inclined to one another by 6.6 (3)°. Two ligands wrap around an Ag—Ag bond of 3.1638 (11) Å, forming a figure-of-eight-shaped complex molecule. Each silver ion is coordinated by two pyridine N atoms and by two carboxamide O atoms of neighbouring molecules, hence forming a three-dimensional framework. The nitrate anion is linked to the framework by N—H⋯O and C—H⋯O hydrogen bonds.The title ligand, C N2,N3-bis(pyridin-4-ylmethyl)pyrazine-2,3-dicarboxamide (L1), is one of a series of ligands synthesized in order to study the superexchange in supramolecular complexes formed using pyrazine carboxamide derivatives and first row transition metal ions  have been described previously. It is very similar to the ligand N2,N3-bis(pyridin-2-ylmethyl)pyrazine-2,3-dicarboxamide (L2), for which a number of transition metal complexes have been described, including some interesting tetranuclear 2×2 grid-like and square complexes 4](ClO4)4}·5CH3OH·4H2O and {[Ni4(L2)4]Cl4}·5CH3CN·13H2O hexane, Huo et al. bis]bis(μ-nitrato)disilver]. They showed that it exhibits highly selective and sensitive luminescence sensing of Cr2O72− ions in aqueous solution. With the rigid tripodal arene-core-based nitrogen ligand, 1,3,5-tris(pyrazol-1-yl)benzene, Shu et al. bisbenzene-N,N′,N′′)trisilver(I) nitrate]. The nitrate counter-anions located in the cationic framework can be exchanged reversibly without destruction of the structure. Hence, this compound can act as a zeolite-like porous material for anion exchange.A search of the Cambridge Structural Database  coordination sites. It is therefore an interesting ligand to study its coordination behaviour with silver nitrate, and herein, we describe the solid state structures of ligand (L1), and the new three-dimensional coordination polymer, polysilver(I)]nitrate] (I).The title ligand has potentially two bidentate  and two monodentate (NL1) crystallized as a dihydrate, and its molecular structure is illustrated in Fig. 1cis to one another, making a dihedral angle of 81.6 (5)°. The terminal pyridine rings (N4/C7–C11) are inclined to one another by 58.5 (4)°. There is an intramolecular N—H⋯N hydrogen bond present, forming an S(5) ring motif . The coordination of the ligand to the silver ions is illustrated in Fig. 2I) are given in Table 2i bonds of the pyrazine ring and the Ag1—Ag1i bond °. The terminal pyridine rings (N4/C7–C11) are inclined to one another by 6.6 (3)°. Two ligands effectively wrap around a Ag—Ag bond of 3.1638 (11) Å, forming a figure-of-eight-shaped molecule, with each silver ion being coordinated by two pyridine N atoms. The silver ions are each further coordinated by the carboxamide O atom, O1, of neighbouring molecules, hence forming a three-dimensional framework, illustrated in Fig. 32O2Agi) then its coordination sphere can be described as distorted trigonal–bipyramidal, with a τ5 value of 0.8  nitrate led to the formation of a three-dimensional coordination polymer (d Table 2. The carL1), molecules are linked by N—H⋯O(water) hydrogen bonds forming chains propagating along the c-axis direction (Table 1L1) molecules by O—H⋯N hydrogen bonds, forming columns propagating along the c axis. The columns are linked by C—H⋯O and C—H⋯N hydrogen bonds, forming a three-dimensional supramolecular structure , the nitrate anion is situated in the cavities of the three-dimensional framework and is linked to the framework by N—H⋯O and C—H⋯O hydrogen bonds (Table 3I) is not essential for forming the three-dimensional structure, although it may act as a template for the formation of the framework bisbenzene-N,N′,N′′)trisilver(I) nitrate] mentioned above  as a three-dimensional coordination polymer, we make here the distinction between a coordination polymer and a metal–organic framework. Both have a three-dimensional framework but there are no cavities, even small ones, in the structure of (I). Hence, it should be classed as a three-dimensional coordination polymer according to the IUPAC recommendations on the ‘Terminology of metal–organic frameworks and coordination polymers’ methyl]xanthene-4,5-dicarboxamide]disilver bis(trifluoromethanesulfonate) chloroform solvate methy]xanthene-4,5-dicarboxamide), that wraps around an Ag—Ag bond forming a similar figure-of-eight-shaped complex. Here the Ag—Ag bond length is 3.134 (1) Å, slightly shorter than the value of 3.1638 (11) Å observed in (I); Table 2L1), N-(4-pyridylmethyl)carbamoyl)benzene, gave only two hits. Both of them are mercury(II) complexes, viz. the binuclear complex bis{μ2-1,2-bis[N-(4-pyridylmethyl)carbamoyl]benzene}tetrakis(trifluoroacetato)dimercury(II) methanol solvate carbamoyl]benzene}dichloridomercury(II) 1,2-dichloroethane solvate] , [N-(2-pyridylmethyl)carbamoyl]benzene, gave zero hits, while that for [N-(3-pyridylmethyl)carbamoyl]benzene gave eight hits. The latter includes the crystal structure of the dihydrate of the ligand itself  was prepared using the same procedure as for ligand (L2) . The solid was collected by filtration, washed with 10 ml of water and dried in air. Recrystallization in ethanol gave colourless plate-like crystals . Spectroscopic data: 1H NMR : 9.33 ; 8.86 ; 8.49 ; 7.39 ; 4.52 . 13C NMR : 165.8, 150.3, 148.9, 147.6, 145.6, 123.0, 42.2. IR : 3273 (s), 3031 (s), 1675 (vs), 1602 (vs), 1564 (vs), 1520 (vs), 1416 (vs), 1364 (s), 1311 (s), 1292 (s), 1220 (s), 1185 (m), 1164 (m), 1124 (s), 1069 (m), 995 (s), 871 (w), 830 (m), 787 (m), 745 (m), 715 (m), 611 (m), 575 (w), 504 (m), 495 (m), 475 (m). Elemental analysis for [C18H16N6O2]·H2O (Mr = 366.39 g mol−1): calculated: C: 59.01 H: 4.95 N: 22.94%; found: C: 59.10 H: 5.05 N: 23.10%.Ligand (I): A solution of (L1)  in 6 ml CHCl3 was introduced into a 13 mm diameter glass tube. It was layered with methanol (ca 2 ml) used as a buffer zone. A solution of AgNO3  in MeOH (6 ml) was then added gently to avoid possible mixing. The glass tube was sealed with a perforated parafilm and left at room temperature. Colourless block-like crystals were obtained after a few days . Elemental analysis for AgC18H16N7O5: (Mr = 518.25 g mol−1): calculated: C: 41.72 H: 3.11 N: 18.92%; found: C: 41.65 H: 3.09 N: 18.85%.Complex (L1), the NH and water H atoms were located in difference-Fourier maps and refined with distance restraints: O—H = 0.85 (2) Å, N—H = 0.88 (2) Å with Uiso(H) = 1.5Ueq(O) and 1.2Ueq(N). In the final cycles of refinement, the water H atoms were treated as riding atoms. For complex (I), the NH H atoms were included in calculated positions and treated as riding: N—H = 0.88 Å with Uiso(H) = 1.2Ueq(N). For both compounds, the C-bound H atoms were included in calculated positions and refined as riding: C—H = 0.95–0.99 Å with Uiso(H) = 1.2Ueq(C). The nitrate anion is positionally disordered about a twofold rotation axis and was refined with fixed occupancies , and all their ADP’s were made equal to that of atom O11. Using a one-circle image-plate diffraction system it is not possible to measure 100% of the Ewald sphere, particularly for triclinic or monoclinic systems. This is the case for ligand (L1), which crystallized in the monoclinic space group Pc and for which only 94.7% of the Ewald sphere was accessible.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989017006387/pj2044sup1.cifCrystal structure: contains datablock(s) L1, I, Global. DOI: 10.1107/S2056989017006387/pj2044L1sup2.hklStructure factors: contains datablock(s) L1. DOI: 10.1107/S2056989017006387/pj2044Isup3.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017006387/pj2044L1sup4.cmlSupporting information file. DOI: 1546645, 1546644CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: