All circulating innate lymphocyte populations were significantly decreased in adult, but not paediatric coeliac donors, when compared with healthy controls. Within the normal small intestine, we noted that Vδ3 cells were the most abundant γδ T cell type in the adult epithelium and lamina propria, and in the paediatric lamina propria. In contrast, patients with coeliac disease showed skewing toward a predominant Vδ1 profile, observed for both adult and paediatric coeliac disease cohorts, particularly within the gut epithelium. This was concurrent with decreases in all other gut lymphocyte subsets, suggesting a specific involvement of Vδ1 cells in coeliac disease pathogenesis. Further analysis showed that γδ T cells isolated from the coeliac gut display an activated, effector memory phenotype, and retain the ability to rapidly respond to in vitro stimulation. A profound loss of CD56 expression in all lymphocyte populations was noted in the coeliac gut. These findings demonstrate a sustained aberrant innate lymphocyte profile in coeliac disease patients of all ages, persisting even after elimination of gluten from the diet. This may lead to impaired immunity, and could potentially account for the increased incidence of autoimmune co-morbidity.Coeliac disease is a chronic small intestinal immune-mediated enteropathy precipitated by exposure to dietary gluten in genetically predisposed individuals. The only current therapy is a lifelong gluten free diet. While much work has focused on the gliadin-specific adaptive immune response in coeliac disease, little is understood about the involvement of the innate immune system. Here we used multi-colour flow cytometry to determine the number and frequency of γδ T cells , natural killer cells, CD56 Depending on the context of their activation, innate lymphocytes can also display immunoregulatory properties, e.g. invariant natural killer T (iNKT) cells can produce IFN-γ or IL-4 depending on the nature of antigen encountered and the cytokine environment [Innate, or unconventional, lymphocytes such as γδ T cells, CD56ironment . The rolironment . Mucosalironment –5. The rironment . The sigironment . The Vδ1ironment –14 and eironment , but datironment –17. Sincironment ,19. Phenironment , and devironment . In this study we used multi-parameter flow cytometry to characterise the frequency and phenotype of a number of novel innate lymphocyte populations in the blood and gut of adult and paediatric patients with CD. By comparing profiles of healthy control donors and CD patients, we were able to identify persistent alterations in innate lymphocyte populations, as a first step toward elucidating the potential roles for these cells in CD pathogenesis.This study was performed in adherence with the Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. The protocol was approved by the Recognised Ethics Committee at Our Lady’s Children’s Hospital, Crumlin (reference GEN/252/12) and the St James’s Hospital and Adelaide, Meath and National Children’s Hospitals  Research Ethics Committee, for paediatric and adult studies, respectively.Blood samples were obtained from 76 adult patients attending the coeliac clinic at St James’s Hospital, Dublin (SJH), and 78 control subjects recruited from healthy, age- and sex-matched research staff. Controls reported no family history of CD and tested negative for tissue transglutaminase antibodies (tTG). Within the coeliac donor cohort, 56 individuals were defined as treated coeliac donors (TCD), i.e. adhering to a gluten free diet, with accompanying tTG negative serology for at least one year prior to sample collection. The remaining 20 coeliac patients were classified as untreated coeliac donors (UCD) - a population of tTG positive patients on a gluten-containing diet. Duodenal biopsies were obtained from 8 TCD, 6 UCD and 7 control subjects. The control cohort comprised patients undergoing routine endoscopy at SJH, who reported no family history of CD, showed minimal histological inflammation, and tested tTG negative. Blood samples and duodenal biopsies were also obtained from 22 paediatric UCD and 38 age- and sex-matched control patients undergoing endoscopy at Our Lady’s Children’s Hospital Crumlin (OLCH).In all cases, CD diagnosis was confirmed according to clinical histological and serological analyses. Patients with a known history or subsequent diagnosis of inflammatory bowel disorders were excluded from recruitment/analysis.All samples were collected with the informed consent of individuals over 18 years of age, or with assent of those under 18 years with consent from guardians, as appropriate.Blood was harvested in EDTA and serum Vacutainer tubes (BD Biosciences).Duodenal biopsies (3-6 sections) were collected into sterile pots containing calcium- and magnesium-free Hank’s Balanced Salt Solution (HBSS) (Sigma-Aldrich) supplemented with 5% v/v foetal calf serum (FCS) (BioWest). Tissue biopsies were processed within 15 minutes of collection.Small intestinal epithelial and lamina propria cells were released from biopsies as previously described . Briefly-/CD56+ lymphocytes, CD56+ T cells as CD3+/CD56+, MAIT cells as CD3+/CD161+/Vα7.2+, and iNKT cells as CD3+/6B11+. Absolute numbers of T cells, B cells and NK cells in unprocessed blood samples were determined by TruCount MultiTEST mAb (BD Biosciences) staining and analysis on a BD FACSCanto flow cytometer. For lymphocyte phenotyping, whole blood was stained directly according to BD FACS Lysing Solution recommendations. Dissociated intestinal cells were resuspended in approximately 50 µl phosphate buffer containing 1% v/v BSA, and 0.02% v/v sodium azide, prior to staining. Fluorochrome-labelled monoclonal antibodies (mAb) used for phenotyping included: CD3-Pacific Blue, γδTCR-PE, Vδ2-PE, CD56-PE/Cy7, Vα7.2-APC, CD161-FITC, CD45RA-FITC, Vα24-Jα18-PE (clone 6B11), CD27-APC , Vδ3 , Vδ1-FITC . Vδ3 mAb was conjugated to APC dye using a Lightening-Link conjugation kit . Cells were stained, fixed in 1% paraformaldehyde (Santa Cruz Biotechnology) and analysed on a CyAn flow cytometer (Beckman Coulter). NK cells were defined as CD36 cells per ml. Cells were stimulated with either medium only or 10 ng/ml phorbol myristate acetate (PMA) and 1 µg/ml ionomycin (both from Sigma-Aldrich), and incubated for 4 hours at 37°C, 5% CO2 in the presence of 10 µg/ml Brefeldin A (Sigma-Aldrich). Cells were then harvested, stained for cell surface markers, fixed in 1% paraformaldehyde (Santa Cruz Biotechnology), permeabilised using 0.2% w/v Saponin (Sigma-Aldrich), incubated with the following intracellular cytokine antibodies: IFN-γ-Pacific Blue and IL-10-APC (BioLegend), IL-17A-PerCP/Cy5.5, IL-21-PE, and IL-22-PECy7 (eBioscience). Cells were analysed on a CyAn flow cytometer (Beckman Coulter). Small intestinal epithelial cells were isolated, washed and resuspended in cRPMI medium, and immediately plated at a density of 1x10Prism GraphPad software was used for data analysis, and differences in control and coeliac donor data were evaluated using a Mann Whitney U test. Statistical advice was provided for this study by Centre for Support and Training in Analysis and Research (CSTAR), University College, Dublin.Flow cytometric analysis of whole blood revealed significant decreases in almost all circulating innate lymphocyte subsets for the adult, but not paediatric, CD cohort when compared to normal controls . Adult c+ cells accounted for the majority of healthy intestinal γδ T cells, in both the epithelium and lamina propria of control donors of all ages, with the exception of the paediatric epithelium, where proportions of Vδ1+ and Vδ3+ cells were similar , and a similar trend was noted in adult donors. We noted that Vδ3 similar . The aveterature –14. In c+ cells in the coeliac gut, we undertook further characterisation of γδ T cells in blood and biopsies taken from paediatric patients (+ cells in paediatric blood and gut (CM), effector memory (TEM) and terminally differentiated (TEMRA) phenotypes were similar in CD patients and controls. However, in the coeliac gut, strong skewing toward a predominant TEM phenotype (CD45RA-/CD27-) was observed in the γδ T cell compartment, with a concomitant reduction in the proportion of naïve and TCM cells. This effect was most striking in the lamina propria, where in some donors almost 100% of γδ T cells displayed the TEM phenotype. Similar findings were observed for non-γδ T cells (data not shown). We also analysed the cytokine producing ability of γδTCR+ cells from the small intestinal epithelium (γδiEL) stimulated in vitro . Taken together, the data provide evidence for the presence of activated and functional γδ T cells in the coeliac gut.Having observed the abundance of Vδ1patients . Memory  and gut . Althougin vitro . Unstimu-/CD56+ lymphocytes) were reduced in the small intestinal epithelium and lamina propria of CD patients (+ cells) as well as non-T cells . For a definitive iNKT cell characterisation however, α-GalCer/CD1d tetramers are the gold standard. However our observations agree with those of Grose and colleagues, who found that Va24+ T cells, Va24 + /Vb11+ T cells and α-GalCer/CD1d tetramer+ iNKT cells were numerically deficient and functionally impaired in coeliac disease [We observed a reduction in MAIT cells in both adult and paediatric blood and gut, similar to observations made in the context of HIV ,26. In H+ target cells and releasing cytokines that promote TH1, TH2 and TH17 responses [It is well documented that Vδ1 cells comprise a major cell population in the normal intestine . Howeveresponses . Vδ1 celesponses . Recogniesponses . Vδ2 cel+ T cells in the CD gut, concurrent with a decrease in circulation, directly opposes findings from other inflammatory bowel disorders, where an increase in Vδ1+ T cells is noted in blood [-/CD45RA-) phenotype. Although initially this phenotype was used to describe Vδ2 cells in blood [Coeliac patients of all ages demonstrated a skewing from a predominant Vδ3 to a predominant Vδ1 phenotype throughout the gut. This was due to an increase in Vδ1 cells and concurrent reduction in Vδ3 cell frequency. It is interesting to note that this increase of Vδ1in blood ,31, but in blood ,32. Our in blood , revealein blood , which iin blood . Furtherin blood , the autin blood . Despitein blood ,36. We pin blood ,38. Actiin blood ,40, antiin blood , and conin blood . Vδ1 celin blood ,40), sugin blood , this mein blood ,45.+, CD3-, all three γδ T cell subsets and MAIT cells . Loss of CD56 expression was observed both in terms of positive cell frequency and expression intensity. Expression of CD56 by CD3- lymphocytes is most frequently used to define NK cells, whereas expression of CD56 by CD3+ lymphocytes identifies a population of T cells capable of potent non-antigen-specific cytotoxicity and cytokine release [+ and CD3- lymphocytes has previously been described in the gut and shown to be associated with a cytotoxic, non-proliferative, TH1 phenotype [+ cells are depleted in inflamed, but not uninvolved, IBD colonic tissue, and in the liver during chronic HCV infection [+ αβ T cells. A similar scenario could potentially occur in CD, where expansion of gluten-reactive αβ T cells in the small intestine has been demonstrated [As well as changes in specific innate subsets, we also noted a profound decline in CD56 expression in the gut of coeliac donors, compared to healthy donors, which extended across all CD56-expressing lymphocytes, including CD3 release . Expresshenotype . CD56+ cnfection . This denstrated . The immnstrated .CD is linked to an increased incidence of autoimmune disorders ,52. One


Objective. By specific knockout of carnitine palmitoyl transferase 1b (CPT1b) in skeletal muscles, we explored the effect of CPT1b deficiency on lipids and insulin sensitivity. Methods. Mice with specific knockout of CPT1b in skeletal muscles (CPT1b M−/−) were used for the experiment group, with littermate C57BL/6 as controls (CPT1b). General and metabolic profiles were measured and compared between groups. mRNA expression and CPT1 activity were measured in skeletal muscle tissues and compared between groups. Mitochondrial fatty acid oxidation (FAO), triglycerides (TAGs), diglycerides (DAGs), and ceramides were examined in skeletal muscles in two groups. Phosphorylated AKT (pAkt) and glucose transporter 4 (Glut4) were determined with real-time polymerase chain reaction (RT-PCR). Insulin tolerance test, glucose tolerance test, and pyruvate oxidation were performed in both groups. Results. CPT1b M−/− model was successfully established, with impaired muscle CPT1 activity. Compared with CPT1b mice, CPT1b M−/− mice had similar food intake but lower body weight or fat mass and higher lipids but similar glucose or insulin levels. Their mitochondrial FAO of skeletal muscles was impaired. There were lipids accumulations  in skeletal muscle. However, pAkt and Glut4, insulin sensitivity, glucose tolerance, and pyruvate oxidation were preserved. Conclusion. Skeletal muscle-specific CPT1 deficiency elevates lipotoxic intermediates but preserves insulin sensitivity. Palmitate oxidation assay was performed with reaction buffer  malic acid, 1.75 mM DTT, 0.07 mM NAD+, 2 mM ATP, 10 Mm Tris-HCl, and 0.07 mM Coenzyme A). The reaction started when 200μM [14C]-palmitate-15% BSA (1 : 6) complex (0.04 μCi/reaction mixture) was added and stopped by 3.5 M perchloric acid after incubation at 37°C for 30 min. CO2-trapping medium  for 14C radioactivity was measured by liquid scintillation to calculate palmitate oxidation rate. After incubation, 14CO2 and 14C-ASPs were measured . All assays were performed in triplicate, and data were normalized to protein content. Calculations were then performed accordingly. As previously described , 25, FAOm/z 264 and 282 (ceramides). Ceramides were quantified by taking the ratios of the integrated intensity for each subspecies to the intensity of C17:0. As previously reported –28, TAGsAs described –31, insu2-6H2O, 0.05 mM EDTA, and 0.05 mM ATP (pH 7.4)), 400 μL homogenate was used and the reaction started when 200 μM [14C]-glucose (0.1 μCi/reaction mixture) was added. The reaction buffer and condition are the same for palmitate oxidation as described above. CO2-trapping medium  for 14C radioactivity was measured by liquid scintillation to calculate glucose oxidation and quantified by weight of the skeletal muscle tissue homogenate.As described previously , 33, sket-test was used to evaluate the statistical significance of differences between knockout and controls. P < 0.05 was regarded as significant. SAS 9.3 (SAS Institute Inc.) was used for data analysis. All data are presented as the mean ± SD. Student's As shown in P < 0.05, resp.). Their lipids levels were higher (P < 0.05), but their glucose and insulin levels were similar.Compared to the mice in CPT1b group, those mice in CPT1b M−/− group had similar daily food intake. However, as shown in As shown in As shown in The relationship of fatty acid oxidation, lipids accumulation, and insulin sensitivity in skeletal muscles has been quite interesting. Imbalanced fatty acid uptake and fatty acid oxidation (FAO) have been linked to insulin resistance in muscles, which in turn worsens and complicates obesity and diabetes, as well as their complications , 34, 35.Some studies showed that increasing fatty acid oxidation alleviated insulin resistant . Our stuβ-HAD and citrate synthase, genes related to transcriptions such as NRF (nuclear respiratory factor), and the major nuclear receptor activator PGC1α [This is very exciting. There may be many pathways involved. First, due to the knockout of CPT1b in skeletal muscles, the major fuel sources were in deficit. This may suggest that signaling pathways related to energy were impaired or injured such as AMPK or mTOR . Second,or PGC1α . ThirdlyIn summary, our study finding is very unique. It suggested that with specific knockout of CPT1b in skeletal muscles, although there were lipids accumulations in skeletal muscles, the insulin sensitivity still remains. This may point towards a great direction for pharmaceutical development.


Campylobacter jejuni carry temperate bacteriophages that can affect the biology or virulence of the host bacterium. Known effects include genomic rearrangements and resistance to DNA transformation. C. jejuni prophage CJIE1 shows sequence variability and variability in the content of morons. Homologs of the CJIE1 prophage enhance both adherence and invasion to cells in culture and increase the expression of a specific subset of bacterial genes. Other C. jejuni temperate phages have so far not been well characterized. In this study we describe investigations into the DNA sequence variability and protein expression in a second prophage, CJIE4. CJIE4 sequences were obtained de novo from DNA sequencing of five C. jejuni isolates, as well as from whole genome sequences submitted to GenBank by other research groups. These CJIE4 DNA sequences were heterogenous, with several different insertions/deletions (indels) in different parts of the prophage genome. Two variants of a 3–4 kb region inserted within CJIE4 had different gene content that distinguished two major conserved CJIE4 prophage families. Additional indels were detected throughout the prophage. Detection of proteins in the five isolates characterized in our laboratory in isobaric Tags for Relative and Absolute Quantitation (iTRAQ) experiments indicated that prophage proteins within each of the two large indel variants were expressed during growth of the bacteria on Mueller Hinton agar plates. These proteins included the extracellular DNase associated with resistance to DNA transformation and prophage repressor proteins. Other proteins associated with known or suspected roles in prophage biology were also expressed from CJIE4, including capsid protein, the phage integrase, and MazF, a type II toxin-antitoxin system protein. Together with the results previously obtained for the CJIE1 prophage these results demonstrate that sequence variability and expression of moron genes are both general properties of temperate bacteriophages in C. jejuni. Campylobacter spp. in the 1960s and 1970s, but it was only in 2005 that whole genome sequencing confirmed their presence in C. jejuni, C. coli, and C. lariCampylobacter jejuni integrated element 1 (CJIE1) prophages Escherichia coli and Salmonella entericaC. jejuni prophages may carry effectors or other proteins that are virulence factors expressed by the host bacterium. If prophages of C. jejuni were found to play roles similar to those of E. coli and Salmonella, an understanding of the contribution of these prophages to virulence and host adaptation could be critical for surveillance, source tracking, and control of the subset(s) of C. jejuni responsible for human disease. The investigations that form the basis of this report were designed to query whether there was sequence variability in a second C. jejuni prophage, CJIE4, and to investigate whether variably carried genes were expressed as proteins and could therefore affect the biology of the host bacterium.Prophages were first demonstrated in C. jejuni RM1221 and CLIE1 from C. lari RM2100 C. jejuni tested and showed rather limited diversity in gene content by comparative genomic analysis using DNA microarrays C. jejuni strain 414 also demonstrated the presence of a prophage homologous to CJIE4, though less than 75% of the RM1221 CJIE4 genes were present C. jejuni S3 CJIE4 was observed by Fouts et al. to exhibit minimal DNA sequence similarity to CJIE1 from isolate RM1221 and greater similarity to both CJIE2 from C. jejuni prophages, consistent with the findings of at least one other group C. jejuni and comparing the results to CJIE4 sequences available in sequence databases. CJIE4 prophage sequences were found to be variable, containing a large indel with differential gene content in the middle of the genome and several other differentially carried genes throughout. Proteins encoded by genes within both major variants of the large central indel were detected, as were additional proteins encoded by conserved prophage genes. However, an examination of the putative functions of these proteins based on homology to known proteins suggested that most or all of the moron proteins carried by CJIE4 prophages may be associated with the function or biology of the phages themselves rather than having an influence on the biology of the host bacterium.Unfortunately, our laboratory has been so far unsuccessful at inducing and propagating Campylobacter and E. coli outbreak in Walkerton, Ontario, Canada The five isolates used for the study were associated with the investigation into the spring 2000 C. jejuni isolates were kept for long-term storage in either 20% skim milk or glycerol peptone water  at −80°C. For use, C. jejuni isolates with a low passage number were retrieved from storage at −80°C, plated to Oxoid Mueller-Hinton agar (Oxoid Inc.) containing 10% sheep red blood cells, and grown for 48–72 h at 37°C under a microaerobic atmosphere .http://pfam.sanger.ac.uk/search?tab=searchSequenceBlock; HHPred: http://toolkit.tuebingen.mpg.de/hhpred; BLAST with default settings . Annotations were checked and corrected using GenDb and the annotated prophage genomes were used to create GenBank submissions.Prophages were sequenced by PCR amplification and DNA sequencing of 2–3 kb fragments as outlined previously The whole genome sequence of isolate 00-2425 is CP006729. The accession numbers for the CJIE4 prophages from the other four isolates are as follows: 00-0949, KF751793; 00-1597, KF751794; 00-2425, KF751795; 00-6200, KF751796; 01-1512, KF751797.Full-length sequences of the CJIE4 homologs from isolates 00-0949 and 00-2425 were used to perform a BLASTn search of the nr database in Genbank. The latest date that sequences were checked was June 3, 2013.C. jejuni were: RM1221, NC_003912.7; S3, NC_017281.1; 140-16, AIPF0100006.1; 1997-4, AIOW01000013.1; 414, ADGM01000016.1; 84-25, AANT02000001.1. In other cases more than one contig was required to obtain coverage of the homologous prophage; in some cases, contigs containing only one or a few loci were assumed to be part of the larger prophage based on synteny with one or more of the five prophages sequenced for this study. Included were C. jejuni strains: DFVF1099, ADHK01000002.1, ADHK01000040.1; 1997-14, AIPA01000003.1, AIPA01000079.1, AIPA01000094.1, AIPA01000120.1, AIPA01000183.1; 51037, NZ_AIPB1000015.1, NZ_AIPB1000046.1, NZ_AIPB1000089.1; 51494, AINZ01000009.1, AINZ01000039.1, AINZ01000066.1, AINZ01000122.1; 87459, AIPE01000015.1, AIPE01000029.1, AIPE01000086.1. A single C. coli sequence from strain 2548  was used.Accession numbers for contiguous full length sequences in 2 concentrations of 2.0 mM, 0.2 mM of each dNTP, 0.5 µM of each primer, and 2.5 U of FastStart DNA polymerase. PCR reactions were performed using a Perkin Elmer 9700 thermocycler.All PCR reactions were done using DNA extracted from bacteria using a Gentra Systems PUREGENE DNA Isolation kit (Qiagen) according to the instructions of the manufacturer. PCR reactions were run using reagents from FastStart Taq DNA Polymerase kits (Roche). For each reaction the following parameters were used: final MgClC. jejuni isolate 01-1512, in which the prophage was inserted between genes encoding homologs of CJE0341c (upstream) and Cj0339c (downstream). Within each primer pair, one primer was located within a CJIE4 gene and one was located in the adjacent genomic DNA in order to unambiguously locate the genomic insertion site. Upstream primers were CJIE4uF 5′ GAG ATC TTT TTG CCT TGG GAC TT 3′ located in the homolog of CJ0341c  and CJIE4uR 5′ TCG ATG ACA TGT GAA CGC TTG AT 3′ located within the homolog of CJE1418 (RM1221 CJIE4). These primers had an optimal annealing temperature of 50.8°C and produced a 907 bp product. Cycle conditions for the primer pair were: initial denaturation at 94°C for 5 min; 35 cycles of 94°C for 30 s, 50.8°C for 1 min, 72°C for 1 min; final extension at 72°C for 7 min; 4°C until reactions were analyzed.PCR primers were developed on the basis of the location of the CJIE4 prophage within the draft whole genome sequence of TCG GTG TAT GGG CTG AAT 3′ located in the intergenic region between homologs of CJE1472 and 1473 (RM1221 homologs) and CJIE4dR 5′ TAA AAT GGG TGA GTT GGT GAG TAA 3′ located within Cj0339 . This primer pair had an optimal annealing temperature of 48.9°C and produced a 1544 bp amplicon. Cycle conditions for the primer pair were: initial denaturation at 94°C for 5 min; 35 cycles of 94°C for 30 s, 48.9°C for 1 min, 72°C for 2 min; final extension at 72°C for 7 min; 4°C until reactions were analyzed. Reaction products were analyzed by submarine electrophoresis using 1.6% agarose gels and stained with GelRed Nucleic Acid Stain (Cedarlane).Primers made to the other end of the CJIE4 prophage were CJIE4dF 5′ Total cellular proteins were prepared, modified, and labelled with iTRAQ reagents as previously described 2), shaking at 600 rpm for 2 min at RT and incubation without shaking for 30 min at RT. After this incubation, the cartridge was washed three times with 100 µl of 50 mM HEPES, pH 8.3.Protein modification and digestion in cartridges was done according to previously published methods Proteins were digested ON with 5 µl (5 µg) trypsin  in the Nanosep 10 cartridges. Following digestion the tryptic peptides were recovered from the cartridge, dried, and suspended in 30 µl 100 mM HEPES, pH 8.3. iTRAQ labels (AB Sciex Pte Ltd.) were suspended in 100% ethanol, added to the peptide mixtures, and incubated ON at RT. After quenching the reactions with sterile MilliQ water the mixtures were dried and stored at −20°C until use.18-reversed phase liquid chromatography on a micro-flow Agilent 1100/1200 series system (Agilent Technologies), using a Waters XBridge C18 guard column  and a Waters XBridge C18 analytical column . Mixed peptides were dried and suspended in LC buffer A , then resolved by a gradient of LC buffer A and buffer B . The gradient started at 3% B from 0–10 min, 8–11% B from 10–17 min; 11–60% B from 17–75 min; 95% B from 75–80 min; and 3% B from 80–170 min at a constant flow rate of 150 µl/min. Fractions were collected across the peptides elution profile (10–75 min), dried and resuspended in 40 µl of nano LC buffer A .iTRAQ-labeled tryptic peptide samples (100 µg) were fractionated by high-pH, C18-reversed phase trap column  with 100% buffer A  at 4 µl/min for a total volume of 30 µl, and then separated on a C18-reversed phase column . Both columns were packed in-house with ReproSil-Pur C18-AQ resin (Dr. Maisch). Peptides were eluted using a linear gradient of 2–20% buffer B  over 120 min at a constant flow rate of 250 nl/min. The total LC/MS/MS run-time was 160 minutes, including the loading, linear gradient, column wash at 95% buffer B, and the equilibration.Each fraction was separately analysed using a nano-flow Easy nLC II (Thermo Fisher Scientific) connected in-line to an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) with a nano-electrospray ion source (Thermo Fisher Scientific). The peptide fractions (6 µl) were loaded onto a Cm/z 300–1700 with a target resolution of 60,000 at m/z 400, and the subsequent fragment ion scans were acquired in the Orbitrap over a dynamic m/z range with a target resolution of 7500 at m/z 400. The lower threshold for selecting a precursor ion for fragmentation was 1000 counts. Dynamic exclusion was enabled using a list size of 500 features, a m/z tolerance of 15 ppm, a repeat count of 1, a repeat duration of 30 s, and an exclusion duration of 15 s, with early expiration disabled.Data were acquired using a data-dependent method, dynamically choosing the top 10 abundant precursor ions from each survey scan for isolation in the LTQ and fragmentation by HCD at 45% normalized collision energy. The survey scans were acquired in the Orbitrap over ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/] and prophage sequences generated in-house. The decoy database option was selected and the following parameters were used: carbamidomethylation (C) and iTRAQ (K and N-terminus) as fixed modifications, oxidations (M) as a variable modification, fragment ion mass tolerance of 0.5 Da, parent ion tolerance of 10 ppm, and trypsin enzyme with up to 1 missed cleavage. Mascot search results were imported into Scaffold Q+ v4.0 (Proteome Software) and filtered using 1.0% Protein FDR; 2 peptides; 0.1% Peptide FDR.All spectra were processed using Mascot Distiller v2.4.1 (Matrix Science), and database searching was done with Mascot v2.4 (Matrix Science). Searches were performed against an in-house built, non-redundant database consisting of NCBI's Genome database of bacteria . Phage genes within strain 414 had 0% abnormal tetranucleotide repeats Comparison of whole genome sequences determined that C. jejuni are characterized by high genetic microdiversity resulting from recombination differences in prophage content C. jejuni are “generalists” that can exhibit phenotypic diversity in, for instance, their interaction with eukaryotic cells regardless of isolation source and in the absence of large differences in gene content In summary, the DNA sequence and protein expression data together suggest that moron proteins encoded by genes within the CJIE4 prophage could potentially alter the homeostasis of infected bacteria. This is consistent with previously published data indicating that at least some


Maximum growth and nitrate uptake exhibit an optimum concentration and threshold tolerance to bicarbonate addition (~9 mM) above which both parameters decreased. Nonetheless, the transient highest cellular lipid and TAG contents were obtained at 18 mM bicarbonate, immediately after combined alkaline pH stress and nitrate depletion (day nine), while oil body and TAG accumulation were highly repressed with low carbon supply (2 mM). Despite decreases in the proportions of EPA and DHA, maximum volumetric and cellular EPA and DHA contents were obtained at this stage due to accumulation of TAG containing EPA/DHA. TAG accounted for 74% of the total fatty acid per cell, containing 55% and 67% of the overall cellular EPA and DHA contents, respectively. These results clearly demonstrate that inorganic carbon availability and elevated pH represent two limiting factors for lipid and TAG accumulation, as well as n-3 LC-PUFA partitioning into TAG, under nutrient-depleted P. lutheri cultures.In most microalgal species, triacyglycerols (TAG) contain mostly saturated and monounsaturated fatty acids, rather than PUFA, while PUFA-enriched oil is the form most desirable for dietary intake. The ability of some species to produce LC-PUFA-enriched oil is currently of specific interest. In this work, we investigated the role of sodium bicarbonate availability on lipid accumulation and  In aubstrate ,50. Bicahydrates ,51. More− to CO2 .i.e., volumetric and cellular total fatty acid, TAG, EPA and DHA contents) were used to estimate EPA/DHA-enriched oil accumulation and productivity during batch cultivation of P. lutheri under continuous light.In this context, the work presented here details the associated effects of sodium bicarbonate addition and nitrate limitation on cell growth, lipid body formation, total and TAG fatty acid composition; additionally, several parameters  demonstrated that bicarbonate addition stopped cell division in the chlorophyte Scenedesmus sp., but not in the diatom P. tricornutum where the cell cycle could be completed [N. salina was reduced by bicarbonate addition, whereas no significant effect was reported on growth of Tetraselmis suecica [P. lutheri to provide an indication of nutrient status and to establish whether there was a correlation between nitrate depletion, growth, and cellular lipid accumulation (P. lutheri using 18 mM bicarbonate exhibited a decrease (~20% in comparison to 9 mM bicarbonate) in final cell density associated with a slight delay in the culture to reach complete nitrate depletion. Results indicate that growth and nitrate uptake of P. lutheri had an optimum and threshold tolerance to bicarbonate addition (~9 mM) above which a decrease in both parameters was recorded. When supplementing cultures with gaseous CO2, Carvalho and Malcata (2005) also described a sensitivity of P. lutheri to inorganic carbon supply with increases in biomass up to a concentration of 1% CO2 in air (v/v), followed by a decrease at higher concentrations [Bicarbonate addition, and associated alkaline pH stress, has been shown to promote lipid accumulation in a number of microalgae but with species-specific responses with respect to cell division ,40,42,43ompleted . Similar suecica . In our mulation . The higmulation A,C,D. Grtrations .2 uptake during growth of photosynthetic microalgae has previously been reported to lead to an increase in pH and a decrease in CO2 partial pressure, with CO2 replacement occurring more slowly than consumption [32−) and insoluble carbonates of metals formation, which are not readily utilized by photosynthetic marine algae and can reduce growth and photosynthesis in some species [P. lutheri, no complete cessation of cell growth was observed with pH rise induced by bicarbonate addition, but a decrease in final cell density with 18 mM bicarbonate, as mentioned previously. Due to the observed salt precipitation, all results in this study are expressed per cell, and not per dry weight as in other studies.The addition of bicarbonate also had significant effects on the alkalinity of the cultures reaching maximum pH levels of pH 9.6 and 9.8 after nine days, applying 18 and 9 mM bicarbonate, respectively B. Howevesumption . At thes species . In P. l−1), compared to cultures with 9 mM bicarbonate addition (3.8 ± 0.5 pg cell−1). Despite the precipitation of salts observed, this result suggests higher inorganic carbon availability with increased bicarbonate supply for de novo synthesis of organic carbon compounds. Interestingly, lipid accumulation was highly repressed at low carbon supply (2 mM bicarbonate), showing almost no changes over time, even under nutrient depletion. As observed in previous studies for several chlorophyte species [P. lutheri also correlated with elevated pH at the time of nitrate depletion. Despite the proven ability of bicarbonate addition to enhance cellular lipid content in P. lutheri, carbon supply seems to be a limiting factor for lipid accumulation. Indeed, Toledos-Cervantes et al. (2013) also reported that lipid accumulation in Scenedesmus obtusiusculus significantly increased at 5% CO2, but not when the species was batch-cultivated at 0.04% CO2 (air without CO2-enrichment) [P. lutheri, Tonon et al. (2002) showed a decrease in the total fatty acid content per cell upon entry into the stationary phase [After full nitrate depletion, the maximum cellular lipid content  . In P. lry phase , which cP. lutheri reached a maximum after nine and 14 days, using 18 and 9 mM bicarbonate, respectively, and subsequent decline was observed, indicating that lipid accumulation ceased and/or cellular lipids were being utilized or degraded. Interestingly, lower in cellular lipid content corresponded to a reduction in pH in the culture media after nine days. During batch culture, harvesting time constitutes, therefore, a critical parameter to obtain the highest cellular lipid content and achieve optimum productivity.Moreover, as suggested in some previous studies ,44,45, l−1).Finally, maximum volumetric lipid production  profiles due to nitrate limitation were accounted for by relative increases in SFA and MUFA levels, especially 16:0 and 16:1 n-7, concomitant with a decrease in the proportion of PUFA . 16:0 and 16:1 n-7 fatty acids increased from 18.5%–19.7% and 17.9%–24.6% to 26.2%–29.9% and 29.3%–30.3% of TFA, respectively; while EPA and DHA decreased from 16.1%–18.7% and 8.6%–9.6% to 9.4%–11.6% and 4.2%–5.3% of TFA, respectively  suggested that the accumulation of TAG with a different fatty acid composition than that of functional and structural lipids in the oleaginous strains, or a shift in lipid class composition  in non-oleaginous strains, could explain these variations upon nitrogen starvation [cf.P. lutheri. Surprisingly, during batch cultivation of P. lutheri under a relative high light (240 µM photons m−2 s−1), EPA and DHA levels were increasing after nitrate depletion in both total and TAG fatty acid extracts [P. lutheri have been reported to increase with light intensity [P. lutheri after nitrate depletion.It is well established that lipid metabolism and fatty acid profiles are highly dependent on nutrient availability ,17,18. Nectively . These r , was observed in association with a decrease in MUFA, mainly 16:1 n-7 (from 24.6% to 17.9%). While only the proportion of SFA  increased, slight decreases in EPA and DHA levels were observed after nitrate limitation. Indeed, Carvalho and Malcata (2005) previously suggested that increased levels of CO2 as inorganic carbon favored the total lipid content, but decreased the amounts of PUFA in P. lutheri [On the other hand, the observed changes in fatty acid composition  that occ lutheri .−1, respectively; and maximum cellular EPA and DHA contents attained 0.9 ± 0.1 and 0.4 ± 0.1 pg cell−1, respectively. Our values are similar to those developed in order to improve the production and the storage of EPA/DHA during P. lutheri batch cultivation [−2 s−1), growth rate during the exponential growth phase was multiplied by 1.5, biomass at the end of the culture was similarly increased and the maximum cellular EPA and DHA contents reached approximately 0.80 and 0.45 pg cell−1, respectively [P. lutheri cells were cultivated at 20 °C and 100 µM photons m−2 s−1; the relatively high cellular EPA and DHA contents might therefore be attributed to the cellular lipid accumulation triggered by bicarbonate addition described previously and not to light and temperature.Despite the observed decreases in the proportions of EPA and DHA (% of TFA) under nitrate limitation, maximum volumetric and cellular EPA and DHA contents  were obttivation . At the ectively . In our Chlamydomonas reinhardtii, which is able to grow mixotrophically or autotrophically, as a model system, acetate (organic carbon) and bicarbonate (inorganic carbon) have been described as limiting factors and central molecules in lipid droplet synthesis under nutrient limitation [P. lutheri, where TAG accumulation, expressed as cellular TAG content, always occurred close to nitrate depletion and was stimulated by bicarbonate addition, reaching 4.8 ± 0.1 pg cell−1 using 18 mM bicarbonate  associated with bicarbonate was dependent on concentration and time of addition [P. lutheri was also highlighted by a higher overall proportion of TAG obtained with 9 and 18 mM  in comparison to low bicarbonate supply . Previously, neutral lipids and glycolipids have been described as the major constituents in P. lutheri cells grown at 16 °C, under 100 µM photons m−2 s−1, using 2.07 mM bicarbonate and harvested in the mid-exponential growth phase, accounting for ~36% and ~54% of the total lipids, respectively [P. lutheri after nitrate depletion could therefore be explained by a membrane re-cycling process, converting some existing membrane polar lipids into TAG, as suggested by Hu et al. (2008) [Several studies have recently suggested that carbon availability is a key metabolic factor controlling oil biosynthesis and carbon partitioning between starch and oil in different microalgal species ,59,60. Umitation ,59,60. Iarbonate A. In theaddition ,38,40. T. (2008) .P. lutheri , using both 9 and 18 mM bicarbonate (data not shown), as described for maximum cellular lipid content obtained in heri cf., and sevP. lutheri, oil droplets revealed by Nile Red staining were much smaller and less abundant in nitrate-depleted cells in the presence of 9 mM bicarbonate compared to cells grown in the presence of 18 mM bicarbonate  [Monodus subterraneus (Eustigmatophyta), the main changes in TAG fatty acid composition as a result of phosphate starvation consisted of an increase in the proportion of 16:1, combined with a decrease in 18:1 n-9 and EPA [P. incisa has so far been described as the only autotrophic microalga able to incorporate ARA into TAG, reaching up to 47% in the stationary phase and exceeding 60% of TFA under nitrogen starvation [P. lutheri in this study  slightly decreased after nitrate depletion. Changes in TAG fatty acid composition of P. lutheri were also observed with increased bicarbonate concentrations, with a strongest effect detected after nitrate limitation  and DHA (~4%) partitioned to TAG were obtained using 9–18 mM bicarbonate before and after nitrate limitation, respectively.In studies reporting on the capability of some species to partition LC-PUFA into TAG after nutrient limitation , cellula of TFA) . In the  and EPA . P. inciarvation ,14. Regais study , similarmitation . The proi.e., alkaline pH) showed an increase in TAG accumulation but a decrease in both membrane lipid classes with unstable membrane lipid fatty acid profiles similar to the TAG, i.e., less unsaturated [P. lutheri after nitrate depletion. At bicarbonate concentrations of 18 mM, the combined increases in cellular TAG content and n-3 LC-PUFA accumulation in TAG lead to significant increases in TAG cellular EPA and DHA contents, reaching a maximum of 0.36 ± 0.03 and 0.14 ± 0.02 pg cell−1, respectively , and betaine lipids [Furthermore, Guckert and Cooksey (1990) also suggested that cells inhibited by stress  at 20 °C and under continuous illumination (100 µM photons m−2 s−1), provided by lumilux cool daylight fluorescent lamps , using a working volume of 1600 mL. F/2-RSE medium, a modified version of Guillard’s (1975) F/2 medium [−1 ReefSalt complemented with: 5.65 mg L−1 NaH2PO4·2H2O, 100 mg L−1 NaNO3, 4.16 mg L−1 Na2EDTA·2H2O, 3.15 mg L−1 FeCl3·6H2O, 9.8 μg L−1 CuSO4·5H2O, 22 μg L−1 ZnSO4·7H2O, 10 μg L−1 CoCl2·6H2O, 0.18 mg L−1 MnCl2·4H2O, 6.3 μg L−1 Na2Mo4·2H2O, 0.1 mg L−1 Thiamine-HCl, 0.5 μg L−1 Vitamin B12, 0.5 µg L−1 Biotin. It is important to note that ReefSalt contains an initial inorganic carbon concentration of ~30 mg L−1 [−1, respectively) was used to investigate the effects of inorganic carbon supplementation . Bicarbonate powder was added after autoclaving, under the laminar hood to prevent contamination, and then pH adjusted to 8.0 by adding 1 M HCl. Inocula were carried out using daily-diluted cultures with fresh medium in order to maintain the cells in exponentially growing stage before starting the experiment. Cultures were agitated by air-bubbling (~0.03%–0.04% CO2). The initial bicarbonate concentration used is closed to 2.07 mM, the usual concentration of bicarbonate added in artificial seawater [6 cell mL−1, over 17 days and carried out with three independent replications for each treatment. Samples were collected on days 4, 9, 14, and 17 to determine growth parameters and for lipid and fatty acid analysis.An axenic strain of Protozoa  at the S2 medium  where fi0 mg L−1 . The medseawater . Each exP. lutheri cells were gently harvested by centrifuging (1200 × g for 10 min) using a Hettich Rotina 38R centrifuge . The pellets obtained were then frozen, and stored at −20 °C prior to analysis.Due to salt precipitation occurring during growth using high initial sodium bicarbonate concentrations , the biomass was not estimated by dry weight. Culture growth and all parameters were estimated on the basis of cell density determined using a Neubauer hemocytometer after immobilizing the cells with Lugol 5% and appropriate dilution. Medium pH was measured using a bench CyberScan pH 510 Meter . et al. (1999) [−1) or medium samples collected previously were diluted 10-fold with distilled water and residual nitrate content was directly determined according to optical density measured at 220 nm using a Cary 50 Scan UV-Visible spectrophotometer .Nitrate concentration was determined according to the modified method reported by Collos . (1999) . StandarP. lutheri cultures were stained with 2 µL of Nile Red (0.5 mg mL−1 in dimethylsulfoxide), incubated at room temperature for 5 min, and immediately observed by fluorescence microscopy .The Nile Red staining method was used to visualize the intracellular lipid bodies as an indicator of TAG formation . AliquotP. lutheri cells were extracted using ultrasounds  in a chloroform/methanol/water  system according to Bligh and Dyer (1959) [et al. (2010) [2SO4 in methanol, and heated until spots appeared.All chemicals used in the experiment were of analytical grade, and were purchased from Fischer Scientific . Total lipids of r (1959) . After p. (2010) . Fish oi2SO4 and heating at 80 °C for 1.5 h with continuous stirring under a nitrogen atmosphere [−1, and then from 200 to 250 °C at a rate of 2 °C min−1. Agilent MSD Productivity ChemStation Software  was used for instrument control, data acquisition and data analysis . Identification of FAME was obtained by co-chromatography with authentic commercially available FAME standards  and FAME of fish oil . Total fatty acid, EPA and DHA contents were quantified by comparison with a known amount of added pentadecanoic acid 15:0  as internal standard.Fatty acid methyl esters (FAME) from total lipids and TAG were obtained by transmethylation of the freeze-dried cells or TAG extracts with dry methanol containing 2% (v/v) Hmosphere . Gas chrn-3 LC-PUFA accumulation induced by nitrate depletion rely mainly on inorganic carbon availability in P. lutheri. Although growth and nitrate uptake present an optimum and threshold tolerance to bicarbonate addition above which both parameters decreased, carbon supply seems to be one of the limiting factor involved in n-3 LC-PUFA-enriched oil production in P. lutheri, besides other primordial factors, such as medium pH, light and temperature. Indeed, lipid and TAG accumulation enhanced by bicarbonate addition at the time of nitrate depletion in P. lutheri was correlated with pH increase during the growth of photosynthetic microalgae. During batch cultivation, harvesting time constitutes also a critical parameter to achieve the best productivity. As a consequence, although an industrial process would require a light/dark cycle and our results are based on cultures exposed to continuous light, which overrides some of the cell cycle control mechanisms, this study contributes to optimized culture strategies applied to develop n-3 LC-PUFA-enriched oil production systems using autotrophic microalgae. Our findings also constitute an important step towards an improved understanding of the mechanisms involved in lipid metabolism regulation, and more specifically oil accumulation and LC-PUFA partitioning in microalgae.The capacity of some algal species to accumulate TAG containing LC-PUFA, particularly during environmental changes, depends on the regulation of various metabolic pathways. In this study, we demonstrated for the first time, to our knowledge, that cellular lipid content, oil body formation and TAG containing


Incubation of HUVEC with H2O2 led to a significant decrease in cell viability and an increase in the percentage of apoptotic cells. The tested drugs, at concentrations of 1–100 μM, significantly inhibited the H2O2-induced toxicity. Also, all donepezil, rivastigmine and galantamine significantly increased the number of vessels in the chorioallantoic membrane when injected into fertilized eggs. In conclusion, AChE inhibitors possess angiogenesis-accelerating properties and have antiapoptotic effects on endothelial cells. These effects of AChE inhibitors may be involved in their beneficial effects on AD.Alzheimer's disease (AD) is one of the most common causes of dementia in the elderly. Recently, a great attention has been paid to the possible role of vascular changes in the pathogenesis of AD. Reduced microvascular density and degeneration of the endothelium are of structural cerebrovascular changes in AD. Acetylcholinesterase (AChE) inhibitors are widely used for the improvement of AD symptoms. Until now, however, the effects of AChE inhibitors on vascular changes including angiogenesis and endothelial cell apoptosis are not fully understood. In the present work, the effects of three AChE inhibitors  were tested on H Alzheimer's disease (AD), a progressive neurodegenerative disorder, is the primary common cause of dementia in the elderly. Intracellular neurofibrillary tangles, amyloid plaques, neuronal loss, and vascular amyloidosis are of characteristic hallmarks of AD . In spitRecently, a great attention has been paid to the possible role of vascular changes in the pathogenesis of AD , 2, 4, 52O2-induced apoptosis, an in vitro model which simulates cell damage in AD [Acetylcholinesterase (AChE) inhibitors, antiglutamic agents, secretase inhibitors, and anti-inflammatory drugs are of available therapeutics for management of AD , 9. Cholge in AD , 20. Als2O2, penicillin/streptomycin solution, 3--2,5-diphenyl-2H-tetrazolium bromide (MTT), and propidium iodide were purchased from Sigma (USA). Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were bought from GIBCO (USA). Fertilized eggs were kindly provided by Morghdaran Toos Co. . Rivastigmine tartrate and galantamine HBr  were donated by Tehran Darou Co. (Iran). Donepezil HCl  was kindly provided by Razak pharmaceutical Co. (Iran).The HUVECs were obtained from Pasteur Institute . Dimethyl sulfoxide (DMSO), Hμg/mL). All cells maintained in humidified atmosphere of 5% CO2 and 95% air at 37°C. Trypsin/EDTA solution was used to passage cells whenever they were grown to confluence. The cells at subconfluent stage were harvested from culture flask and seeded overnight in 96-well culture plate (15000 cells/well) after checking the viability with trypan blue exclusion technique. Then, to determine the effect of test drugs on cell viability, the medium was changed by a fresh one containing donepezil , rivastigmine  and galantamine  in the presence or absence of H2O2 (1 mM), and the cells were incubated for 24 h.The HUVECs were cultivated in high-glucose DMEM supplemented with FBS (10%), penicillin (100 units/mL), and streptomycin  at 4°C overnight in the dark [The cells were seeded overnight in 12-well culture plate (75000 cells/well) and treated for 24 h with tested drugs. Then floating and adherent cells were harvested and incubated with 750 the dark . Then, tFertilized eggs were incubated at 37°C and 70% relative humidity in a forced draught incubator. At day 8, a window opening is punctured on each egg and 0.01–10 nmol/egg of donepezil, rivastigmine, or galantamine was injected into the chorioallantoic sac. The control eggs received sterile PBS only. Then, the injection holes were closed by wax, and the eggs were maintained in the incubator. At day 12, the eggs were opened and the chorioallantoic membrane vasculatures were photographed using a stereo microscope equipped with a digital camera (Canon EOS 40D with Canon EF 100 mm f/2.8 USM macrolens). The angiogenic response was evaluated by counting the vessel density using Photoshop CS2 and image measurement software.P values were less than 0.05. All experiments were performed three times in duplicate. The results were presented as the mean ± SD.Statistical comparison was made by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Differences were considered significant when 2O2 significantly decreased cell viability from 100 ± 3% (control) to 64 ± 5% (P < 0.001). Incubation with donepezil significantly attenuated the H2O2-induced cell death (μM of donepezil was 94 ± 17% (P < 0.05 versus untreated cells) and 99 ± 5% (P < 0.01), respectively. Also, the presence of 1, 10, and 100 μM of galantamine in the cell medium increased the cell viability from 64 ± 5% (untreated cells) to 112 ± 19% (P < 0.05), 199 ± 19% (P < 0.001), and 144 ± 18% (P < 0.001), respectively, , 10 μM , or 100 μM  of rivastigmine was significantly higher than that of untreated cells  of cells were apoptotic (P < 0.001) reduced by 1 μM (20.8 ± 2%), 10 μM (21.4 ± 4.8%), and 100 μM (19.6 ± 0.1%) of donepezil  decreased in the cells treated with 1 μM (30 ± 3%), 10 μM (25 ± 2.8%), and 100 μM (26 ± 1.1%) of galantamine  and 153 ± 4% (P < 0.05), respectively, (P < 0.05) and rivastigmine at 0.1 nmol/egg , 1 nmol/egg , and 10 nmol/egg  significantly increased angiogenesis  of donepezil, rivastigmine, and galantamine [AChE inhibitors are of the most currently used therapeutics for symptomatic improvement in AD . The bendoses 10– mg/day oc and subsequent activation of caspase-3, the extrinsic pathway begins with a cell death receptor and associated stimulation of caspase-8 which in turn can activate caspase-3. Activated caspase-3 targets substrates that promote DNA fragmentation and cell death. H2O2 can activate both intrinsic and extrinsic apoptosis pathways in endothelial cells, leading to activation of caspases-3 and subsequent apoptosis [2O2 disturbs balance between the production of reactive oxygen species and cellular antioxidant defenses  and thereby damages macromolecules such as lipids, proteins, and nucleic acids, which trigger apoptotic pathways [It is well documented that structural cerebrovascular changes  are contributed to the pathogenesis of AD , 7. Recepoptosis . Excess pathways , 29. 2O2 and lipid peroxidation have been shown to mediate amyloid beta neurotoxicity [2O2-induced apoptosis of endothelial cell may be involved in their beneficial effects on AD.Increased levels of Htoxicity . Also, otoxicity . It has toxicity . Therefoβ efflux from brain and enhances angiogenesis. Deposition of amyloid beta accelerates the process of blood vessel branching. Over time, therefore, highly branched vessel networks appear in AD brain. While early angiogenesis restores blood flow and reduces amyloid beta, the resulting hypervascularization impairs perfusion efficiency [in vivo situation [Our data also showed that donepezil, rivastigmine, and galantamine are able to increase angiogenesis. At present, however, the role of angiogenesis in pathogenesis of AD is not yet fully understood. Previous studies have shown that cognitive impairments in aging individuals are frequently associated with vascular diseases . Also, cficiency , 32, 33.ficiency . With thficiency . In the ituation . AlthougTaken together, in the present study, we demonstrated that donepezil, rivastigmine, and galantamine promoted angiogenesis and also protected endothelial cells against oxidative stress-induced cytotoxicity. Therefore, these effects of AChE inhibitors may be involved in their beneficial effects on AD.


Lactobacillus paracasei N1115 is a new strain with probiotic properties isolated from traditional homemade dairy products in Inner Mongolia, China. Here, we report the complete genome sequence of L. paracasei N1115, which shows high similarity to the well-studied probiotic Lactobacillus rhamnosus GG, and 3 structures turned out to be inversions, according to the colinearity analysis of the BLAST alignment. Lactobacillus paracasei N1115 has been isolated from traditional dairy products in Inner Mongolia, China. A patent has been applied for this strain to be a probiotic since it exhibits high-level resistance to acid and bile stresses and it stimulates macrophages to produce interleukin 10 (IL-10), IL-6, and tumor necrosis factor alpha (TNF-α) .denovo (http://soap.genomics.org.cn/soapdenovo.html)  tool, 96.2% of the reads were mapped to assembly sequences. Most of the gaps within the scaffolds were ﬁlled by local assembly and overlap extension with paired-end reads. We also filled the gaps between scaffolds by sequencing PCR products using an ABI 3730 capillary sequencer.High-molecular-weight genomic DNA was used to construct three genomic libraries, with 0.5-kb (914 Mb), 2-kb (250 Mb), and 6-kb (250 Mb) insert sizes, respectively. Using the Illumina HiSeq 2000 system, 1.4 Gbp clean data were given, which provided 420-fold genome coverage. Using SOAPLactobacillus rhamnosus ATCC 53103 (3.010 Mb) and Lactobacillus casei ATCC 334 (2.925 Mb). The complete genome of N1115 contains 3,063 predicted gene sequences with 2,584,095 bp  and four plasmids . The overall G+C content is 46.45%, whereas the four plasmids have G+C contents of 43.26% on average. This genome size is slightly larger than the complete genomes of ,095 bp 26, coveri.59% G+C 55 bp). TA comparative genome analysis was made between the assembly sequence and reference sequence, and 3 structural variations were found. Based on the colinearity analysis of the BLAST alignment, 3 structural variations turned out to be inversions (an inversion is a chromosome rearrangement in which a segment of a chromosome is reversed end to end). All 3 inversions were verified by PCR amplification.L. casei, and no known pathogenic genes were identiﬁed. A comparative genomic analysis was performed with the published genomes of L. casei ATCC 334 and L. rhamnosus GG. In conclusion, the genome sequence provides new avenues to further explore the gene-based functional and probiotic mechanisms of N1115. In addition, comparative and functional genomics analyses might also be carried out to trace the origin and evolution of this bacterium.A significant comparative genome analysis showed that N1115 carries most of the core genes of L. paracasei N1115 chromosome and plasmid pN1, pN2, pN3, and pN4 sequences have been deposited in GenBank under accession no. CP007122, CP007123, CP007124, CP007125, and CP007126, respectively.The


The G-protein coupled receptor 55 (GPR55) is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance) in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior. An alternative is to examine mice lacking GPR55. Studies with GPR55 loss-of-function mutant mice or cultured cells suggested a role for GPR55 in pain To reveal potential physiological roles of GPR55 in CNS function, we first examined the abundance of GPR55 in specific brain regions and then examined anatomical structures from GPR55 knockout brains. Next, a panel of electrophysiological and behavioral assays was employed to examine synaptic function and plasticity, as well as behaviors using GPR55 null mutant mice and their littermates. These studies suggest that GPR55 plays a role in regulating motor coordination and pain sensations.ad libitum. The animals used for testing were backcrossed onto the C57BL/6 strain for 8 generations. For all experiments, heterozygous females and heterozygous males were mated to generate GPR55 KO and wild-type (WT) littermate controls. All genotypes were generated in numbers close to the expected Mendelian ratios. After weaning, two to four mice were housed together. Animal procedures have been approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine (protocol # AN-3327) and Indiana University (protocol # 10-004) and were conducted in compliance with the U.S. Department of Health and Human Services (assurance #: 3823-01 for BCM and 4094-01 for IU).GPR55 knockout (KO) mice were acquired from TIGM (Texas A&M Institute for Genomic Medicine)  GCG CAT CGC CTT CTA TC –3′ and 5′ – TCA AGC TAC GTT TTG GGT T –3′ (expected PCR product size is 301 bp) were used. For detection of WT allele, the following pair 5′ – GCC ATC CAG TAC CCG ATC C –3′ and 5′ – GTC CAA GAT AAA GCG GTT CC –3′ (expected PCR product size is 441 bp) were used. PCR cycle conditions were: 5 min at 95°C, 36 cycles of three steps , then 5 min at 72°C using standard PCR reagents.Animals were genotyped as previously described Mice used for quantitative real-time RT-PCR (qPCR) were sacrificed at five months of age . Anesthetized mice were decapitated, tissue harvested and immediately placed into RNAlater  and stored as per manufacturer’s instructions. Prefrontal cortex was taken to be the anterior 3 mm of cortex, and “cortex,” refers to the remaining cortex, minus the hippocampus. Mice at 2 months of age were processed for histology and immunohistochemistry. Mice were deeply anesthetized with an intraperitoneal injection (3 ml/kg) of a rodent anesthetic cocktail containing ketamine 37.6 mg/ml, xylazine 1.92 mg/ml and acepromazine 0.38 mg/ml. Following establishment of anesthesia, mice were transcardially perfused with PBS, pH 7.4, followed by 4% PFA in PBS, pH 7.4. The brains were then post-fixed with the same fixative overnight at 4°C, washed with PBS and stored in 0.1% sodium azide/PBS at 4°C until processing.http://www.ncbi.nlm.nih.gov/tools/primer-blast) and the corresponding mouse gene. The following mixtures of two primers was used for detection of cDNAs: for ABHD6, 5′–CCT TGA TCC CAT CCA CCC CGG A–3′ and 5′–CCC GGA CAC ATC AAG CAC CTG G–3′; for DGLα, 5′– CAC AGA GGC ACC TGG TTG GGC–3′ and 5′– TCC GCC ATT TGG GCT TGG TGC–3′; for DGLβ, 5′–GGC GAC TGT TGC AGA GCC AGA G–3′ and 5′–GCA TGA TGG CTA ACA GGG CCG C–3′; for FAAH, 5′–GCC TCT GTT TCC TCG GCT GGC–3′ and 5′–ACC CCC GCA GGG AAG TCC AG–3′; for MGL, 5′–TCT TCC TCC TGG GCC ACT–3′ and 5′–AAA GTA GGT TGG CCT CTC TGC–3′.Primers for selected components of the endocannabinoid system were designed using Primer-Blast  and genomic DNA was removed with DNase  following the manufacturer’s instructions. RT-PCR was performed using a one-step, Sybr Green amplification process .Quantitative PCR was performed using a Stratagene Mx3000P thermocycler. Primers for glutaraldehyde-3-phosphate dehydrogenase (GADPH) or β-actin were used as internal controls for each brain region with the threshold cycle set within the linear range (10 fold above baseline). Once the standard critical threshold (Ct) was set, the relative expression levels for genes for each brain region were determined. Data analysis and statistics were performed using Excel  and Prism  software.2O2 in PBST, washed extensively with PBST and blocked for one hour with 3% normal goat serum in PBST at room temperature. Sections were then incubated with primary antibodies diluted in PBST/1% normal goat serum/2% BSA at 4°C overnight. The next day, sections were washed with PBST, and incubated with biotin-conjugated goat anti-rabbit antibodies  in PBST at room temperature for two hours. Following this incubation, sections were washed with PBST three times for 10 minutes each, then incubated with avidin/biotin complex  for two hours at room temperature. Sections were then washed, and immuno-signals detected with a DAB kit (Vector Labs). Color development was observed under a dissecting microscope. Sections were washed with PBS, mounted, and air-dried overnight before counterstaining with Nissl. Immunofluorescence staining was similar to the procedure described above, with the following modifications: the quenching step with hydrogen peroxide was omitted, and Alexa 488-conjugated goat anti-rabbit antibodies and Cy3-conjugated goat anti-mouse antibodies  were used. Sections were counterstained with DAPI and mounted with Vectashield (Vector Labs). The following primary antibodies were used: rabbit anti-tyrosine hydroxylase , rabbit anti-vesicular glutamate transporter 2  and mouse anti-NeuN . Bright field images were taken with an Olympus BX51 upright microscope under consistent light conditions using an Olympus DP70 CCD camera with Olympus DPC controller software with 2x/0.08 Plan Apo and 4x/0.16 U Plan Apo objectives . Fluorescent images were taken with a Zeiss AxioImager M1 system with 5x/0.16 Zeiss objectives, using AxioVision software. All images were processed in Adobe Photoshop CS2 for brightness/contrast, orientations, and background corrections to better illustrate the staining patterns.Brains were serially sectioned in the coronal or sagittal plane into 100 µm thick sections using a Leica VT-1000 vibrating microtome . One set of sections was mounted on slides, air-dried overnight, and subjected to Nissl staining. For immunohistochemical staining, free-floating sections were washed with PBS/0.01% Triton X-100 (PBST) and permeabilized with 0.2% Triton X-100 in PBS at room temperature for 20 minutes. Free-floating sections were then incubated with 30% methanol and 3% H4 was purchased from Tocris Bioscience . N-arachidonoyl ethanolamide , 2-arachidonoyl glycerol (2-AG), 2-linoleoyl glycerol (2-LG), N-oleoyl ethanolamide (OEA), N-palmitoyl ethanolamide (PEA), N-linoleoyl ethanolamide (LEA), N-arachidonoyl glycine (NAGly), N-palmitoyl glycine , N-arachidonoyl serine (AraSer), N-oleoyl serine (OlSer), Prostaglandin E2 (PGE2), and Prostaglandin F2α (PGF2α) were purchased from Cayman Chemical . HPLC-grade water and methanol were purchased from VWR International . HPLC-grade acetic acid and ammonium acetate were purchased from Sigma-Aldrich .Anadamide-dLipid extracts of tissues were performed as previously described 1 cannabinoid receptor-sensitive depolarization-induced suppression of excitation (DSE) Hippocampal neurons isolated from the CA1–CA3 region of WT or GPR55 knockout P1–P2 mouse pups were cultured for 8–10 days on microislands and recordings performed as previously described 2, 7 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 7 D-glucose saturated with 95% O2 and 5% CO2. 400 µm thick transverse hippocampal slices were prepared with a vibrating microtome Series 1000  and recovered at 31±0.5°C for an hour in an interface chamber. Field excitatory postsynaptic potential (fEPSP) were recorded at 31±0.5°C in an interface chamber perfused at 1 ml/min with artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, and 14.83 D-glucose. Extracellular stimuli were administered along the Schaffer collaterals using Formvar-insulated, bipolar nichrome electrodes controlled by a stimulus isolator . An ACSF-filled glass-recording electrode was placed in stratum radiatum to record the field potential changes. Electrophysiological traces were amplified with AC-coupled amplifier , digitized using a Digidata 1320A , and acquired with pClamp 10 software (Molecular Devices).All data acquisition and analysis was carried out blinded to genotype. For field potential recordings, 3 month-old mice were anesthetized with isoflurane and their brains were removed and immersed immediately in ice-cold cutting solution (in mM): 220 sucrose, 2.5 KCl, 0.5 CaClTo assess baseline synaptic transmission, input-output relationships were examined by measuring the rising slope of the fEPSP evoked by 100 µs pulses over various stimulus intensities (1 V to 10 V). The stimulation intensity that evoked a fEPSP whose slope was 30–40% of the maximum fEPSP slope, determined by the input-output recording experiment, was used for the following recording paradigms. A paired-pulse recording paradigm was conducted with paired stimuli separated with inter-stimulus intervals of 10, 20, 50, 100, or 200 ms. Stimulus pairs were delivered at 0.05 Hz and five trials were averaged at each inter-stimulus interval given in a random order. The paired-pulse ratio was calculated by dividing the slope of the second fEPSP by the slope of the first fEPSP. Long-term potentiation (LTP) was induced by either a theta burst stimulation (TBS) consisting of 10 trains of five stimuli at 100 Hz, repeated 5 times with an inter-train interval of 5 sec, or a high-frequency stimulation (HFS) protocol, which consisted of 2 trains of 100 pulses at 100 Hz that were 20 sec apart 3, 25 D-glucose, 11.6 sodium ascorbate, 7 MgSO4, 3.1 sodium pyruvate, 2.5 KCl, 1.25 NaH2PO4, and 0.5 CaCl2. Brains were sectioned in a coronal plane at 350 µm thicknesses using a Leica VT-1000 vibrating microtome. During recording, slices were maintained in recording chamber with 32–33°C oxygenated ACSF perfused at 2 ml/min. Voltage-clamped spontaneous EPSCs (sEPSCs) were recorded from CA1 pyramidal neurons using a Multiclamp 700B amplifier (Molecular Devices) at 10 kHz sampling rate with 4 kHz Bessel filter using pClamp 10. High-seal (GΩ) and low-series (<30 MΩ) resistances were monitored throughout the experiment to ensure high-quality recordings.For whole-cell patch-clamp recordings, acute hippocampal slices were prepared from 6–7 week old male mice. Brains were removed and immersed immediately in oxygenated ice-cold cutting solution (in mM): 110 choline-chloride, 25 NaHCOSpontaneous EPSCs were recorded at −60 mV. Recording pipettes (4–7 MΩ tip resistance) made from borosilicate glass were filled with intracellular solution . Five-minute recording traces were analyzed for their frequency and amplitude distributions using MiniAnalysis . The root mean square (RMS) of the background noise was computed for each set of data. The detection threshold for an event was set to three times the RMS value.Current-clamped whole-cell recordings were also conducted to examine excitability and membrane properties. A series of current injection steps (range: −100 pA to +400 pA with 50 pA per step) were given to evoke action potentials (APs). Current pulse durations were 600 ms for AP train recording. All summary data are presented as means ± SEM. Student’s t-test was used to determine statistical significance. Prism 3.0 (Graph-Pad Software) was used for all statistical analyses.Male and female mice at 2–5 months of age were subjected to selected tests from the test battery originally described by Crawley and Paylor The elevated plus-maze was made of four perpendicular runways (7×25 cm) elevated 40 cm off the ground. Two arms were enclosed by 15 cm white walls and two arms were open, except for a small 5 mm rim. The test animals were placed in the center of the elevated maze facing one of the two open arms, and left to explore for 10-minutes. The number of entries, distance traveled and time spent in the open and closed arms were recorded using the ANY-maze tracking system . The number of rearings and groomings in the open and closed arms, and the number of head dips in the open arms were also scored by the experimenter. Data were analyzed using two-way (gender×genotype) ANOVA.Each mouse was placed into the center of a clear Plexiglas chamber (40 cm×40 cm×30 cm) with photo beams to record horizontal and vertical movements of the mouse. Activity was recorded over a 30-min period using a computer-operated VersaMax Animal Activity Monitor System . Testing was performed in the presence of overhead bright lights (∼750 lux of illumination) and white noise (55 dB). Data were collected in 2 min intervals and the following measures were analyzed: total distance traveled (cm), vertical activity, time spent in center zone and the center to total distance ratio. These ratios were calculated by dividing the center distance (distance traveled in the arena center: 22.5 cm×22.5 cm) by total distance traveled. Total distance traveled data in 10 min intervals were analyzed using a three-way (gender×genotype×time) ANOVA with repeated measures. All other data for the 30 min period were analyzed using two-way (gender×genotype) ANOVA.Prepulse inhibition of the acoustic startle response was measured as described in Paylor and Crawley Acoustic startle response amplitude data were analyzed using two-way (gender×genotype) ANOVA. PPI data were analyzed using a three-way (gender×genotype×prepulse sound level) ANOVA with repeated measures. One mouse did not meet our criterion for minimum startle response to the 120 dB sound stimulus (100) and therefore it was excluded in the analysis.Motor coordination and skill learning were tested using an accelerating rotarod . Mice were placed on a rotating drum (3 cm in diameter), which accelerated from 4 to 40 rpm over a 5 min period. The time spent walking on top of the rod until the mouse either fell off the rod, or slipped and held onto the rod to ride completely around was recorded. Mice were given four trials on 2 consecutive days with a maximum time of 300 s (5 min) per trial and a 60 min inter-trial rest interval. Rotarod data were analyzed using a three-way (gender×genotype×day) ANOVA with repeated measures.Each mouse was placed onto a wire grid, and the screen was inverted so that the mouse was hanging upside down from the grid several inches above a plastic covered foam pad. The latency to fall with a cutoff time of 60 sec was measured. Data were analyzed using two-way (gender×genotype) ANOVA.For the wire-hang test the mouse was held by the tail and allowed to grasp with its forepaws in the middle of a single 3-mm plastic coated wire suspended 15 inches above a plastic-covered foam pad and released. For the dowel test, the mouse was placed length-wise onto a plastic dowel suspended several inches above a plastic-covered foam pad. Two dowel sizes were used in succession: 3/8-in and ¼-in. Latency to fall was measured with a 60-sec cutoff time for wire-hang, and a 120-sec cutoff time for dowel test. Data were analyzed using two-way (gender×genotype) ANOVA.For this test, each mouse was held by the tail and allowed to grasp the bar of the Chatillon-Ametek grip strength meter with both its forepaws and then the mouse was pulled away from the bar until it released the bar. The test was repeated 5 times for each mouse, and the maximum force generated for each pull was recorded. The highest and lowest scores from individual mouse were removed and the grip strength values were acquired by averaging the scores from three pulls. Data were analyzed with student’s t-test.The test apparatus was enclosed in a clear Plexiglas chamber (20 cm×20 cm×28 cm); 1.6 mm diameter rods were spaced 6 mm apart and elevated 1 cm above a metal plate. In this assay, each mouse was required to walk and balance on thin (1.6 mm) parallel rods spaced 6 mm apart. Ataxia and locomotor activity were recorded simultaneously for 10 min. Footslips were detected when a paw touched a metal plate below the parallel rod floor, completing a circuit that was scored by the system . Locomotor activity was measured by the ANY-maze tracking system. The number of errors (footslips) per distance traveled (cm) was calculated and analyzed using two-way (gender×genotype) ANOVA.t-test.Each male mouse was placed for 6 min in a glass beaker  filled with water . Water was changed between mice. The duration of immobility during the last four minutes of a six minute trial was recorded. Minimal movements made to balance the body and keep the head above the water were scored as immobility. Data were analyzed using Student’s Freezing behavior in a conditioned fear paradigm was measured as described previously minus percent freezing in the pre-CS phase. Data were analyzed using two-way (gender×genotype) ANOVA for the total duration.Mice were tested for contextual and cued fear conditioning 24 h after conditioning. For the context test, mice were placed back into the original test chamber for 5 min and freezing behavior was recorded. One to two hours later, mice were tested for responses to the auditory CS in a new environment. For the CS test, white Plexiglas inserts were placed on the sides and floor of the chamber to alter the shape, texture and color of the chamber. Vanilla extract was placed in the chamber behind the insert to alter the odor. Transfer cages were altered (no bedding) and red house lights replaced the normal white house lights. Mice were placed into this new chamber and freezing was recorded for 3 min during this ‘pre-CS’ phase. The auditory CS was then presented for another 3 min and freezing was recorded. Data for the CS test were calculated as the percent freezing during the CS Mice were tested for analgesia-related responses using a hot-plate apparatus . The hot-plate was enclosed in a clear Plexiglas chamber and preheated to 55±0.1°C. Each mouse was placed gently onto the hot-plate and the time to initiate a hind limb response was recorded. The same mice were tested on hot-plate preheated to 50±0.1°C on a separate day. Typical nociceptive responses are licking or shaking the hindpaw, or jumping. Mice were immediately removed after showing a response, with a cut-off time of 45 sec. Data were analyzed using two-way (gender×genotype) ANOVA.P≤0.05.Statistical analysis was conducted using SPSS , Prism 3.0 (Graph-Pad Software) or SigmaPlot . All data were analyzed using two-way ANOVA (gender×genotype), three-way ANOVA with repeated measures (gender×genotype×time), or student’s t-test (genotype) if only male mice were tested. All data are presented as mean ± SEM. The level of significance was set at In human brain, Northern blotting detected GPR55 mRNA in the caudate and putamen, but not in the frontal cortex, hippocampus, thalamus, pons, or cerebellum Anandamide  and 2-arachidonoyl glycerol (2-AG) are two major endogenous ligands of cannabinoid receptors 1, 59 = 0.105, p = 0.747, gender: F1, 59 = 144.210, p<0.001; GPR55 KO mice are fertile and show no obvious alteration in physical characteristics (appearance of fur and whiskers). There were no significant differences in body weight between male or female GPR55 KO and their WT littermates (genotype: FDepolarization-induced suppression of excitation (DSE) is a well-characterized form of endocannabinoid (eCB)-mediated synaptic plasticity. DSE sensitivity and strength can be quantified by constructing a “dose-response” curve where the suppression of excitatory postsynaptic potentials to progressively longer depolarizations is determined. To examine whether GPR55 deletion affects eCB-dependent DSE, autaptic culture neurons were prepared from GPR55 KO and control pups. The DSE dose response curves were very similar in neurons cultured from GPR55 KO or WT mice Fig. 2A.GPR55 activation inhibits the potassium M-current in HEK293 cells expressing GPR55 ; WT, 28 recordings from six animals; KO, 31 recordings from seven animals). Similar input–output relationships were found when comparing fEPSP slope with fiber volley amplitude between GPR55 knockout mice and control mice . These data indicate that synaptic transmission is grossly normal in the GPR55 KO Schaffer collateral pathway.To further explore the potential contribution of GPR55 in regulating synaptic function and plasticity, field potential recordings were conducted in the stratum radiatum of CA1 with stimulation of the Schaffer collateral pathway in acute hippocampal slices prepared from GPR55 KO and their WT littermate mice at 3 months of age. We first analyzed general synaptic transmission by examining the input-output relationship of synaptic responses in the CA1 area over a range of stimulus intensities ). To examine whether long-term synaptic plasticity was defective in GPR55 KO mice, both theta-burst stimulation  and high frequency stimulation protocols  were used to induce the formation of long-term potentiation (LTP) at Schaffer collateral synapses. Strong and persistent LTP was induced with either TBS or HFS in GPR55 KO CA1 . Taken together, deleting GPR55 function has no impact on the types of short-term and long-term plasticity examined.Next, short-term plasticity was evaluated with paired stimuli separated by a 10, 20, 50, 100, or 200 msec inter-stimulus intervals Fig. 3B.To address the behavioral effects of GPR55 deletion, we subjected 2–5 month old male and female GPR55 KO mice and their WT littermates to a comprehensive battery of behavioral tests.1, 35 = 0.772, p = 0.386, gender: F1, 35 = 0.092, p = 0.764). However, there was a significant genotype effect but no gender effect on time to hindlimb response at higher temperature   but no significant interaction effects at 55°C . To compare to the previous report, the data were analyzed separately for male and female mice. Similar to previous report, it was the female GPR55 KO mice that showed a significant reduction in withdrawal latency at 55°C when compared to WT littermates . Thus, loss of GPR55 perturbs thermal nociception.Staton et al. = 0.607) Table 2.1, 54 = 0.391, p = 0.535; gender F1, 54 = 0.973, p = 0.329) or in the number of entries to open arms . No significant gender×genotype interaction effect was observed in either parameter. In addition, no overall differences between the genotypes and genders were found in other measures of anxiogenic- or anxiolytic-like behaviors monitored, such as number of head-dips in the open arms (exploratory behavior), number of rearings, and number of groomings . There was no overall difference in the total number of entries between GPR55 KO and WT mice either, another indication that the absence of GPR55 activity has no impact on spontaneous activity in the elevated plus maze .To explore the possible role of GPR55 in regulating anxiety levels in mice, we performed both elevated plus maze and open field tests. The elevated plus maze assay is a well-validated behavioral test to measure anxiety in rodents roomings Table 2.1, 59 = 2.645, p = 0.109). GPR55 KO mice showed a trend towards decreased horizontal activity throughout the 30 min time period, but this was not statistically significant . Both GPR55 KO mice and WT littermates decreased their exploratory activities as testing progressed , indicating habituation to the novel environment. There was no genotype×interval interaction effects . For vertical activity, there was a significant gender difference but the activity levels were comparable between GPR55 KO and littermate control mice in both male or female mice . There was no significant gender×genotype interaction effect .The open field paradigm allows for simultaneous assessment of novelty-induced exploratory activity and anxiety levels 1,59 = 0.61, p = 0.438, gender: F1,59 = 0.401, p = 0.529) and time spent in center zone . Integrating the results of these studies, GPR55 KO mice showed essentially normal baseline anxiety levels and slightly reduced reactivity to a novel environment, but habituated normally.The ratio of center distance to total distance traveled  and time spent in the center zone measured in the open field assay provide measures of anxiety-related responses to a bright and open arena 21 = 0.459, p = 0.651) in immobility time between GPR55 KO  and WT . These data suggest that GPR55 KO mice do not exhibit overt depressive behaviors.Cannabinoids are also known to influence depressive behaviors 1, 59 = 9.292, p = 0.003) and genders , but no genotype×gender interaction effects , suggesting that GPR55 KO mice performed significantly worse than littermate controls on the rotarod. There was an overall significant time effect , but no significant gender×time , genotype×time , or gender×genotype×time  interaction effects. This suggested that both male and female GPR55 KO and littermate control mice improved significantly during training, indicating that GPR55 KO mice were able to learn as the time they stayed on a rotating rod significantly increased over two consecutive days of testing. In summary, GPR55 KO mice exhibited significant motor coordination deficits but their motor performance improved with repeated training.GPR55 is expressed in the basal ganglia and cerebellum Fig. 1, 1, 49 = 0.927, p = 0.59, gender: F1, 49 = 2.162, p = 0.152). In the grip strength test, each mouse was allowed to grasp onto a bar and the maximum force generated while pulling the mouse away from the bar was measured. There was no significant difference between GPR55 KO mice and littermate controls . The results of these assays suggest that GPR55 KO mice have normal grip strength. We also tested the mice on two different dowel assays to assess their ability to remain on a narrow beam. Two beam sizes (3/8 and ¼ inch rods) were used. No significant differences were found between GPR55 KO and WT littermates . There was no genotype×gender effect on the number of footslips per cm traveled . For clarity, male and female data were combined and shown in 31 = −2.75, p = 0.01). Taken together, these results show that GPR55 KO mice exhibit an ataxia-like phenotype due to motor coordination deficits.Next, we challenged GPR55 KO mice with the parallel rod footslip assay to further examine their motor coordination. This test allows the simultaneous measurement of ataxia and locomotor activity 1, 59 = 12.942, p = 0.001, gender: F1, 59 = 8.706, p = 0.005). There was no genotype×gender interaction effect . For clarity, male and female data were combined and shown in 60 = 3.0, p = 0.004). For PPI, there was a main effect of prepulse level  as expected. Thus, as the prepulse level increases, there is greater suppression of the startle response. We found no significant difference in the percentages of PPI between GPR55 KO mice and WT littermates  or genotype×gender×prepulse level interaction effects . In summary, GPR55 KO mice exhibit normal sensory-motor integration.The prepulse inhibition of acoustic startle reflex (PPI) is the phenomenon in which a weak non-startling sound suppresses the startle response to a strong acoustic startle stimulus presented immediately after the pre-stimulus. As the prepulse level increases, there will be greater suppression of the startle response. PPI provides an operational measure of sensory-motor gating processes in humans and mice . Similar levels of freezing behavior for auditory cues were found between GPR55 KO and littermate control mice . Thus, GPR55 doesn’t seem to be critical for the learning and memory of cue- or context-dependent fear-conditioning.To explore the role of GPR55 in learning, we employed the conditioned fear paradigm associating contextual or auditory cues with mild foot shock. It is believed that contextual and auditory-cue conditioned fear are mediated by hippocampal and amygdala-dependent processes, respectively GPR55 has been implicated in diverse biological processes including analgesia Motor coordination is controlled by various brain regions, including the motor cortex, basal ganglia, and cerebellum The regulation of Purkinje cell activity is important for motor behavior and motor learning Muscle strength, motivation to move, vestibular dysfunction, proprioceptive feedback, as well as individual differences in anxiety can potentially contribute to the deficits in the rotarod and parallel rod assays Mice with a perturbation in the auditory function often display vestibular impairment in addition to hearing loss The regional distribution of GPR55 in the brain is controversial. Using quantitative PCR we found that GPR55 mRNA was expressed in the following order (from high to low abundance): striatum>hippocampus>forebrain>cortex>cerebellum, whereas in a previous report the expression levels were found in the following order: frontal cortex>striatum>hippocampus = cerebellum Staton et al. Gpr55 gene. This exon contains the entire coding region of GPR55 protein. The absence of GPR55 transcript in GPR55 KO brains of the line used in this study confirmed the complete removal of GPR55 function The GPR55 knockout mice used in Staton et al. Two very recent brief reports found that GPR55 modulates synaptic strength at Schaffer collateral/CA1 synapses during period of high synaptic activity Most preclinical studies of GPR55 suggest that GPR55 antagonists might be therapeutically beneficial. For example, GPR55 deletion ablates hyperalgesia following partial nerve ligation and injection of complete Freund’s adjuvant