While previous studies have shown that AT2R is expressed by nociceptors in human DRG (hDRG), and that EMA401 inhibits capsaicin responses in cultured hDRG neurons, the expression and levels of its endogenous ligands AngII and AngIII in clinical neuropathic pain tissues, and their signalling pathways, require investigation. We have immunostained AngII, AT2R and the capsaicin receptor TRPV1 in control post-mortem and avulsion injured hDRG, control and injured human nerves, and in cultured hDRG neurons. AngII, AngIII, and Ang-(1-7) levels were quantified by ELISA. The in vitro effects of AngII, AT2R agonist C21, and Nerve growth factor (NGF) were measured on neurite lengths; AngII, NGF and EMA401 effects on expression of p38 and p42/44 MAPK were measured using quantitative immunofluorescence, and on capsaicin responses using calcium imaging.The clinical efficacy of the Angiotensin II (AngII) receptor AT2R and TRPV1 in hDRG and in vitro. AngII staining by image analysis showed no significant difference between control (n = 12) and injured (n = 13) human nerves. AngII levels by ELISA were also similar in control human nerves , injured nerves , and painful neuromas ; AngIII and Ang-(1-7) levels were undetectable (<0.03 and 0.05 pmol/g respectively). Neurite lengths were significantly increased in the presence of NGF, AngII and C21 in cultured DRG neurons. AngII and, as expected, NGF significantly increased signal intensity of p38 and p42/44 MAPK, which was reversed by EMA401. AngII mediated sensitization of capsaicin responses was not observed in the presence of MAP kinase inhibitor PD98059, and the kinase inhibitor staurosporine.AngII immunostaining was observed in approximately 75% of small/medium diameter neurons in control (n = 5) and avulsion injured (n = 8) hDRG, but not large neurons i.e. similar to TRPV1. AngII was co-localised with AT2R ligand in human peripheral nerves is AngII, and its levels are maintained in injured nerves. EMA401 may act on paracrine/autocrine mechanisms at peripheral nerve terminals, or intracrine mechanisms, to reduce neuropathic pain signalling in AngII/NGF/TRPV1-convergent pathways.The major AT Neuropathic pain has a prevalence of 1–8% in the general population, and is a major unmet medical need due to limited efficacy and tolerability of currently available drug treatments. Hyperexcitability and abnormal sprouting of primary afferent sensory nerve fibres underpin peripheral neuropathic pain [2R) was identified as a novel target for neuropathic pain: several small molecule AT2R antagonists with >1000-fold selectivity over the AT1R receptor produced dose-dependent analgesia in multiple rodent neuropathic pain models, with the analgesia abolished in mice null for the AT2R [2R antagonist, inhibited neurite outgrowth and capsaicin responses in cultured human and rat DRG neurons, in an in vitro model of sensitization [2R antagonist, EMA401, for 4 weeks produced significant pain relief above placebo and was well-tolerated [The angiotensin II (AngII) type 2 receptor  is a biologically active heptapeptide formed endogenously from either AngI or AngII, with vasodilatory and antiproliferative activities that oppose the constrictive and proliferative effects of AngII [1 receptor blockers [Systemic AngII is known to be derived from the action of kidney derived renin on AngI in the circulation. However, local renin angiotensin system (RAS) components have been described in brain , heart, of AngII , 11. Cirblockers –14.2R and TRPV1 in clinical tissues, and quantified the levels of AngII, AngIII and Ang-(1-7) by ELISA. The effects of AngII, the AT2R antagonist EMA401 and the synthetic AngII agonist EMA1087 (Compound 21) [2R and TRPV1 expression were also studied in cultured human DRG neurons using immunofluorescence, and signalling pathways using calcium imaging.We have studied the expression of AngII, ATound 21)  on calci2R antibody showed that AngII and AT2R were co-localised in some small/medium diameter (≤50 µm) cells ; neurite lengths were significantly increased in rDRG neurons treated with combined AngII and NTFs , or AngII alone , or EMA1087 (C21) alone , compared with vehicle treated controls ,  in the absence of added drugs was reduced due to repeat stimulation (tachyphyllaxis) . Capsaicin responses were significantly enhanced in the presence of 100 nM AngII, compared with responses in the absence of AngII ; this enhancement was not observed in the presence of the MAPK inhibitor PD98059   levels were below the detection limits, in accord with other studies [Quantitative ELISA studies in human tissues showed that the major AT studies . Previou studies , and loc studies , 9. In s studies . These sIt is important to note that we have measured AngII levels in human nerves and DRG by ELISA in frozen tissue extracts; as AngII may degrade when extracted in frozen tissues, freshly extracted tissues need to be studied in future for AngII, other putative endogenous RAS ligands, and their metabolites. The levels obtained in our study are higher than those reported in other studies of rat and human trigeminal ganglia , 9, and 2R and AngII expression in injured human or rodent sensory neurons is unknown, and may be influenced by target-organ derived or intrinsic factors. In our previous study [2R levels were reduced in human nerve segments proximal to injury , but they were preserved or high in chronic painful neuromas, which comprise regenerating nerve fibres. These AT2R receptor level changes parallel those of key pain receptors and ion channels regulated by target-derived growth factors e.g. TRPV1, Nav 1.7 and Nav1.8 by NGF after nerve injury. The mode of analgesic action of EMA401 may involve inhibition of augmented AngII/AT2R signalling in NGF convergent pathways, a postulate which is supported by our in vitro results, described below. The lack of CNS penetration of EMA401, and the time-course of its clinical effects in patients with post-herpetic neuralgia  argue against AngII acting as a classical neurotransmitter in dorsal spinal cord.Injury and associated inflammation may increase local AngII, contributing to sensitization of nociceptors, particularly in sprouting nerve fibres. The molecular regulation of ATus study , AT2R le2R and TrkA, which underlies neuronal differentiation of NG108 cells [Co-expression of AngII, AT2R and TRPV1 was observed in cultured small diameter hDRG neurons using immunofluorescence, in accord with the hDRG tissue findings. AngII was co-localised in cultured DRG neurons with AT2R and TRPV1, as illustrated in Figure 08 cells , sensiti2R activation appears to have a synergistic effect with TrkA, other pathways such as PI3K [2R in mediating neurite outgrowth in vitro via estrogen activation [2R antagonists in such animal models, and in patients with chronic pain, particularly those more prevalent in females.In our previous study , neurons as PI3K  may alsotivation , 21, andtivation . These m2R agonist, caused neurite outgrowth in our assay is in agreement with the neurite promoting effects of C21 previously described in the neuroblastoma-glioma hybrid NG108 cells, that was blocked by the AT2R receptor antagonist  [2 receptor, and potential efficacy in pain states associated with abnormal nerve sprouting. Conceptually, while promoting AT2 receptor mediated neuronal outgrowth could be beneficial after certain pathologies (e.g. stroke), aberrant or collateral neuronal regeneration with hypersensitivity may underpin some painful conditions in the periphery mediated via AT2R [Our finding that AngII and EMA1087 (C21), a small molecule AT123,319) , 23. PD1123,319) , and EMAvia AT2R , in accovia AT2R . An impovia AT2R , with ne2R is a GPCR, the AngII mediated increase in cAMP [αs mechanism. The consequent TRPV1 sensitization was not observed in the presence of the MAP kinase inhibitor, PD98059 in hDRG and rDRG neurons in this study. AngII effects have been associated with increased neuronal excitability and promotion of neurite outgrowth via multiple mechanisms of AT2R activation, depending on the cell type [As AT in cAMP  is in keell type . cAMP isell type , which iell type , but notOur results showed that both AngII and its analogue EMA1087 (C21) caused TRPV1 sensitization, suggesting that like AngII, C21 has a pro-nociceptive effect. While the effects of EMA1087 are more similar to AngII for neurite outgrowth but less so in sensitization effects, EMA1087 did show significantly enhanced responses as did AngII. EMA1087 is a synthetic molecule and AngII a peptide, which could explain differential effects in assays. AngII appeared to result in greater sensitization of human neurons compared with rat neurons, possibly reflecting a species specific difference.1R and AT2R, and activation of a number of signalling pathways including p44/42 MAPK, p38 MAPK, c-JUN and NF-κB [1R, AT2R and LOX-1, and IL-1β in cardiomyocytes, with activation of MAPKs, c-Jun and NF-kB, suggesting a positive feedback between AngII and inflammation [2R and TrkA. Other pathways need investigation, such as GDNF signalling, which may have similar additive effects.Previous studies have shown pro-inflammatory effects of AngII in a variety of tissues including kidney, heart and blood vessel wall, by up-regulating the expression of both ATnd NF-κB , 27. Angammation . CD68 poammation . There iammation , 29–31 aammation –34; AngI+ channel activity in hippocampal neurons, that was reversed by the AT2R antagonist PD123,319 [m. ulcerans [AngII has been reported to increase KD123,319 . In a reulcerans . While tulcerans .2R are co-expressed in nociceptive human sensory neurons, and the levels of AngII, the major endogenous ligand in human peripheral nerves, are preserved after injury. AngII induces p38, p42/p44 mitogen activated protein kinase (MAPK) activation, neurite outgrowth in adult rat DRG neurons, and sensitization of adult rat and human DRG neurons that is blocked by EMA401. Hence increased AngII/AT2R signalling in DRG neurons secondary to peripheral nerve injury may have a key role in chronic pain mechanisms, including neuropathic pain. The mode of EMA401 analgesic action appears to involve inhibition of augmented AngII/AT2R induced p38 and p42/p44 MAPK activation, and hence inhibition of DRG neuron hyperexcitability and sprouting of DRG neurons. EMA401 is likely to be most effective in conditions of hypersensitivity associated with abnormal nerve sprouting, where AngII may synergize or augment NGF mechanisms. Selective AT2R antagonists represent a new class of analgesics for improved relief of neuropathic pain.AngII and ATA range of tissues was used in this study obtained with consents and approvals as described previously , includiTissues were supported in optimum cutting tissue (OCT) medium . Tissue sections (15 µm thick) were collected onto coated glass slides and post-fixed in 4% w/v paraformaldehyde in 0.15 M phosphate buffered saline (PBS) for 30 min (for frozen section only). Endogenous peroxidase was blocked by incubation in methanol containing 0.3% w/v hydrogen peroxide for 30 min. After rehydration with PBS buffer, sections were incubated overnight with primary antibody using a range of dilutions  as previously described . SectionAntibody-immunoreactive, nucleated neurons in sensory ganglia (DRG) were counted and their diameter assessed using a calibrated microscope eyepiece graticule and expressed as % total; image analysis (% area) of nerve sections has been described previously .Frozen tissue samples of control normal nerve (n = 31), injured nerves (n = 7), and painful neuromas (n = 12), were weighed and extracted in boiling 0.5 M acetic acid for 10 min. Extracts were then concentrated and de-salted by applying 0.2 ml extract to an activated Sep-Pak column, washed and desalted with water containing 0.1% TFA. Bound peptides were eluted from the columns using 1 ml of 80% acetonitrile containing 0.1% TFA. The eluate was evaporated to dryness using a speed-vac overnight. Each vial was reconstituted in 1× assay buffer supplied by the manufacturer (see below).A human specific AngII/AngIII immunoassay kit  was used according to the manufacturer’s instructions (but note AngIII specific immunoassay kit described below showed undetectable levels of AngIII in these nerve extracts). AngII standard (0.04–25 ng/ml) and specimens (50 µl) were added in duplicate and the mean used for subsequent analysis. The absorbance at 450 nm in each well was measured using a plate reader. Standard curve was plotted using a log scale and the concentration of human AngII in each specimen determined using Excel software.In order to determine the recovery of AngII after extracting in boiling in acetic acid and concentrating using a Sep PaK, 200 µl of standard (at 1 nmol/ml) was added to 800 µl of boiling acetic acid for a further 10 min, and after cooling 200 µl was applied to Sep Pak and dried overnight as above. This was reconstituted with 200 µl of assay buffer and 50 µl used for assay in duplicate.A human specific AngIII immunoassay kit  that reacts 100% to AngIII and with no significant cross-reactivity with analogues, was used according to the manufacturer’s instructions. AngIII standard (6.17–500 pg/ml) and specimens (50 µl) were added in quadruplicate and the mean used for subsequent analysis. The absorbance at 450 nm in each well was measured using a plate reader. Standard curve was plotted and the concentration of human AngIII in each specimen determined using Excel.A human specific Ang-1-7 immunoassay kit , reacting 100% to Ang-(1-7) and no significant cross-reactivity with analogues, was used according to the manufacturer’s instructions. Ang-(1-7) standard  and specimens (50 µl) were added in quadruplicate and the mean used for subsequent analysis. The absorbance at 450 nm in each well was measured using a plate reader. Standard curve was plotted and the concentration of human Ang-(1-7) in each specimen determined using Excel.hDRG were obtained from five patients with brachial plexus avulsion undergoing nerve repair surgery, excised as a necessary part of surgical repair i.e. redundant tissue; ganglia were enzyme digested, mechanically dissociated and plated on collagen and laminin coated MatTek dishes (MatTek Corp USA), in Ham’s F12 medium containing 10% HIFCS , penicillin/streptomycin (100 μg/ml each), NTFs , as previously described [Cultures were fixed in 4% PFA for 30 min, permeabilised with chilled methanol , rehydrated in PBS and incubated in primary antibodies to goat anti AT2R , rabbit anti TRPV1 , or goat anti β tubulin (1:200 Abcam), mouse anti AngII (Serotec 1:200), rabbit anti PGP9.5 , mouse anti Gap43 , and visualised with secondary antibodies , donkey anti mouse (Alexa 488), donkey anti rabbit (Alexa 350), and donkey anti goat (Alexa 546), incubated for 1 h at room temperature, and mounted on glass slides in Citifluor mounting medium with the antifade agent DABCO . TIFF images were acquired using widefield fluorescence optics with an upright Olympus microscope BX43, and cooled CCD camera (Coolsnap), using uv illumination, and Cellsens software . Cell counts were obtained for neurons expressing AT2R, AngII and TRPV1, and neuronal diameters were measured using Cellsens software.48 h after plating, hDRG neurons were loaded with 2 μM Fura2 AM, and responses to capsaicin, AngII, and EMA1087 (C21), were imaged as before . In eachBilateral DRG from all levels of 6 adult female Wistar rats were isolated and plated as before , in coll48 h after plating, duplicate dishes were treated with AngII (10 nM), AngII + EMA401 (10 and 100 nM respectively), 100 nM EMA401, NGF (100 ng/ml) or vehicle treated (control 0), for 30 min at 37°C, then fixed with 4% PFA for 30 min for immunostaining.Following fixation, and 3 min permeabilisation with methanol at −20°C, the neurons were incubated in rabbit polyclonal antibody to phospho-p38MAPK pThr180 , or rabbit monoclonal antibody to phospho –p44/42 , for 1 h at room temperature, visualised with secondary antibody Alexa fluor 546 goat anti rabbit , incubated for 45 min, and mounted in glycerol containing antifade agent DABCO (Sigma UK) on glass slides. TIFF fluorescence images were acquired with an upright Olympus BX43 widefield fluorescence microscope with the same acquisition settings for all groups, using Cellsens software . Signal intensity was measured after background substraction, from at least 120 neurons in each group, from at least three experiments using Image J software (N.I.H. USA), and normalised to controls.21) or vehicle treated, for 48 h at 37°C. Cultures were fixed and immunostained with mouse Gap43 , visualised with FITC conjugated Alexa Fluor goat anti mouse , for 45 min each, and mounted on glass slides. TIFF images were acquired with an Olympus upright microscope as above. The longest neurite lengths were measured for calculating the average from approximately 50 neurons in each group and normalised to vehicle treated controls.rDRG neurons were prepared as above in BSF2 medium, and 48 h later duplicate dishes were treated with AngII (10 nM), NTFs , 10 nM EMA1087 (Compound t test was used to compare groups, and P < 0.05 was considered statistically significant.Student’s


N = 10) were created by the interaction of factors in study: type of resin (self-cured (SC) or heat-cured (HC)) and reinforcement method , unidirectional glass fiber (Uni), short glass fiber (Short), unidirectional and short glass fiber (Uni-Short), thermoplastic resin fiber (Tpl), and steel wire (SW)). Reinforced bars (25 × 2 × 2 mm) were tested in flexural strength (0.5 mm/min) and examined by scanning electron microscopy (SEM). Data (MPa) were submitted to factorial analysis, ANOVA, and Tukey and T-student tests (a = 5%) showing significant interaction (P = 0.008), for SC: Uni (241.71 ± 67.77)a, Uni-Short (221.05 ± 71.97)a, Ind (215.21 ± 46.59)ab, SW (190.51 ± 31.49)abc, Short (156.31 ± 28.76)bcd, Tpl (132.51 ± 20.21)cd, Control SC (101.47 ± 19.79)d and for HC: Ind (268.93 ± 105.65)a, Uni (215.14 ± 67.60)ab, Short (198.44 ± 95.27)abc, Uni-Short (189.56 ± 92.27)abc, Tpl (161.32 ± 62.51)cd, SW (106.69 ± 28.70)cd, and Control HC (93.39 ± 39.61)d. SEM analysis showed better fiber-resin interaction for HC. Nonimpregnated fibers, irrespective of their length, tend to improve fracture strength of acrylics.The aim of this study was to evaluate the flexural strength of acrylic resin bars by varying the types of resin polymerization and reinforcement methods. Fourteen groups ( Heat- or self-polymerized acrylic resins are generally composed of polymethyl methacrylate (PMMA). They are used for complete dentures, provisional restorations, or even aesthetic surgery corrections , 2. PMMAThe use of metal and fiber reinforcements produces beneficial results –7. MetalThe effectiveness of fiber reinforcement is influenced by many variables including the quantity of fibers , 15 and Unidirectional long fibers generate orthotropic mechanical properties inside composites, producing the reinforcement effect in one specific direction . On the The fibers adhesion to the polymer matrix and the fibers impregnation with the resin affect the degree of reinforcement , 20, dueIt is expected that, after silanization, the reinforcement effect of pure nonimpregnated glass fibers would be similar to industrialized glass fibers. Also, it is hypothesized that fibers would produce better reinforcement than metal wire. The aim of this study was to evaluate the flexural strength differences of acrylic resin bars related to different resin polymerization and reinforcement method.n = 10 per group) were created with the combination of studied variables: type of acrylic resin (heat- (HP) or self-polymerized (SP)) and reinforcement method , unidirectional pure glass fiber (Uni), short pure glass fiber (Short), unidirectional and short pure glass fiber (Uni-Short), thermoplastic resin fiber (Tpl), and steel wire (SW)). The number of samples per group was based on a similar previous study [The materials used in this study are listed in us study  with theA condensation silicon impression material  mold was constructed from a stainless steel pattern to produce standardized rectangular specimens with dimensions of 25 mm (±2.0) × 2 mm (±0.1) × 2 mm (±0.1) and 0.11 g (±0.01), according to ISO 4049/2000 . All reiThe silicon mold was filled with a thin layer of low viscosity acrylic resin and, right above this layer, the reinforcements were positioned and fully covered with a second layer of acrylic resin, following the powder/liquid ratio recommended by the manufacturer. All reinforcements were oriented in the direction of the long axis of the specimen. The mold was covered with a clean glass slab to remove excess resin and kept at room temperature (25°C) for 20 minutes under 9.8 N load until polymerization of the resin was completed. The heat-polymerized acrylic resin specimens were polymerized in a crockpot curing  under 380 MPa pressure, at 120°C for 15 minutes.2 for 40 seconds each. Control specimens were fabricated without any reinforcement (0.109 g (±0.01)). Specimens were finished with 600, 1000, and 1200 grit silicon carbide paper  under constant water stream. All specimens were stored in distilled water at 37°C for 24 hours before testing.Specimens containing industrialized preimpregnated glass fibers were light polymerized by irradiating 3 different areas at their top surface  with a LED light source  at 850 mW/cmPL/wb2, where “P” is the maximum load at fracture, “L” is the distance between the supports (20 mm), “w” is the sample thickness, and “b” is the height. The samples' thickness and height were measured with a digital caliper.Specimens were positioned on a 3-point bending flexural strength testing apparatus  with two supports 20 mm apart and tested at a crosshead speed of 1 mm/min. The load at fracture was recorded in Newtons and flexure strength (FS) was calculated in MPa with the following equation: FS = Random samples were selected from each group and analyzed with a SEM. The samples, fixed on metal stubs, were placed in an ultrasonic bath of deionized water for 10 minutes and then sputtered with gold (1 cycle of 120 s), under vacuum, in a sputtering device . The surfaces were analyzed by SEM , focusing on the fracture features, integrity, and homogeneity along the interfaces between reinforcement material and acrylic resin. Samples were examined under magnification varying from ×20 to ×10,000. The unit operated at 20 kV, WD = 15–18 mm and with a spot size range of 25 pA to 100 pA. t-test was used. For all tests, groups were considered statistically different at α = 5%.Statistical analysis was performed with Kolmogorov-Smirnov test of normal distribution and two-way ANOVA (2 × 6) followed by Tukey's honestly significant difference (HSD) test with a general linear model procedure in SSPS17.0  to analyze the interaction between polymerization type and reinforcement method. One-way ANOVA followed by Tukey's HSD test was used within each acrylic resin group to compare effectiveness of different reinforcements. For pairwise comparisons of resin types within each reinforcement group Student'sP = 0.008) and for the type of resin (P = 0.0001) but not for the reinforcement method (P = 0.728). The results of combination of studied variables, type of acrylic resin (heat- (HP) or self-polymerized (SP)) and reinforcement method , unidirectional pure glass fiber (Uni), short pure glass fiber (Short), unidirectional and short pure glass fiber (Uni-Short), thermoplastic resin fiber (Tpl), and steel wire (SW)), are presented in Statistical analysis showed significant interaction between factors  release less residual monomers than the SP ones once high temperatures promote higher degree of conversion and reduced powder (PMMA)/liquid (MMA) ratio in the mixture, affecting flexural strength . BesidesThe oxygen inside voids inhibits the polymerization of acrylic resins and the porosities can increase water sorption by polymeric matrix with a detrimental effect on mechanical properties in a long-term evaluation . The resComparisons of flexural strength (FS) among the groups showed interaction between the factors in the study. For SP groups all reinforcements improved FS with the exception of Short and Tpl groups, and for HP groups Tpl and SW did not show improved FS. In agreement with this study, a previous report  found siFiber's critical length is a measure of minimum fiber length required for maximum stress transfer within the polymer matrix. Working with a bisGMA resin, the critical fiber length was established between 0.5 and 1.6 mm  and for Tpl groups showed similar FS compared to controls, irrespective of the acrylic resin. Dental floss is composed by thermoplastic resin fibers showing presence of wax around fibers, which resulted in poor adhesion Figures  and reduIn general, acrylic resin reinforcement with glass fibers produced improved fracture strength. Provisional or even definitive prosthesis can successfully employ fiber reinforcement in order to assure better longevity and ease of repair . The useFiber reinforcement significantly increases fracture strength of acrylic resins and this is related to the resin polymerization method.A better interaction between fibers and resin results in higher flexural strength. Heat-polymerized resin tends to produce better wetting of fiber.Nonimpregnated fibers, irrespective of their length, tend to improve flexure strength of acrylics.Steel wire reinforcement may reinforce self-polymerized acrylics but its micromechanical interaction does not seem to be effective.According to the results and limitations of the present study it is possible to conclude the following.


Pseudomonas aeruginosa ATCC 33988 is highly adapted to grow in jet and diesel fuel, with a defined regulation of adaptive genes and metabolization of n-alkanes. The draft genome of strain ATCC 33988 is 6.4 Mb in size, with 5,975 coding sequences and 66.3% G+C content, and it is highly similar to that of the clinical strain P. aeruginosa PAO1. Pseudomonas aeruginosa ATCC 33988, also known as P. aeruginosa QMB 1592, was isolated from a fuel storage tank in Ponca City, OK. This strain efficiently metabolizes n-alkanes and grows extremely well in Jet-A fuel and F-76 diesel (P. aeruginosa ATCC 33988 is highly adapted to the fuel environment and is able to regulate multiple metabolic pathways to enhance its resistance to fuel (P. aeruginosa ATCC 33988 and its high capacity for hydrocarbon degradation compelled us to define its genetic makeup by sequencing its genome.P. aeruginosa ATCC 33988 was sequenced on a Roche 454 GS Junior platform using a whole-genome shotgun (WGS) approach, resulting in 388,306 reads. The sequence reads were assembled with the Roche de novo Assembly software. The assembly reported 224 large (>500 bp) contigs, with an N50 of 68,712 bp. The longest contig was 235,965 bp. The draft genome sequence is 6,410,803 bases in length, with a G+C content of 66.3%, which is similar to the 66% G+C content of the P. aeruginosa PAO1 strain. Rapid genome annotation using the RAST annotation server (n = 716 CDSs), carbohydrates (n = 458), cofactors (n = 360), vitamins and pigments (n = 360), protein metabolism (n = 279), cofactors and membrane transport (n = 307), cell wall and capsule (n = 220), fatty acids, lipids, and isoprenoids (n = 212), stress response (n = 193), iron acquisition and metabolism (n = 146), virulence, disease, and defense (n = 142), metabolism of aromatic compounds (n = 125), and miscellaneous (n = 88) were the most abundant subsystems. These results support that this strain is well adapted to cope with biotic and abiotic stresses, including fuel contamination.P. aeruginosa ATCC 33988 with other P. aeruginosa strains within the RAST database identified P. aeruginosa 19BR as its closest neighbor, with a score of 501, followed by P. aeruginosa 213BR, with a score of 490. P. aeruginosa PAO1 (taxonomy ID 208964.1) was the twelfth closest neighbor, with a score of 343.A comparison of P. aeruginosa ATCC 33988 transcriptome shows high similarity with the PAO1 strain, with hybridization to >5,460 probe sets contained within the PAO1 GeneChip Pae_G1a , efflux pumps and porins , biofilm formation , and iron acquisition  were at least 99% identical to those in PAO1. Further analyses showed alkB1 and alkB2 have two synonymous single-nucleotide polymorphisms (SNPs) compared to the alkB1 and alkB2 genes of the PAO1 strain.The  Pae_G1a . A DNA sJPQQ00000000.This whole-genome shotgun project has been deposited in DDBJ/EMBL/GenBank under the accession no.


Bacillus atrophaeus strain 930029. Strain 930029 shows evidence of drift, based on a comparison to the corresponding source strain publicly available today.We report here the draft genome sequence of  Bacillus atrophaeus (formerly Bacillus globigii and Bacillus subtilis niger) (Bacillus anthracis), a high-priority biological warfare and bioterrorism threat. Multiple strains of B. atrophaeus have shown evidence of interlaboratory strain variation (B. atrophaeus strain 930029 as a simulant for biodefense preparedness efforts in France motivated in-depth characterization by whole-genome sequencing (WGS).The aerobic spore-forming s niger) , 2 has as niger) . Among tariation . The impde novo assembled using Velvet version 1.2.07 into 131 contigs >1,000 bp, which suggests that this strain has a genome size of at least 4.05 Mbp, with a G+C content of 43.24%. The N50 contig length was 66,438 bp, with the longest being 192,113 bp. The average coverage for the contig sequence of strain 930029 was 101×. Using BioNumerics version 7.1, the sequence reads were mapped to the complete reference genome sequence of B. atrophaeus 1942 (accession no. CP002207), and a 16-kb deletion (positions 208228 to 224482) was identified in strain 930029. Multiple gene deletions affect hypothetical proteins and two genes encoding for RapK phosphatase and for an inner spore coat protein. Multiple (23 silent) single-nucleotide polymorphisms (SNPs) were also detected. Historically, strain 930029 was acquired from Collection de l’Institut Pasteur (CIP)  as CIP77:18 (batch “kia”) in 1993. The publicly available strain kindly provided in 2013 by CIP (batch “15409_3”) has been also sequenced; 6,528,908 reads for a total of 1,193,477,765 bp were generated. The reads were de novo assembled into 151 contigs >1,000 bp, which suggests that this strain has a genome size of at least 3.89 Mbp, with a G+C content of 43.74%. The N50 contig length was 64,872 bp, with the longest being 273,228 bp. The average coverage for the contig sequence was 69×. This strain is not missing the 16-kb fragment previously identified in strain 930029 and is discriminated from strain 930029 based on 19 SNPs. Therefore, strain 930029 presents features different from the two other isolates of the B. atrophaeus species, 1942 and CIP77:18, such as the conspicuous absence of genes involved in extracellular peptide signaling and endospore development. Despite this deletion, strain 930029 is easy to grow and sporulate in vitro. This raises the question of strain variations during long-term propagation and of standardization issues for tests and evaluations in biodefense.WGS of strain 930029 was sequenced by using an Illumina MiSeq personal instrument and on paired-end reads with a read length of 300 nucleotides (nt). A total of 5,568,114 reads were generated, for a total of 1,236,558,746 bp. The reads were CUWS00000000.The draft genome sequence for strain 930029 has been deposited at GenBank under the accession no.


Supplemental Digital Content is available in the text Previous studies of small cohorts have implicated several circulating cytokines with progression of chronic hepatitis B (CHB). However, to date there have been no reliable biomarkers for assessing histological liver damage in CHB patients with normal or mildly elevated alanine aminotransferase (ALT).The aim of the present study was to investigate the association between circulating cytokines and histological liver damage in a large cohort. Also, this study was designed to assess the utility of circulating cytokines in diagnosing liver inflammation and fibrosis in CHB patients with ALT less than 2 times the upper limit of normal range (ULN).P < 0.001). Patients with significant fibrosis had higher levels of IL-8 (P = 0.027), transforming growth factor alpha (TGF-α) (P = 0.011), IL-2R (P = 0.002), and CXCL-11 (P = 0.032) than the group without significant fibrosis. In addition, 31.8% and 29.1% of 151 patients with ALT < 2 × ULN had at least moderate inflammation and significant fibrosis, respectively. Multivariate analysis demonstrated that CXCL-11 was independently associated with at least moderate inflammation, and TGF-α and IL-2R independently correlated with significant fibrosis in patients with ALT < 2 × ULN. Based on certain cytokines and clinical parameters, an inflammation-index and fib-index were developed, which showed areas under the receiver operating characteristics curve (AUROC) of 0.75 (95% CI 0.66–0.84) for at least moderate inflammation and 0.82 (95% CI 0.75–0.90) for significant fibrosis, correspondingly. Compared to existing scores, fib-index was significantly superior to aspartate aminotransferase (AST) to platelet ratio index (APRI) and FIB-4 score for significant fibrosis.A total of 227 CHB patients were prospectively enrolled. All patients underwent liver biopsy and staging by Ishak system. Patients with at least moderate inflammation showed significantly higher levels of CXCL-11, CXCL-10, and interleukin (IL)-2 receptor (R) than patients with less than moderate inflammation  was superior to APRI and FIB-4 for the diagnosis of significant fibrosis in patients with normal or mildly elevated ALT. The limitations of liver biopsy have led to the development of noninvasive assessments for histological liver damage. Nevertheless, there are no reliable biomarkers or noninvasive models for assessing liver inflammation and fibrosis in chronic hepatitis B (CHB) patients with normal or mildly elevated ALT.Alanine aminotransferase (ALT) has been widely used for assessing hepatitis activity. However, patients with normal or mildly elevated serum ALT are not necessarily free from liver damage or liver-related mortality.8 Cytokines are a large family of molecules, including T helper type 1 cells (Th1) cytokines , which enhance the cellular immune responses, Th2 type cytokines , which favor antibody responses, and Th17 cytokines  that contribute to the proinflammatory reaction.9 It has been proved in recent experimental studies that an imbalance in cytokine production plays a key role in the development of liver damage, necro-inflammation, and subsequent fibrosis. Specifically, IL-1 and TNF-α have been suggested to be highly proinflammatory cytokines, which contribute to hepatocyte apoptosis and immume cell activation. In addition, IL-20, IL-13, and IL-33 have been defined as profibrogenic cytokines that promote activation and proliferation of hepatic stellate cells.10 IL-22, which is produced primarily by Th17 and IL-22+ cells, has been reported to be largely increased in liver tissue of HBV-infected patients with liver cirrhosis.12 IL-17 signaling has been shown to promote production of IL-6, IL-1, and TNF-α by inflammatory cells and to directly induce production of collagen type I in hepatic stellate cells by activating the signal transducer and activator of transcription 3 (Stat3) signaling pathway.13Recently, emerging evidence has suggested that cytokines and regulatory molecules are fundamental mediators in the host adaptive immune response to HBV and viral clearance.18 In a cohort of African-American injection drug users with chronic hepatitis C, CXCR3 ligands  were shown to contribute to liver cirrhosis and to discriminate advance liver fibrosis from mild fibrosis.19 These studies, however, have included relatively few CHB patients with liver biopsy. Thus, larger CHB cohort studies are needed to further understand of the association between circulating cytokines and the pathological state of HBV-infected liver.A few studies have also tried to reveal the possible correlation between circulating cytokines and histological liver damage in CHB patients. The current literature indicates that circulating Th17 cells increase histological activity index (HAI) via IL-17, IL-10, and INF-γ, and that IL-10, TNF-α, and transforming growth factor (TGF)-beta are correlated with fibrosis stages in CHB patients.The aim of the current study was to investigate the association of circulating cytokines with liver inflammation and fibrosis in a large cohort. Also, this study was designed to assess the utility of circulating cytokines in predicting histological liver injury in CHB patients with normal or <2 × ULN ALT levels.9/L]) × 100; FIB-4 = (age × AST)/(platelet count × 109/L × ALT1/2).21 The reference value for ALT was 0–40 IU/L.22A total of 235 patients were prospectively enrolled in this study. Inclusion criteria consisted of: positive hepatitis B surface antigen for at least 6 months; treatment naive; age between 18 and 65 years; negative serum levels for anti-HAV IgM, anti-HCV, anti-HEV IgM/IgG, anti-EBV IgM, and anti-CMV IgM; and off potential transaminase-lowering agents such as bicyclol for at least 2 weeks prior to blood sampling biochemistries. Patients were recruited from 24 hospitals in mainland China between October, 2013 and September, 2014. Exclusion criteria were: hepatitis C virus or human immunodeficiency virus coinfection; presence of other causes of chronic liver diseases such as alcoholic, autoimmune, genetic, drug-induced, and nonalcoholic fatty; and pregnancy. Patients with hepatocellular carcinoma or decompensated cirrhosis were also excluded. Clinical data and chemical parameters were documented, and serum samples were taken at the time of liver biopsy. Noninvasive assessments for fibrosis, aspartate aminotransferase to platelet ratio index (APRI), and FIB-4 score were calculated as follows: APRI = .1–1.7 × 108 IU/mL) was measured by COBAS AmpliPrep/COBAS TaqMan as previously described.22 Qualitative detection of hepatitis B core antigen (HBeAg) and HBeAb were also performed using appropriate Roche Elecsys assays according to the manufacture's instructions.Serum HBV DNA  were measured by Human cytokines/Chemokine panel I . Six cytokines and chemokines  were measured by Luminex screening system . The results were analyzed by a Luminex 200 system  according to the manufacturer's instructions. The coefficient of variation between the duplicate wells was controlled within 10%, and 23 For analysis, HAI ≥ 5 was defined as moderate/significant inflammation, and F ≥ 3 as significant fibrosis.25Ultrasonographic-guided liver biopsies were routinely processed in each institute according to a standardized protocol after receiving the patient's written informed consent. Specimens were fixed, paraffin-embedded, and stained with hematoxylin–eosin and Masson's trichrome. A minimum of 2.0 cm of liver tissue with at least 11 portal tracts were required for diagnosis. Pathological interpretations were conducted in the Department of Pathology at the You An Hospital affiliated to Capital Medical University. Each section was assessed by 2 pathologists, blindly and independently. When discrepancies occurred, the samples were reviewed by experienced pathologists who were also responsible for the reassessment of 10% random samples. All pathologists were blinded to the protocol. HAI and staging of liver fibrosis were assessed according to Ishak criteria.U test. A binary backward stepwise logistic regression analysis was conducted to determine the independent variables of liver inflammation and fibrosis. Receiver operating characteristic curves were created for the assessment of the predictive scores for staging inflammation and fibrosis. The predictive value of variables for severity of liver damage expressed as the area under the receiver operating characteristic curves curve (AUC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. All P-values reported are 2-sided, and P < 0.05 was considered to be statistically significant. The classification accuracy of variables for diagnosis was validated by leave-one out cross-validation (LOOCV).Statistical analyses were performed using SPSS ver. 16.0 . Quantitative variables were expressed as the mean ± standard deviation (SD). Categorical variables were compared using the Chi-square test, and continuous variables were compared using the Mann–Whitney A total of 235 patients were enrolled in this study, and 8 patients were excluded due to inadequate liver tissue. The remaining 227 cases were analyzed, which included 151 with ALT < 2 × ULN and 76 with ALT ≥ 2 × ULN. Patients were also stratified by HBV DNA levels and HBeAg status. In patients with ALT ≥ 2 × ULN, 48 (63.2%) patients had at least moderate inflammation and 23 (30.3%) had significant fibrosis. Of 151 patients with ALT < 2 × ULN, 31.7% and 29.1% showed at least moderate inflammation and significant fibrosis, respectively. Clinical characteristics of the study population were shown in Table P > 0.05). Biochemical markers ALT (P < 0.001), AST (P < 0.001), alkaline phosphatase , γ-glutamyl transpeptidase , and total bilirubin  were significantly elevated in patients diagnosed with at least moderate inflammation . Cytokines and chemokine levels also correlated with HAI scores. Patients with moderate or severe inflammation had significantly higher levels of CXCL-11 (71.77 ± 75.06 pg/mL), CXCL-10 (73.08 ± 55.81 pg/mL), and IL-2R (689.47 ± 313.85 pg/mL) than the patients with no/mild inflammation  Multivariate analysis indicated that CXCL-11  and GGT  were independently associated with moderate and severe inflammation , HBV DNA/HBeAg status, and liver inflammation . Patients with significant fibrosis had higher levels of IL-8 (P = 0.027), TGF-α (P = 0.011), CXCL-11 (P = 0.032), and IL-2R (P = 0.002) than patients with no significant fibrosis . Multivariate analysis showed that levels of TGF-α , IL-2R , CXCL-10 , and PLT  were independently associated with significant fibrosis (Table Platelet counts (PLT) were significantly decreased and GGT increased in patients with significant fibrosis. Age, gender, BMI, HBV DNA/HBeAg status, and ALT, AST, ALP, and TBil levels were not associated with fibrosis stages . Multivariate analysis indicated that CXCL-11, CXCL-10 (P = 0.053), AST, GGT, and HBeAg/HBV DNA were independently associated with moderate or severe inflammation. However, P values for CXCL-10, GGT, and AST were not significant (P > 0.05)  Table .Based on the variables above, we developed an inflammation-index by binary logistic regression. Inflammation-index = exp(g× 1)/[1 + exp(g× 1)]P = 0.043), TNF-α , CXCL-10 , IL-2R , and TGF-α  were higher in patients with significant fibrosis than in those without. Multivariate analysis showed TGF-α, IL-2R, platelets, and HBeAg/HBV DNA were independent predictors for significant fibrosis in patients with ALT < 2 × ULN /[1 + exp(g × 2)]The inflammation-index had an areas under the receiver operating characteristics curve (AUROC) of 0.75 (95% CI 0.66–0.84) for diagnosing moderate or greater inflammation in patients with ALT < 2 × ULN. Using a cutoff of ≥0.46, moderate or greater inflammation could be diagnosed with a sensitivity of 48.9%, specificity of 91.2%, NPV of 80.2%, PPV of 71%, and accuracy of 78.2%. Fib-index, APRI, and FIB-4 showed AUCs of 0.82 , 0.74 , and 0.67 , respectively, for diagnosing significant fibrosis in patients with ALT < 2 × ULN. Applying a cutoff of ≥0.44, significant fibrosis could be diagnosed by fib-index with sensitivity of 63.6%, specificity of 90.6%, NPV of 85.7%, PPV of 73.7%, and accuracy of 82.7% .To validate these noninvasive models for predicting moderate or greater inflammation and significant fibrosis, LOOCV was performed. LOOCV showed that 74.5% cross-validation grouped cases were correctly classified by inflammation-index and 81.3%, 74.3%, and 64.2% classified by fib-index, APRI, and FIB-4, respectively, in patients with ALT < 2 × ULN .In the total population, the inflammation-index had an AUC of 0.76 for moderate inflammation, while fib-index, APRI, and FIB-4 showed AUCs of 0.78, 0.66, and 0.63 for significant fibrosis, respectively (Supplemental Table S2, 26 However, the exact mechanisms by which cytokines mediate liver inflammation and fibrogenesis are not fully understood. INF-γ is well known to be the main mediator for control of HBV infection.8 In the progression to HBV-induced inflammation, INF-γ may be overproduced to clear HBV infection, but may be exhausted in the late stages of chronic inflammation. The inability to control the viral infection leads to the recruitment of inflammatory infiltrates into the liver parenchyma by IFN-γ-inducible CXCL-10 and -11 chemokines.27 Some of the results in the current study could be explained by the fact that INF-γ showed a declining trend while CXCL-10 and CXCL-11 levels increased in patients with moderate and severe inflammation compared to those without.This study showed that selected circulating cytokines were associated with liver inflammation and fibrosis. This implies that there are likely various mechanisms of liver inflammation and fibrosis during the progression of chronic HBV infection. Previous studies have shown that cytokines initiate downstream signaling pathways by binding to specific receptors expressed on the target cells, and which subsequently secrete various cytokines.28 TGF-α is a ligand for the epidermal growth factor receptor, which is active in many biological processes, and is a central mediator in the initiation and maintenance of fibrosis in many diseases.29 In in-vitro studies, TGF-α has been found to attenuate hepatic fibrosis through upregulation of MMP-1 and inhibition of type I procollagen peptide synthesis.31 To the best of our knowledge, this is the first study to demonstrate that circulating TGF-α levels were independently associated with liver fibrosis. IL-2R is primarily secreted by activated T-helper lymphocytes and is widely expressed on activated T lymphocytes, regulatory T cells, B cells, and monocytes.32 Some studies have examined IL-2R in chronic liver disease and found increased IL-2R in hepatic cirrhosis.36 However, the performance of IL-2R in diagnosing liver inflammation and fibrosis had not been determined. In the present study, circulating IL-2R levels increased in both moderate inflammation and significant fibrosis in CHB patients.The current study also showed that circulating IL-8, TGF-α, and IL-2R levels were increased in patients with significant fibrosis. IL-8 has been proven to play a role in recruitment and activation of hepatic macrophages by CXCR1 in human liver cirrhosis.37 A recent meta-analysis concluded that 20.7% of 830 CHB patients with ALT levels ≤40 IU/L had significant fibrosis.38 The current study showed that 31.7% and 29.1% of 151 patients with ALT levels <2 × ULN had moderate inflammation and significant fibrosis, respectively. It should be noted that patients with significant fibrosis or moderate/severe necro-inflammation should be treated immediately according to current guidelines.7 Therefore, the use of serum ALT levels underestimates the proportion of patients who should urgently receive antiviral therapy. There are no reliable noninvasive markers as surrogate for ALT to assess histological liver damage in patients with ALT levels <2 × ULN. In this study, we assessed the relationship between circulating cytokines and liver inflammation and fibrosis in patients with ALT levels <2 × ULN. Our results indicated CXCL-11 as a biomarker independent of other biochemical parameters and known risk factors for moderate greater inflammation. We also found IL-2R and TGF-α to be independent indicators for significant fibrosis.Serum ALT is the most widely used parameter to screen for and to monitor patients with liver disease. However, patients with HBV infection, a disease in which apoptosis is likely the dominant mechanism of cell death, can have normal or only minimally elevated serum ALT levels.40 Studies on APRI and FIB-4 have been validated in patients with normal and mildly elevated ALT, showing AUROCs of 0.71 and 0.72, respectively.41 We employed the 2 scores in our study to evaluate significant fibrosis, obtaining AUROCs of 0.74 and 0.67. Compared to the existing scores, the fib-index performed better in diagnosing significant fibrosis for patients with ALT < 2 × ULN.The APRI and FIB-4 scores derived from patients with HCV infection are the 2 most widely studied noninvasive tools for assessing liver fibrosis in CHB patients.42 Although a validation set was not designed in this study, LOOCV was utilized to detect the reliability of inflammation-index and fib-index. The LOOCV approach has the advantage of producing model estimates easier and with less bias in smaller samples.43 Another limitation of our study is lack of longitudinal data; this will require future work. Finally, not all the cytokines which could be detected were measured.The current study has limitations such as a lack of a validation group. A proper internal validation is necessary for the development of a reliable and reproducible prognostic model for external validation.In conclusion, the present study supports the concept that cytokines contribute significantly to the development of liver inflammation and fibrosis in CHB. Various cytokines have been found to be associated with liver inflammation and fibrosis, which implies that there are likely various mechanisms by which liver inflammation and fibrosis occur and immune cells become activated. Cytokines CXCL-11, INF-γ, and IL-17A are associated with liver inflammation, whereas CXCL-10, IL-2R, TGF-α, and IL-8 are correlated with liver fibrosis in chronic HBV-infected patients. In chronic HBV-infected patients with ALT < 2 × ULN, CXCL-11 was a marker of moderate or severe inflammation independent of other biochemical parameters and known risk factors. IL-2R and TGF-α were independent predictors for significant fibrosis. An IL-2R and TGF-α-based score fib-index was superior to the existing scores APRI and FIB-4 for predicting significant fibrosis in chronic HBV-infected patients. This represents a promising tool for non-invasive diagnosis of fibrosis in patients with normal and mildly elevated ALT levels.


Brachycephalus  is described from Serra do Quiriri in the municipality of Campo Alegre, Santa Catarina, southern Brazil. Specimens were collected from the leaf litter between from 1,263 and 1,318 m above sea level. The new species is distinguished from all its congeners by the combination of the following characters: snout–vent length 9.9–13.1 mm; skin on head and dorsum without dermal co-ossification; snout mucronate in dorsal view; dorsum rugose; general color brown, with a narrow orange vertebral stripe. The region where the new species is located is also shared with other endemic anuran species and has experienced strong anthropogenic impacts,suggesting that immediate actions should be taken to ensure their long-term preservation.A new miniaturized toadled of the genus  Brachycephalus Fitzinger, 1826   is known to harbor exceptionally high species richness, being considered as one of the world’s biodiversity hotspots . Howeverhalidae) . Despiteed e.g., .Brachycephalus have been recently divided into three species groups : ephippium , didactylus , and pernix Pombal, Wistuba & Bornschein, 1998. The latter included the species of Brachycephalus that shared their bufoniform body shape with the ephippium group, but that lacked their characteristic dermal ossification, namely B. pernix Pombal, Wistuba & Bornschein, 1998, B. brunneus Ribeiro, Alves, Haddad & Reis, 2005, B. izecksohni Ribeiro, Alves, Haddad & Reis, 2005, B. ferruginus Alves, Ribeiro, Haddad & Reis, 2006, B. pombali Alves, Ribeiro, Haddad & Reis, 2006, and B. tridactylus Garey, Lima, Hartmann & Haddad, 2012. However, a single additional study more than doubled the number of species in the pernix group ; head length, from tip of snout to angle of jaw (HL); head width—greatest width of head located between angles of jaw (HW); eye diameter (ED); nostril diameter (ND); interorbital distance between anterior corners of the eyes (IOD); internostril distance between inner margins of nostrils (IND); eye-nostril distance from anterior corner of the eye to posterior margin of nostril (END); thigh length (THL); and tibia length (TBL).All specimens collected for this study were deposited in the Coleção Herpetológica do Departamento de Zoologia, Universidade Federal do Paraná, Curitiba, Paraná, Brazil (DZUP). In addition, we examined specimens from the following collections: Célio F. B. Haddad collection, deposited in the Departamento de Zoologia, Universidade Estadual Paulista, Campus de Rio Claro, São Paulo, Brazil (CFBH), DZUP, Museu de História Natural Capão da Imbuia, Curitiba, Paraná, Brazil (MHNCI), Museu Nacional, Rio de Janeiro, Rio de Janeiro, Brazil (MNRJ), Museu de Zoologia da Universidade de São Paulo, São Paulo, São Paulo, Brazil (MZUSP), and Museu de História Natural, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil (ZUEC). A list of the examined specimens is provided in http://zoobank.org/.” The LSID for this publication is: 5267B6A8-373D-4DD2-9F7F-9D41FB6933E0. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new name contained in the electronic version is effectively published under that Code from the electronic edition alone. This published work and the nomenclatural act it contains has been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “Collection permits for this study were issued by ICMBIO (SISBIO 1911426). We classified vegetation in collection sites according to the classification of Holotype. DZUP 172 , municipality of Campo Alegre, state of Santa Catarina, southern Brazil, on 03 February 2011 by MRP, Marcos Ricardo Bornschein & Dulce Carvalho.DZUP 172 , adult fParatopotypes. DZUP 171, 173–6, collected with the holotype. DZUP 524-30 , collected by MRP, LFR & Marcos Ricardo Bornschein on 20 January 2015.Diagnosis.Brachycephalus quiririensis is distinguished from all of the species in the genus by the following combination of characters: body robust, bufoniform, adult size SVL 9.9–13.1 mm, rough dorsum  and have leptodactyliform body shape.h dorsum ; generalh dorsum . A repreDescription of Holotype. Body robust, bufoniform; head slightly wider than long; head length 31% of snout-vent length; snout short, with length almost equal to eye diameter, snout mucronate in dorsal view; nostrils protuberant, directed anterolaterally; canthus rostralis indistinct; lips nearly sigmoid; loreal region slightly concave; eye slightly protuberant in dorsal and lateral views; ED 34% of head length; tympanum indistinct; vocal sac not expanded externally; vocal slits present; tongue longer than wide, with posterior half not adherent to floor of mouth; choanae relatively small and round; vomerine odonthophores absent; arm and forearm relatively slender; arm approximately as long as forearm; tip of finger I and II slightly rounded, tip of finger III pointed; finger I and IV very small, vestigial; relative lengths of fingers IV <I < II < III; subarticular tubercles absent; inner and outer metacarpal tubercles absent; legs short, moderately robust; thigh length 34% of snout-vent length, tibia length 79 % of thigh length; toes II–III short, relatively distinct; toe I externally absent, toe V vestigial; relative length of toes V < II < III < IV; subarticular tubercles and inner metatarsal tubercles absent; outer metatarsal tubercle distinct, large, ovoid; rough dorsum, without co-ossifications; glandular warts large, circular to oval, present and spaced in dorsum and legs; head and arms smooth; sides of the body granular; large glandular warts circular and spaced on the sides of the body; belly granular; circular glandular warts spaced and distributed from belly to legs and arms, chin smooth.Coloration of Holotype. In life, dorsum, head, sides of the body, dorsal region of arms, thighs, and legs brown; dorsal region of snout and head orange, continuing into an orange stripe along the vertebral column and forming two spots near the tips of the sacral vertebra; chin, ventral portion of arms, thighs, legs, and ventral regions of hand and feet orange; belly with orange spots against a the brownish coloration that extends from the sides of the body; iris black : SVL 11.12 ± 1.03 (9.9–13.1); HL 3.73 ± 0.31 (3.3–4.4); HW 4.23 ± 0.42 (3.7–5.0); ED 1.18 ± 0.10 (1.1–1.4); ND 0.21 ± 0.04 (0.2–0.3); IOD 2.25 ± 0.17 (2.0–2.6); IND 1.26 ± 0.11 (1.1–1.4); END 0.64 ± 0.07 (0.6–0.8); THL 3.83 ± 0.37 (3.1–4.7); TBL 3.23 ± 0.30 (2.9–3.8); THL/SVL 35 ± 2% (32–37%); TBL/THL 85 ± 6% (70–93%); HL/SVL 34 ± 1% (31–36%); ED/HL 32 ± 2% (28–34%); HL/HW 88 ± 5% (79–93%). There is variation among individuals in the breadth of the orange stripe along their backs, as well as different degrees of darkness in the brown regions  and refers to type locality.Distribution.Brachycephalus quiririensis is known from the type locality, as well as an additional nearby site (rby site  in the mrby site .Remarks. Individuals of B. quiririensis were found on leaf litter of a cloud forest (“Floresta Ombrófila Densa Altomontana” sensuB. quiririensis hidden in the leaf litter, some of which vocalizing. The vegetation in the area has experienced severe disturbances in recent years, particularly fire and extensive cattle ranching. Moreover, two highly endemic bufonid species of the genus Melanophryniscus are found in the region (B. quiririensis. Strong measures should be taken by the environmental protection agency of the State of Santa Catarina to ensure the long-term preservation of these species. In addition, B. quiririensis should be integrated into the “Plano de Ação Nacional para a Conservação dos Anfíbios e Répteis Ameaçados da Região Sul do Brasil,” an initiative organized by the Instituto Chico Mendes/Ministry of the Environment that seeks to organize conservation initiatives for the conservation of the herpetofauna of southern Brazil.10.7717/peerj.1179/supp-1Appendix S1Click here for additional data file.


Oenocarpus bacaba and Oenocarpus bataua, are very often used in the diet of local communities, but the biological activities of their roots and leaflets remain poorly known. Total phenolic content (TPC) and antioxidant activity of root and leaflet extracts from Oenocarpus bacaba and Oenocarpus bataua were assessed by using different chemical assays, the oxygèn radical absorbance capacity (ORAC), the 2,2-diphenyl-l-picrylhydrazyl (DPPH) free radical-scavenging capacity and the ferric-reducing ability of plasma (FRAP). Cellular antioxidant activity and cytotoxicity were also measured in Normal Human Dermal Fibroblasts. The polyphenolic composition of Oenocarpus extracts was investigated by LC-MSn. Oenocarpus leaflet extracts were more antioxidant than root extracts, being at least as potent as Euterpe oleracea berries known as superfruit. Oenocarpus root extracts were characterized by hydroxycinnamic acids (caffeoylquinic and caffeoylshikimic acids), while leaflet extracts contained mainly caffeoylquinic acids and C-glycosyl flavones. These results suggest that leaflets of both Oenocarpus species could be valorized as a new non-cytotoxic source of antioxidants from Amazonia, containing hydroxycinnamic acids and flavonoids, in the pharmaceutical, cosmetic or agri-food industry.Native palm trees fruit from the Amazonian rainforest,  According to their fragment ions at m/z 191, 179 and 173 in negative mode − ions at m/z 335, [M + Na]+ at m/z 359, and showed UV spectra (λ = 298 and 320 nm) corresponding to caffeoylshikimic acids .In the m/z 317, 291, 179, 161 and 135 in negative mode − at m/z 431 and [M + H]+ at m/z 433 and losses of 90, 120 and 150 Da, characteristics of hexose residues. Compound 17 was confirmed to be 6-C-glucosyl apigenin due to fragment ions at m/z 311 (aglycone + 42) and m/z 341 (aglycone + 72) in negative mode − at m/z 593, and losses of 90 and 120 Da characteristic of hexose derivatives were in concordance with a 6,8-di-C-hexosyl apigenin. Compound 8 and 9 (Mr = 674) yielded [M − H]− at m/z 673 and [M + K]+ at 713. The MS2 spectra showed a first loss of 80 Da, characteristic of sulfates, followed by typical losses of hexose residues , were indicative of 6,8-di-C-hexosyl apigenin sulfates − ions at m/z 563 in the negative mode. Fragment ions corresponding to neutral losses of 60, 90, 120 and 150 Da, were characteristic of pentose and hexose residues and suggested 6,8-di-C-pentosyl-hexosyl apigenin derivatives. Compounds 12 and 16 were identified as 6-C-pentosyl-8-C-hexosyl apigenin isomers, due to the higher intensity of the fragment [M − H-60]− ion (m/z 503) resulting from the fragmentation of the pentose moiety on position 6, which fragments preferentially over sugars on position 8 − at m/z 447, [M + H]+ at m/z 449 and showed UV spectra (λ = 270 and 340 nm) characteristics of flavones. Fragment ions at m/z 327 (aglycone + 42) and m/z 357 (aglycone + 72) in negative mode, and typical losses of 90 and 120 Da clearly indicated C-glycosyl luteolin derivatives [2 data clearly indicated compound 11 as 8-C-glucosyl luteolin and compound 13 as 6-C-glucosyl luteolin. This was confirmed using synthetic standards.Compounds ivatives . RetentiO. bataua and O. bacaba leaflet extracts (7–17) was similar between the two Oenocarpus species (from 500 to 1400 µg/g DM), except when O. bataua leaflets were extracted with acetone where a 2- to 3-fold increase in flavone content (3500 µg/g DM) was measured compared to O. bacaba. In fact, C-glycosyl flavones are ubiquitous flavonoids in plants, especially within the monocotyledons including Arecaceae, the palm tree family [O. bataua and O. bacaba leaflet extracts were similar, characterized mainly by the presence of C-glycosyl apigenin accounting for 60% to 80% of all flavones. This flavonoid pattern is less frequent within the Arecaceae family, where C-glycosyl luteolin derivatives are more common [O. bataua and O. bacaba.C-glycosyl flavones and caffeoylquinic acids were the main constituents of extracts a. The ove family . The flae common . Thus, COenocarpus species. The CQA content was 5–10-fold higher in O. bataua than in O. bacaba leaflet extracts (200–1400 µg/g DM against 40–100 µg/g DM), no matter the extraction solvent , while O. bataua roots showed a mixed CQA-CSA profile, with a CQA content accounting between 20%–70% of all HCA  were correlated with high contents of 3-CQA and 4-CQA rather than with CSA content . The dried matter was then stored at −20 °C to limit degradation until analysis.Leaflets and adventitious roots from v/v) and methanol/water (M) , freeze-dried powders (2.5 g) were extracted four times in 50 mL of solvent under sonication  at room temperature. Then, each extract was centrifuged  and supernatants were combined after filtration. Organic solvents were evaporated in a rotary evaporator using a bath at 40 °C. Aqueous extracts were lyophilized. Finally, dried extracts were weighed to determine their yield and dissolved in the extraction solvent at a concentration of 40 mg/mL.Three solvents were selected for extraction of palm roots and leaflets: water (W), acetone/water (A) , 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and glacial acetic acid were obtained from Fluka Sigma–Aldrich . 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) and quercetin came from Acros Organics . K2HPO4, KH2PO4 and FeCl3 came from Fisher Scientific . Gallic acid and iron(II) heptahydrate sulfate were purchased from Alfa Aesar . Folin–Ciocalteu reagent , dimethyl sulfoxide (DMSO), Na2CO3, ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-Na2) and hydrochloric acid came from Carlo Erba reagents . Hydrogen peroxide and sodium acetate trihydrate came from Chimix .Analytical grade quality methanol, acetone and ethanol were used . Fluorescein disodium salt, 2,4,6-tris(2-pyridyl)-Oenocarpus extracts was determined according to the Folin-Ciocalteu procedure with some modifications of the protocol developed by Arnous et al. [2CO3) solution was added and incubated for 2 h at room temperature in the dark. The UV absorbance was recorded at 750 nm using a Cary® 50 UV-Vis spectrophotometer . Gallic acid was used as a reference standard. The TPC was expressed in µg gallic acid equivalent per milligram of dried matter (µg GAEq/mg DM).The total phenolic content of s et al.  or V. L.s et al. . Briefly source and coupled to a Pro Star Dynamax LC system  and a Pro Star 335 PDA (Agilent). Extracts were filtered using 0.2 µm PTFE filters. 20 µL of samples were injected onto a Kinetex PFP column, (100 × 4.6) mm, 2.6 µm (Agilent) using a gradient of 1% formic acid (A) and acetonitrile (B) at 25 °C. The gradient was as follows: 5%–10% B in 10 min, 10%–20% in 10 min, 20% held for 10 min, 20%–100% in 5 min, 100% held for 5 min, and returned to initial conditions for 9 min to re-balance the column. The flow rate was 1 mL/min. The total run time per sample was 60 min.Liquid Chromatography Mass Spectometry (LC-MS/MS) analysis was performed on an ion trap  equipped with an electrospray ionizationm/z 120–750), both in negative mode and positive mode to identify the molecular ions and then in TurboDDS™ mode (Data Dependent Scanning) to acquire MS2 spectra. Helium was used as the damping and collision gas at 0.8 mL/min. Under negative ionization mode, the operation parameters were as follows: nebulizer gas: air, nebulizer gas pressure 50 psi, drying gas pressure 25 psi, drying gas temperature 350 °C, needle voltage −5000 V, spray shield voltage −600 V, spray chamber 50 °C, capillary voltage 100 V. Under positive ionization mode, the operation parameters were as follows: nebulizer gas nitrogen, nebulizer gas pressure 50 psi, drying gas pressure 25 psi, drying gas temperature 350 °C, needle voltage 5000 V, spray shield voltage 600 V, spray chamber 50 °C, capillary voltage 70 V. Enhanced mass spectrum was used as a survey scan to trigger information dependent acquisition of MS/MS spectra. The criteria for the data-dependent acquisition (IDA) were set for the most two intense peaks, which exhibited more than 5000 counts. The identification of compounds was performed according to their MS2 fragmentation, using standards when available.Analyses were first performed using Full scan mode . 5-CQA and 6-C-glucosyl apigenin (isovitexin) were used as calibration standards to quantify hydroxycinnamic acids (caffeoylquinic acids CQA and caffeoylshikimic acids CSA) and flavones, respectively.Chemical antioxidant properties were assessed using DPPH, FRAP and ORAC assays as described in our previous paper, Leba et al. . These a® 50 UV-Vis spectrophotometer . The DPPH radical scavenging activity was expressed in μmoles Trolox equivalent/g of dried matter (TEq μmol/g DM).DPPH radical scavenging activity of the extracts were measured using the method adapted from Brand-Williams et al. . Five di3 (20 mM in water) freshly prepared. 3000 µL of this FRAP solution was mixed with 300 μL of distilled water and 100 μL of reference standard (Fe(II) as Fe2SO4) or test sample at three different concentrations in triplicates. The absorbance was measured at 595 nm after 30 min at 37 °C using a Cary® 50 UV-Vis spectrophotometer . Results were expressed in mmol Fe(II) equivalent/g of dried matter (mmol Fe(II) Eq/g DM).The FRAP assay was performed using the original method of Benzie and Strain , with soThe ORAC procedure was done according to the method of Ou et al.  with som2 as recommended by the manufacturer. The fibroblasts were used for cytotoxicity assay and cellular antioxidant assay as described below.Normal Human Dermal Fibroblasts cells (NHDF) were purchased from PromoCell . NHDF cells were cultured in growth medium (RPMI GlutaMAX™ (89%), supplemented with 5% FBS , and 1% of antibiotics. Cells were maintained at 37 °C and cultured in 5% COFirst, cytotoxicity assays in NHDF cells were performed to determine the maximum concentration of palm tree extracts to use for the CAA assay so as to ensure the viability of the NHDF cells. The cytotoxicity was measured using the 3--2,5-diphenyltetrazolium bromide (MTT) method adapted from Ferreira Silva et al. . Briefly4 cells by well on a 96-well microplate in 100 μL of growth medium by well. Twenty-four hours after seeding, the growth medium was removed and the wells were washed with PBS. Triplicate wells were treated for 1 h with 100 μL of palm tree extract at 10, 25, 50 and 100 µg/mL plus 50 μM DCFH-DA dissolved in treatment medium and kept in the dark. Quercetin was used as a reference standard. Each plate included triplicate control and blank wells: control wells contained cells treated with 50 µM of DCFH-DA; blank wells contained cells treated only with growth medium. Then the wells were drained from the treatment medium and 100 μL of AAPH at 250 µM was added in each well (excepted in blank wells). Immediately after the AAPH addition, the plate was placed into a plate-reader Dynex Magellan Biosciences  at 37 °C and the fluorescence was recorded during 30 min. Emission was measured at a wavelength of 538 nm with an excitation wavelength of 485 nm. After blank subtraction from the fluorescence readings, the area under the curve of fluorescence versus time was integrated to calculate the CAA value at each concentration of palm extracts tested as follows: CAA unit: 100 − (∫SA/∫CA) × 100 where ∫SA is the integrated area under the sample fluorescence versus time curve and ∫CA is the integrated area from the control curve. The median effective dose EC50, was determined for palm extracts from the median effect plot of log (ƒa/ƒu) versus log (concentration), where ƒa is the fraction affected and ƒu is the fraction unaffected by the treatment. The EC50 values were expressed in μg/mL. The EC50 of palm extracts were then converted in µmol Quercetin Eq/g DM and μmol Quercetin Eq/100g of FW using the equation log (ƒa/ƒu) = f (log(concentration)) for comparison with literature data.As all palm extracts were not toxic or weakly toxic to NHDF cells at a concentration of 100 µg/mL, this concentration was therefore chosen as the maximum concentration for the CAA assay using NHDF cells. The CAA assay was done according to Wolf and Liu  with somp < 0.05.Results were presented as the mean of three biological replicates and standard error (SE) or standard error of the mean (SEM). Comparisons between palm extracts in the CAA, ORAC, FRAP and DPPH assays were performed using an ANOVA followed by multiple comparisons using Fisher’s least significant difference test. Differences were considered to be significant when O. bataua and O. bacaba, and to a lesser extent root extracts, were highly potent in the chemical antioxidant assays  and this translated well to the cellular antioxidant assay with Normal Human Dermal Fibroblasts. These results suggest that extracts were able to work through a mechanism of free radical scavenging in a cellular environment. Oenocarpus leaflets were characterized by a complex flavonoid pattern, composed mainly of C-glycosyl apigenin derivatives, which are quite rare among Arecaceae species and may be used as phytochemical markers of Oenocarpus. Hydroxycinnamic acids were identified in both organs, as caffeoylquinic acids in leaflets, as caffeoylquinic and caffeoylshikimic acids in root extracts. CQA and flavones were the main drivers of antioxidant activity in the Oenocarpus roots and leaflets, respectively. The antioxidant activity of leaflet extracts was very promising, being at least as high as E. oleracea berries known as superfruit. These extracts could be valorized as a new non-cytotoxic source of antioxidants for dermatological or pharmaceutical applications.Leaflet extracts of


Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by impairment of memory function. The 5XFAD mouse model was analyzed and compared with wild-type (WT) controls for aberrant cortical excitability and hippocampal theta oscillations by using simultaneous video-electroencephalogram (EEG) monitoring. Seizure staging revealed that 5XFAD mice exhibited cortical hyperexcitability whereas controls did not. In addition, 5XFAD mice displayed a significant increase in hippocampal theta activity from the light to dark phase during nonmotor activity. We also observed a reduction in mean theta frequency in 5XFAD mice compared to controls that was again most prominent during nonmotor activity. Transcriptome analysis of hippocampal probes and subsequent qPCR validation revealed an upregulation of Plcd4 that might be indicative of enhanced muscarinic signalling. Our results suggest that 5XFAD mice exhibit altered cortical excitability, hippocampal dysrhythmicity, and potential changes in muscarinic signaling. In addition to the presence of Aβ plaques and NFTs in the brain, considerable neuron loss is also a prominent feature of AD, the mechanisms of which still remain unclear. Importantly, familial AD (FAD) mutations in genes for amyloid-β precursor protein (AβPP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2) implicate Aβ as an initiating factor in AD pathogenesis. These FAD mutations increase the release of Aβ42 from AβPP, the latter being sequentially cleaved by the β- and γ-secretase enzymes to release the Aβ. The latter plays a central role in the pathophysiology of AD and ultimately leads to neuronal cell loss, for example, in the septum, entorhinal cortex, and hippocampus [Alzheimer's disease (AD) is an irreversible, progressive brain disorder slowly destroying learning and memory skills. The histopathology of AD is characterized by two hallmark lesions, extracellular amyloid-pocampus , 4.β-site APP cleaving enzyme 1 (BACE1) that is responsible for the generation of Aβ plaques [β42 production, that is, three human APP mutations  and two mutant PSEN1 . Individually, each FAD mutation enhances Aβ42 generation, but together they act synergistically to predominantly accumulate Aβ42. Thus, 5XFAD mice already exhibit intraneuronal Aβ accumulation at 1.5 months, Aβ deposition at 2 months, and memory deficits at 4 months of age [β plaques [Transgenic mouse models have proven to be valuable tools in investigating the etiopathogenesis of AD –11. Stud plaques –14. Ther plaques , 16. Amos of age , 17–20.  plaques , 21, 22. plaques , 23; the plaques . It is nIntense efforts were carried out to characterize the transcription and expression profile in 5XFAD mice compared to WT. Using quantitative mass spectrometry to investigate proteome-wide changes in 4-month-old 5XFAD mice , alteratβ level and the appearance of plaques [β-induced neuronal hyperexcitability [Although neuropathological changes in AD have been well described previously, direct effects on systemic electrophysiological alterations have received less attention in the past. Currently, the impact of brain oscillation analysis as a novel tool in early diagnosis and prediction of disease progression is strongly discussed as functional impairments in AD can occur even without any significant neuronal loss and therefore could be independent of plaque formation –31. Rece plaques , 33, but plaques , 35. Alt plaques , 36. Thetability –44. Neurtability –53. Intetability , 54–57 wtability . The reaIn this study we combined seizure analysis, time-frequency-based theta analysis, and transcriptome data to elicit systemic cortical and hippocampal network alterations in the 5XFAD model of AD. PSEN1 . The 5XFAD mice were obtained from The Jackson Laboratory  [This study was performed in Tg 6799Vas (5XFAD) transgenic mice with a B6/SJL background overexpressing mutant forms of human APP  and mutantns, USA) . Five WTAll animal procedures were performed according to the Guidelines of the German Council on Animal Care and all protocols were approved by the Local Institutional and National Committee on Animal Care . The authors further certify that all animal experimentation was carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC). Specific effort was made to minimize the number of animals used and their suffering.http://dx.doi.org/10.1155/2015/781731). Electrodes were fixed using glass ionomer cement  and the scalp was closed using Over and Over sutures . To avoid hypothermia, supplemental warmth was given to the animal during the whole surgical procedure. A detailed description of the implantation procedure is given in [Mice were anesthetized using ketamine/xylazine (100/10 mg/kg i.p.) and the radiotelemetry transmitter ; specifications: weight 3.9 g, volume 1.9 cc, input voltage range ± 1.25 mV, and channel bandwidth 1–50 Hz) was implanted into a subcutaneous pouch on the back of the animals. The EEG electrodes of both transmitter channels were stereotaxically positioned using a computerized 3D stereotaxic StereoDrive . The differential epidural electrode of channel 1 targeting the primary motor cortex (M1) was positioned at the following coordinates referring to the bregma craniometric landmark: (+)-lead, cranial +1 mm, and lateral of bregma 1.5 mm (left hemisphere). For deep, intracerebral brain recordings targeting the hippocampal CA1 region the differential electrode of channel 2 was positioned as follows: (+)-lead, caudal −2 mm, lateral of bregma 1.5 mm (right hemisphere), and dorsoventral (depth) 1.5 mm. For both channels, epidural reference electrodes were placed on the cerebellar cortex at bregma −6 mm, lateral of bregma 1 mm (left hemisphere), and bregma −6 mm, lateral of bregma 1 mm (right hemisphere), respectively  and hematoxylin-stained for visualization of the branch canal. Two controls and one 5XFAD mouse did not meet strict EEG electrode placement criteria and were thus excluded from the analysis.Seven controls and six 5XFAD mice were originally implanted. To verify the correct electrode placement targeting the CA1 region, brains were extirpated postmortem and fixed in 4% paraformaldehyde. Afterwards, brains were cut to 60 Ten days after radiotransmitter implantation, simultaneous video-EEG recordings from the motor cortex (M1) and the hippocampal CA1 region were performed for 48 h in all animals from both study groups using Dataquest ART 4.2 software (DSI) at a sampling rate of 500 Hz with no a priori filter cut-off.μV for the dynamic range. In case of the absolute threshold the maximum amplitude was fixed at 1000 μV with a threshold value of 200 μV. In both seizure protocols the minimum spike duration was determined at 1 ms and the maximum spike and short/slow-wave duration at 100 ms. Spike trains were detected with a minimum train duration of 0.5 s including at least 4 individual spikes. Spike intervals within a train were ranging between 0.05 and 0.3 s. The interval between individual spike trains was determined at 1 s. Seizure protocols contained the total number of seizure episodes and spikes, spike frequency, total spike train duration, shortest and longest spike train durations. Data were calculated and plotted throughout the figures as mean ± standard error of the mean (SEM). Significance was calculated using Student's t-test which included pretesting for normal distributions via the Kolmogorov-Smirnov test.For further analysis data were exported to NeuroScore 2.1 (DSI). Qualitative and quantitative seizure analysis was performed using the NeuroScore seizure detection module. Spike parameters including dynamic and absolute threshold, spike duration, and spike intervals were adapted for different seizure protocols. Dynamic thresholds were based on multiplication of the root mean square (RMS) values of the EEG signals one minute prior to EEG segments being analyzed. The threshold ratio (minimum threshold) was defined as 2 while the maximum ratio was defined as 15. The minimum amplitude value of spikes was 100 Urethane  was freshly dissolved in 0.9% NaCl and systemically (i.p.) administered at 800 mg/kg to induce atropine-sensitive type II theta oscillations. Four control mice from the original study group  and four 5XFAD animals from the original study group  were used for this approach. CA1 recordings under baseline conditions (30 min duration) and under urethane (30 min duration 15 to 45 min after injection) were used for analysis of urethane induced hippocampal theta oscillations.b is the bandwidth parameter, c the center frequency, and i the imaginary unit [Complex EEG analysis was performed for spontaneous 48 h recordings at a sampling rate of 500 Hz. Data segments with a length of 60 min each were extracted from the 48 h total recordings. Data segments were analyzed using complex Morlet wavelets to calculate both frequency and amplitude of oscillations. The complex Morlet wavelet is defined as ary unit . In our ary unit .Amax: maximum amplitude):Theta-positive oscillatory segments of 2.5 s duration were defined as follows (activity . Some exμg) and subsequently hybridized to the arrays before scanning in a GeneChip 3000 7G scanner (Affymetrix). Normalization to the median of all samples, background correction, and statistical analysis were performed with GeneSpringGX software (Agilent technologies). An implemented GC-RMA algorithm was applied on all chips to summarize probe level information. Microarray data were analyzed using unpaired t-tests. An uncorrected significance level P < 0.05 was adopted in all instances. Differentially regulated transcripts with a fold change (FC) greater than 1.6 were then subject to hierarchical clustering analysis in order to visualize gene expression changes across groups. Array data are available in the GEO database under GSE50521. DAVID [Total RNA (250 ng) was prepared from the hippocampi of three independent control mice  and three independent 5XFAD animals  that were not involved in any other experimental procedures before using RNeasy Lipid Tissue Mini Kit (Qiagen). Microarray experiments were carried out using Mouse Exon ST arrays (Affymetrix). Arrays were washed and stained according to the manufacturer's recommendations. Labeled and purified cDNA was fragmented  was carried out using anchored-oligo(dt)18 and hexamer primer in a two-step RT-PCR approach  and qPCR reaction protocol was based on LightCycler 480 SYBR Green I Master (Roche). The qPCR was performed in a Light Cycler 480 System (Roche) thermocycler. The following cycler protocol was used for all primer pairs : 95°C  and between-subjects factor, that is, genotype (control mice versus 5XFAD mice) was applied. The Simes-Hochberg “step-up” procedure was used to correct for multiple testing, if necessary [P-level of 0.05.Further statistical analyses concerning duration, frequency, amplitude of theta segments, and comparisons between groups and experimental conditions were done with IBM SPSS Statistics, Version 22 . The Kolmogorov-Smirnov test was used to test for normal distributions. Student's ecessary . This prn = 5 v. 35.32 ± 2.40 g, n = 5, df = 8, t = 3.757, and P = 0.006). The same holds true for a characteristic clasping phenotype involving a simultaneous retraction of both fore- and hindpaws [As reported previously, 5XFAD mice exhibited a reduced body weight in comparison to their WT littermates  and WT (n = 5) mice  EEG recordings were obtained in 5XFAD ( 5) mice . EEG rec 5) mice II wherea 5) mice I. Video n = 5) and WT mice (n = 5) was analyzed using simultaneous video-EEG recordings for a total duration of 48 h  on hippocampal tissue revealed 1421 transcripts differentially regulated between 5XFAD mice and WT controls. The statistical analysis applied an uncorrected significance level of δ dependent pathway [+ channels as well as Kcnma1 K+ channels were not changed in transcription. Interestingly, microarray analysis suggested potential alterations in the muscarinic signal transduction pathway including Plcb1, Plcd4, Prkcb, Cacna1e, and Cacna2d1 that might be relevant for theta oscillations [We performed qRT-PCR on hippocampi from three independent WT controls and three independent 5XFAD mice to determine gene expression changes. Among the differentially expressed genes in the microarray assay, we chose eight genes for qRT-PCR analysis based on their involvement in the theta-genesis pathway. The selected genes and their qPCR primers are listed in  pathway . The Scnllations . ValidatPrior to analysis of the hippocampal oscillatory behavior, we analyzed motor activity in control and 5XFAD mice as active exploratory behavior is associated with a different type of theta entity as compared to, for example, alert immobility . MovemenAs expected, however, there was an increase in motor activity from light to dark phase in both genotypes Figures . Based ot = 1.928, P = 0.090, t = 3.189, P = 0.013, To determine whether hippocampal theta oscillations were indeed altered in 5XFAD mice, we performed spontaneous 48 h video-EEG recordings from the CA1 region of the hippocampus from both controls and 5XFAD mice. Using a time-frequency approach, theta duration, theta frequency, and theta amplitude were calculated. First, theta duration was analyzed for both motor and nonmotor activity. No significant changes were observed for controls compared to 5XFAD mice during the total observation period ). The same holds true for the nonmotor activity ) and nonmotor phase. This finding correlates with previous observations in 5XFAD but also TgCRND8 mice [n = 4) and WT mice (n = 4). Pharmacodynamically, urethane has a multitarget character capable of inducing atropine-sensitive type II theta. Theta oscillations were analyzed for 30 min baseline and 30 min postinjection episodes (n = 4) as in 5XFAD mice  and the factor “experimental condition/urethane effect” exhibited significance  = 7.270, P = 0.036). The “genotype” factor exhibited a statistical trend  = 5.256, P = 0.062, F = 7.691, P = 0.032) with increase in controls  and 5XFAD mice . The factor “genotype” exhibited a statistical trend  = 5.148, P = 0.064, F = 13.536, P = 0.010) on theta oscillation frequency for the total analytical period (6.57 ± 0.61 Hz to 5.48 ± 0.36 Hz (n = 4) in controls versus 6.18 ± 0.31 Hz to 5.11 ± 0.33 Hz (n = 4) in 5XFAD mice, F = 8.036, P = 0.030) also holds true for the nonmotor episodes (6.60 ± 0.63 Hz to 5.44 ± 0.35 Hz (n = 4) in controls versus 6.14 ± 0.53 Hz to 5.15 ± 0.36 (n = 4) in 5XFAD mice (Besides analysis of spontaneous theta activity, we also investigated urethane induced theta oscillations in 5XFAD (episodes . The durFAD mice .β formation in certain mouse models can differentially alter cholinergically induced rhythmicity according to the structure of Aβ plaques [βPP [β release [βPP signaling is important for the development of hippocampal excitability [β as a pathogen [βPP [β are cytotoxic inducing the appearance of aberrant excitatory neuronal network activity in vivo and triggering complex molecular and cellular patterns of compensatory inhibitory and excitatory mechanisms in the hippocampal circuitry [Alzheimer's disease is a complex neurodegenerative disorder accompanied by cognitive impairment that ultimately leads to dementia. In the present study, we investigated transcriptional alterations and the EEG phenotype of 5XFAD mice. This mouse model harbors five early-onset FAD mutations and displays substantial amyloid plaques and neurodegeneration , 23. Our plaques  and ther plaques . Experim plaques . Reduced plaques –76. Inteues [βPP  and BACE release , also catability . In somepathogen . Chronicgen [βPP  revealedgen [βPP , 31. In gen [βPP , 31, 79.gen [βPP , 81. Congen [βPP , 82. Intgen [βPP . The mulircuitry , 85.β peptides underlying AD triggers synaptic degeneration, circuit remodeling, and abnormal synchronization within the same networks. Because neuronal hyperexcitability amplifies the synaptic release of Aβ, seizures create a vicious spiral that accelerates cell death and cognitive decline in the AD brain [The toxic accumulation of AAD brain . While dHow can seizure activity correlate with altered theta oscillations in the hippocampus? Complex cognitive operations depend on a sophisticated coordination of activity across a plethora of neuronal groups. One of the most intriguing mechanisms of neuronal coordination and communication is through neuronal synchronization by brain oscillations . Theta ov2.3 voltage-gated Ca2+ channels are involved in both the generation of cellular correlates of ictal discharges, that is, after depolarisation and plateau potentials [v2.3 cascade [Previous studies have shown that the muscarinic signal transduction cascade  is severtentials , 95, andtentials , 96, 97. cascade ; howeverOur study demonstrates that 5XFAD mice exhibit both nonconvulsive seizure activity and altered theta architecture, the latter being related to atropine-sensitive type II theta. Based on hippocampal microarray analysis we hypothesize that altered muscarinic signaling might play a role in the pathophysiology of both alteration. Although some caution is warranted in interpreting results from the aggressive 5XFAD model, our results support the view that pharmacological interference with muscarinic signaling is a potential valuable target in AD treatment. Finally, our study suggests that alterations in theta characteristics might serve as a diagnostic and prognostic biomarker in AD in the future.Methodological aspects of stereotaxic EEG electrode placement, analysis of theta amplitude, and microarray analysis in WT and 5XFAD mice.