This screening system lays the foundation for high-throughput discovery of novel ligands to the cell surface.The screening of living cells using high-throughput microarrays is technically challenging. Great care must be taken in the chemical presentation of potential ligands and the number of collisions that cells make with them. To overcome these issues, we have developed a glass slide-based microarray system to discover small molecule ligands that preferentially bind to one cell type over another, including when the cells differ by only a single receptor. Chemical spots of 300 ± 10 μm in diameter are conjugated covalently to glass slides using an arraying robot, and novel near-infrared fluorophores with peak emission at 700 nm and 800 nm are used to label two different cell types. By carefully optimizing incubation conditions, including cell density, motion, kinetics, detection,  Extracellular membrane (ECM) receptors are of significant importance to the development of new therapeutic agents, being the molecular targets for more than 60% of clinical drugs ,2. To diA simple alternative approach is direct panning of living cells over ligand-spotted microarrays, where each spot is a single, defined chemical entity. We have previously described functionalized microarray slides capable of rapid and high-throughput screening of over 5000 different chemical compounds binding to living bacteria, including quantitation of binding parameters ,20,21. IN‑hydroxysuccinimide modified polyethylene glycol (PEG-NHS) for both conjugation of potential ligands and presentation of ligands far from the glass surface pentane-1,5-dioic acid (GPI), 2-(3-(5-amino-1-carboxypentyl)ureido)pentanedioic acid (KUE), beta Ala-Gly (β-AG) and all trimeric ligands were in house compounds, which were synthesized as previously described [i.e., 200-μm of clear space between any two spots). After incubating for 3 h at room temperature in air, the slides were immersed in deionized water to deactivate unreacted NHS groups. After washing with ethanol, the slides were dried under a nitrogen stream and stored in a dust free environment before use.All chemicals were American Chemical Society grade or higher and commercially available unless noted otherwise. We previously provided details on the microarray slide, which uses  surface . All ligescribed ,23. The escribed  using a 2 at 37 °C. Human prostate cancer cells, LNCaP and PC3, were cultured in RPMI supplemented with 10% FBS and 100 units/mL P/S under 10% CO2 at 37 °C. All cells were plated into 100-mm culture dishes  in 10 mL of pre-warmed culture media. When the cells reached 70%–80% confluence at a density of 2–5 × 106 cells/dish, either ESNF10 (700 nm NIR fluorophore) [vs. 30 rpm), incubation time (30–180 min), presented ligand concentration at the time of spotting (0.1–1.0 mM), and applied cell density (0.2 × 106−8 × 106 cells), were systematically optimized. After incubation, the slides were gently washed with cell culture media before scoring.Melanoma cell lines including M21, M21-L, and B16 were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin/streptomycin (P/S) under 5% COrophore)  or IR786Living cells bound to chemical spots were imaged using a Nikon TE2000 epifluorescence microscope equipped with a 75 W Xenon light source and an Orca-ER  camera ,26. Two vβ3-positive M21 cells (positive control) labeled with the 700 nm NIR fluorophore ESNF10 and integrin αvβ3-negative M21-L cells (negative control) labeled with the 800 nm NIR fluorophore IR786 were panned over the surface of our SMM  being defined by the PAAm control spots .To validate the assay, integrin α our SMM A. PAAm,  our SMM B. Thus tResults of the assay using the integrin-binding peptide cRGDyK as the ligand spot are shown in 6 cells per slide. To maximize the specificity of binding, we compared incubation in the presence or absence of 10% serum, and with or without negative control cells  and are shown in 4 integrin αvβ3 receptors per cell  on their surface, while M21-L cells have no detectable integrin αvβ3 receptors (1 × 103) [vβ3 has a type I transmembrane topology and binds the cyclic peptide ligand cRGDyK with an affinity of approximately 50 nM. LNCaP cells have approximately 2 × 105 prostate-specific membrane antigen (PSMA) receptors per cell on their surface, while PC3 cells have no detectable PSMA [3 melanocortin 1 receptors (MC1R) receptors per cell on their surface, while LNCaP cells have no detectable MC1R [6 panned cells per slide), all three cell-ligand interactions are detectable with our SMM screening system with relatively high specificity and with sensitivity proportional to Bmax.To explore the usefulness of our SMM screening system, we tested three cell-ligand interactions, which all differed in terms of cell type, receptor type, ligand type, and B1 × 103) . Integrible PSMA . PSMA hable MC1R . MC1R ismax, motion might not only be possible but could improve specificity by reducing non-specific interactions. Similarly, we only explored the presence or absence of 10% serum, but in some model systems, a higher or lower concentration could be optimal.It should be noted, however, that many of the system parameters are inter-dependent and should be re-optimized for a particular model system. For example, in the presence of extremely high affinity and BKD), and therefore the ratio of affinity to Bmax, might have a profound impact on the sensitivity of the SMM screening assay. To explore this relationship we utilized a series of PSMA ligands previously reported by our group (KD = 2 µM), GPI (KD = 9 nM), β-AG trimer (KD = 60 nM), and GPI trimer (KD = 0.4 nM). The SMM was probed with PSMA-positive cells (LNCaP) and PSMA-negative cells (PC3), using the optimized parameters described for max is low, even high affinity interactions may result in only a few cells bound per spot [The results from ur group A, which per spot ,31.We have developed a SMM glass slide-based system for the rapid and efficient screening of ligands that bind to the surface of living mammalian cells. If careful attention is paid to critical parameters, the system appears to function well over a wide range of ligand affinities, receptor topologies, and cell types of interest. It is hoped that this system can be applied to the screening of diverse and complex chemical libraries to find lead candidates for improved diagnostic and therapeutic drugs.


Traditional Chinese medicines (TCMs), as an important part of natural products, have been reported to exert protective effect against oxidative stress in mitochondria. In this study, we screened SIRT1 activators from TCMs and investigated their activities against mitochondrial damage. 19 activators were found in total by in vitro SIRT1 activity assay. Among those active compounds, four compounds, ginsenoside Rb2, ginsenoside F1, ginsenoside Rc, and schisandrin A, were further studied to validate the SIRT1-activation effects by liquid chromatography-mass spectrometry and confirm their activities against oxidative damage in H9c2 cardiomyocytes exposed to tert-butyl hydroperoxide (t-BHP). The results showed that those compounds enhanced the deacetylated activity of SIRT1, increased ATP content, and inhibited intracellular ROS formation as well as regulating the activity of Mn-SOD. These SIRT1 activators also showed moderate protective effects on mitochondrial function in t-BHP cells by recovering oxygen consumption and increasing mitochondrial DNA content. Our results suggested that those compounds from TCMs attenuated oxidative stress-induced mitochondrial damage in cardiomyocytes through activation of SIRT1.Sirtuin type 1 (SIRT1) belongs to the family of NAD It has been reported that, in mammals, SIRT1 plays a critical function in cellular metabolism and response to oxidative stress [Sirtuin type 1 (SIRT1) belongs to the family of class III histone deacetylases (HDAC) that consume NADe stress .α and multiple transcription factors [Recently, researchers have found that SIRT1 activators can protect mitochondrial function from oxidative-induced mitochondrial damage in various types of cell through regulating PGC-1 factors –9, which factors , 11. It  factors , can ext factors –15. Ther factors . Astragalus membranaceus [ Panax ginseng [ Panax notoginseng [Natural products have historically been regarded as an important resource of therapeutic agents in pharmaceutical discovery over the past century . Traditiranaceus , 20, Pan ginseng , 22, andoginseng , 24 haveoginseng . However in vitro assay to screen SIRT1 activators was performed to discover bioactive compounds from TCMs, and the lead compounds were validated by liquid chromatography-mass spectrometry (LC-MS) analysis. Effects of identified SIRT1 activators on mitochondrial function were further investigated in cardiomyocytes exposed to tert-butyl hydroperoxide (t-BHP). ATP content, intracellular ROS formation, and activity of Mn-SOD were measured. Moreover, oxygen consumption and mitochondrial DNA content of cardiomyocytes were used to evaluate the effects of those SIRT1 activators on mitochondrial function.The aim of present study is to discover SIRT1 activators from TCMs and validate their activities against mitochondrial damage. A sensitive2 was purchased from National Institute for Food and Drug control .SIRT1 protein (human recombinant) and lysyl endopeptidase were purchased from Cayman Chemical (USA). Ginsenoside F1, ginsenoside Rc, and schisandrin A were purchased from Shanghai Winherb Medical Technology Company (China). Ginsenoside Rbμg/mL streptomycin . All the cells were grown in 5% humidified CO2 atmosphere at 37°C.H9c2  were cultured in DMEM  containing 10% fetal bovine serum , 100 U/mL penicillin, and 100 μM) in the presence of the tested compound (50 μM). Fluorescence intensity was recorded by a TECAN infinite F200 microplate reader with excitation wavelength 320 nm and emission wavelength 465 nm.The measurement of SIRT1 activity and effects of compounds on SIRT1 activation were performed by a previously reported fluorescent method . Brieflyhttp://dx.doi.org/10.1155/2016/4206392. The inhibition or activation of SIRT1 was calculated by the following equation:A total of 195 constituents of TCMs were screened by the assay to evaluate their regulatory effects on SIRT1 activity. The detailed information related to the chemicals and their CAS number were illustrated in Supplementary Material available online at Is and Ic represented the fluorescence intensity of tested sample group with various test compounds and control group without test compounds. Is0 and I0 represented the fluorescence intensity of tested sample and control when incubated without SIRT1 protein. To explore the dose-related effects of these compounds, several active compounds, including ginsenoside Rb2, ginsenoside Rc, and schisandrin A, were further tested with the concentrations of 1, 10, 25, and 50 μM.μg/mL) was added to TPE-GK(Ac)YDD (20 μM) and NAD+  for 1 h incubation at 37°C in the presence or absence of ginsenoside Rb2, ginsenoside F1, ginsenoside Rc, and schisandrin A (50 μM), respectively. Samples were boiled at 100°C for 10 min to degenerate SIRT1 and terminate reaction.In order to validate activation of SIRT1, SIRT1 . The acquisition parameters for LC/ESI-MS were as follows: nebulizing gas, high-purity nitrogen (N2); collision gas, high-purity helium (He); capillary voltage: −15 V; capillary temperature: 350°C; ion spray voltage: −3 kV; tube lens offset voltage: −30 V; mass range: m/z 100−1500. Chromatographic separation was performed by a reversed-phase ZORBAX SB-C18 analytical column. The mobile phase included water containing 0.1% (v/v) formic acid (A) and acetonitrile (B). The flow rate was 0.6 mL/min. A gradient program was carried out as the following profile: 0 min, 50% B; 5 min, 50% B; 30 min, 95% B; 40 min, 95% B.Samples were analyzed by Agilent 1100 LC system  and Finnigan LCQ Deca XP2, ginsenoside F1, ginsenoside Rc, and schisandrin A (20 μM) for 24 h before being exposed to t-BHP (300 μM) for 1 h. Intracellular ATP content was measured by CellTiter-Glo® Luminescent Assay kit (Promega) according to the instruction of manufacture. Intracellular ROS content was measured by DCFH-DA probe (5 μM) whilst fluorescence intensity was recorded by a TECAN infinite F200 Multifunction microplate with excitation wavelength 485 nm and emission wavelength 535 nm. The changes of ATP content and ROS accumulation were calculated by comparing the luminescent or fluorescent signal of the treated cells with that of untreated H9c2 cells.H9c2 cells were seeded in 96 wells at the density of 4,000/well. The cells were preincubated with ginsenoside Rb5/mL. H9c2 cells were preprotected for 24 h by ginsenoside Rb2, ginsenoside F1, ginsenoside Rc, and schisandrin A (20 μM) before being exposed to t-BHP (300 μM) for 1 h. The cells were lysed and the concentration of total protein was measured by BCA assay kit. Mn-SOD activity in total protein was measured by Mn-SOD assay kit .Manganese superoxide dismutase (Mn-SOD) was an antioxidative enzyme, which protected against ROS-induced damage . To meas6 H9c2 cells were seeded in 100 mm culture plate. After being grown to stable attachment, cells were preincubated with ginsenoside Rb2, ginsenoside F1, ginsenoside Rc, and schisandrin A at final concentrations (20 μM) for 18 h before being exposed to t-BHP (100 μM) for 1 h. Cells were washed with PBS twice, subsequently collected by trypsinization followed by centrifugation, and resuspended in fresh medium. Respiratory activity was measured with a Clark-type oxygen electrode . An aliquot (1 mL) of suspended cells (2 × 106 cells/mL) was placed in the air-tight liquid-phase oxygen electrode chamber. The system was maintained at 37°C. After equilibration, the slope of oxygen consumption in H9c2 cells was measured. Every 1 × 106 cells oxygen consumption was calculated as the basic respiration rate of each group. The experiment was also performed in the presence of SIRT1 inhibitor, EX527 at final concentration of 20 μM to investigate whether the effect of ginsenoside Rb2 can be prevented by SIRT1 inhibitor.2 × 102, ginsenoside F1, ginsenoside Rc, and schisandrin A at final concentrations (20 μM) before being exposed to t-BHP (300 μM) for 1 h. Cells in normal condition were used as control group. Total DNA was extracted using Mammalian Genomic DNA Miniprep Kits . DNA was quantified by measuring A260 values, and 50 ng of total DNA was used for PCRs by GenElute™ QuantiFast SYBR Green PCR Kit . Primers specific to the mitochondrial-encoded Atp6 gene  were used to assess mitochondrial DNA copy numbers. Primers designed against the nuclear-encoded Rpl13 gene  were used for normalization. CT values were obtained automatically. The number of mitochondrial genes was calculated by 2CT−ΔΔ method.Real-time PCR was used to determine relative quantities of mitochondrial DNA content in H9c2 cells exposed to t-BHP and cells incubated with SIRT1 activators. Cells were preincubated for 24 h with ginsenoside Rbp values of less than 0.05 were considered statistically significant.All values were expressed as the means ± SD. One way ANOVA was used to analyze differences among groups. Statistical analysis was performed using GraphPad Prism.  Panax ginseng and Ophiopogon japonicas were regarded as Tonifying herbs, whilst Schisandra chinensis was Astringent herb and Sophora flavescens came from heat-clearing medicinal. 3 (60%), ginsenoside Rb3 (28%), ginsenoside F1 (22%), and ginsenoside F2 (45%) from Panax ginseng, gypenoside XVII (43%) and notoginsenoside Ft1 (40%) from Panax notoginseng, polyphyllin I (24%), polyphyllin III (32%), polyphyllin VI (31%), and polyphyllin VII (32%) from Paris polyphylla, liriopesides B (65%) and Liriope muscari baily saponins C (57%) from Liriope muscari, ophiopogonin D′ (54%) from Ophiopogon japonicas, saikosaponin A (25%) from Bupleurum chinense, schisandrin B (30%) from Schisandra chinensis, and anisodine hydrobromide (60%) from Anisodus tanguticus. It was obvious that most of identified activators belonged to Tonifying herb. Moreover, potential inhibitors of SIRT1 almost belonged to herbs with heat-clearing efficacy.To screen SIRT1 activators, we chose 195 compounds from TCMs with different efficacy, which were defined according to the TCMs theory. For example,2 showed 8% to 152% activation with the concentration range of 1~50 μM. Ginsenoside Rc exerted 88% activation at the concentration of 50 μM. Schisandrin A showed 28% activation at the concentration of 50 μM.Representative compounds were chosen to validate their activation effects on SIRT1 activity. Ginsenoside Rbμg/mL) and NAD+ (3 mM) for 1 h. The reaction product was identified by LC-MS based on the molecular weight. As shown in 2, ginsenoside F1, ginsenoside Rc, and schisandrin A, the peaks of deacetylated product were significantly elevated  analysis was employed. The specific substrate of SIRT1, TPE-GK(Ac)YDD , was incelevated . Our fin2, ginsenoside F1, ginsenoside Rc, and schisandrin A attenuated the decrease of oxygen consumption. 2/mL/min), t-BHP treated cells (1.10 ± 0.25 nmol O2/mL/min), and ginsenoside Rb2 treated cells (1.43 ± 0.25 nmol O2/mL/min). The results suggested that these SIRT1 activators recovered the oxygen consumption rate in t-BHP injured cardiomyocytes. In the presence of SIRT1 inhibitor EX527 [2 was blocked, which indicated that the effect of ginsenoside Rb2 to recover the oxygen consumption rate was SIRT1 dependence  for 1 h, activity of Mn-SOD was decreased. Preprotection of cells by ginsenoside Rb2, ginsenoside F1, ginsenoside Rc, and schisandrin A enhanced the activity of Mn-SOD compared with t-BHP group.Mn-SOD was one of the antioxidative enzymes in mitochondria that assured mitochondrial oxidative stress resistance. As shown in 2, ginsenoside F1, ginsenoside Rc, and schisandrin A of mitochondrial protection or biogenesis, cells were injured by t-BHP after compounds preprotection, and then mitochondrial DNA content was analyzed. As shown in 2, ginsenoside F1, ginsenoside Rc, or schisandrin A significantly elevated the mitochondrial DNA content. Our findings suggested that these natural SIRT1 activators facilitated mitochondrial biogenesis.To verify the effect of ginsenoside Rb3, ginsenoside Rb2, ginsenoside Rb3, ginsenoside F1, and ginsenoside Rc from Panax ginseng, ophiopogonin D′ from Ophiopogon japonicas, and schisandrin A and schisandrin B from Schisandra chinensis. Interestingly, those herbs consisted of a traditional Chinese formula named Shengmai San, which have been clinically used for the treatment of coronary heart diseases [2, ginsenoside F1, ginsenoside Rc, and schisandrin A, were validated by LC-MS analysis and we found their effects against mitochondrial oxidative damage in further study. Our findings were in accordance with previous reports on other cell lines. Ginsenoside Rc was reported to suppress oxidative stress in HEK293T cells [2O2 in intestinal epithelial cells [In the present study, we screened and identified 19 SIRT1 activators, such as ginsenoside Rgdiseases , 31 and diseases , 33. Fou3T cells , whilst al cells .Mitochondrial dysfunction has been one of mechanisms in organ injuries and diseases due to its influence on ATP formation, metabolism, and apoptosis . Our fin2, ginsenoside F1, ginsenoside Rc, and schisandrin A, exerted significant activities against t-BHP induced oxidative damage in cardiomyocytes. Our findings provided useful evidence to illustrate the cardioprotective effects of TCMs with Tonification effects and led to a new insight into the scientific illustration of TCMs theory.In summary, we identified 19 SIRT1 activators from TCMs. Four active compounds, ginsenoside RbA total of 195 constituents of TCMs were screened by the assay to evaluate their regulatory effects on SIRT1 activity. The Supplementary Material illustrated the detailed information related to the chemicals and their CAS number. And the Supplementary Material also provided the orders, activation/inhibition rates and function of 195 constituents in the heatmap of Figure 1.


A novel variant strain of porcine epidemic diarrhea virus (PEDV) emerged on pig farms in South Korea during late 2013. Genomic DNA isolated from a K14JB01 strain identified in a diarrheal pig showed high sequence similarity to PEDV strains prevailing in the United States in 2013. This is the first study to identify the complete genome sequence of a novel variant PEDV in South Korea. Coronaviridae; it is an enveloped, single-stranded RNA virus that causes enteritis, vomiting, and watery diarrhea (–Porcine epidemic diarrhea virus (PEDV) belongs to the family diarrhea . PEDV wadiarrhea , 3, and diarrhea –7. In 20iarrhea –. FrequenIn January 2014, fecal samples were collected from pigs with diarrhea in Gyeonggi Province, South Korea, and viral RNA was extracted using the RNeasy Minikit  according to the manufacturer’s instructions. cDNA was prepared using an OneStep reverse transcription (RT)-PCR kit . Twenty sets of primers were designed (based on the USA/Iowa/18984/2013 strain) to cover the full-length PEDV genome. The amplified PCR products were cloned into the pGEM-T Easy Vector  and then sequenced at the Cosmogentech Institute using an ABI Prism 3730xi DNA sequencer. All fragments were then assembled and edited using Clustal X 1.83  in length, excluding the 3′ poly (A) tail, and is arranged as follows: a 5′ untranslated region (UTR) comprising 292 nt, an open reading frame 1a (ORF1a) and ORF1b encoding a replicase of 20,345 nt, a spike (S) gene comprising 4,161 nt, an ORF 3 comprising 675 nt, an envelope (E) gene comprising 231 nt, a membrane (M) gene comprising 681 nt, a nucleocapsid (N) gene comprising 1,326 nt, and a 3′ UTR comprising 333 nt.The complete genome of PEDV strain K14JB01 showed high nucleotide sequence homology (99.7 to 99.8%) with variant U.S. strains  identified in 2013, and with Chinese strains  identified in 2011 and 2012 (99.1 to 99.4%). However, K14JB01 showed relatively low sequence homology (96.3 to 97.6%) with Korean vaccine strains (SM98 and DR13) and the CV777 strain.The ORF1a and ORF1b genes of K14JB01 show 99.8% homology (at the nucleotide level) with those of USA/Iowa/16465/2013; in addition, the S genes are 99.6% similar, the ORF3 genes are 100% identical, the M genes are 99.9% similar, and the N genes are 99.9% similar.In summary, PEDV strain K14JB01 identified from a diarrheal pig in South Korea shows high nucleotide sequence homology with variant strains circulating in the United States and China. The complete genome sequence data of K14JB01 will be the basis of our understanding of the genomic characterization and will contribute to the elucidation of PEDV in South Korea.KJ623926.The complete genome sequence of PEDV strain K14JB01 was deposited in GenBank under accession no.


T, RITF807 and RITF211, isolated from root nodules of Acacia melanoxylon grown in Ganzhou city, Jiangxi Province, China, had been previously defined, based on amplified 16S rRNA gene restriction analysis, as a novel group within the genus Bradyrhizobium. To clarify their taxonomic position, these strains were further analysed and compared with reference strains of related bacteria using a polyphasic approach. According to 16S rRNA gene sequence analysis, the isolates formed a group that was closely related to ‘Bradyrhizobium rifense’ CTAW71, with a similarity value of 99.9 %. In phylogenetic analyses of the housekeeping and symbiotic gene sequences, the three strains formed a distinct lineage within the genus Bradyrhizobium, which was consistent with the results of DNA–DNA hybridization. In analyses of cellular fatty acids and phenotypic features, some differences were found between the novel group and related species of the genus Bradyrhizobium, indicating that these three strains constituted a novel group distinct from any recognized species of the genus Bradyrhizobium. Based on the data obtained in this study, we conclude that our strains represent a novel species of the genus Bradyrhizobium, for which the name Bradyrhizobium ganzhouense sp. nov. is proposed, with RITF806T ( = CCBAU 101088T = JCM 19881T) as the type strain. The DNA G+C content of strain RITF806T is 64.6 mol% (Tm).Three slow-growing rhizobial strains, designated RITF806 Acacia melanoxylon R. Br.) has its origin in the temperate forests of eastern Australia but it is a versatile and highly adaptive tree species that occurs naturally across a wide range of Australian forest ecosystems , glnII (~680 nt) and atpD (~530 nt), with the amplifying primers and conditions described by nodC (~900 bp) and nifH (~800 bp) using primer pairs nodCF540/nodCR1160 and nifHF/nifHR and the protocol of The three test strains and reference strains were obtained from the Research Institute of Tropical Forestry and the State Key Laboratory of Agrobiotechnology. They were maintained on YMA medium  at 4 °C mega 5.1.All PCR products were sequenced with a BigDye terminator v3.1 kit using an ABI-PRISM 3730 Genetic Analyzer (ABI) with protocols recommended by the manufacturer. Gene sequences of type strains were obtained from the GenBank database. Neighbour joining (NJ) and maximum-likelihood (ML) phylogenies were inferred with T, RITF807 and RITF211 represented a member of the genus Bradyrhizobium and had 99.9 % sequence similarity to ‘Bradyrhizobium rifense’ CTAW71T  indicated that strains RITF806 CTAW71T , Fig. 1.recA, glnII and atpD). For each of the three genes, the phylogenetic relationships were consistent between the NJ and ML trees (data not shown). In the multilocus sequence analysis (MLSA), the ML tree reconstructed on the basis of the combined sequences of the three housekeeping genes , and ‘B. rifense’ CTAW71T for the glnII gene (97.6 % similarity) and for the atpD gene (96.2 % similarity) (T was most similar to ‘B. rifense’ CTAW71T and B. diazoefficiens USDA 110T according to the MLSA (95.8 % similarity).The three strains were found to have nearly identical sequences for three housekeeping genes (ng genes  had the ng genes . The seq species . The mosilarity) . The novnodC and nifH genes, the novel strains examined in the present study always formed a distinctive lineage that differed from all defined species. In the nodC phylogenetic tree , the three test strains had identical sequences, which were most similar to that of Mesorhizobium sp. CCBAU 33477 (79.5 % similarity), which had been isolated from effective nodules of Astragalus sinicus in Southern China (data from GenBank). For the nifH gene, the highest similarity (around 90 %) was to Bradyrhizobium lablabi CCBAU 23086T , isolated from root nodules of Lablab purpureus and Arachis hypogaea grown in the Anhui and Sichuan provinces of China . However, they can provide valuable information about rhizobial species . The DNA–DNA relatedness of RITF806T to RITF807 and RITF211 was 85.23 and 78.79 %, respectively, indicating that both strains belong to the same species. The DNA–DNA relatedness between RITF806T and reference strains Bradyrhizobium cytisi CTAW11T, B. huanghuaihaiense CCBAU 23303T, B. diazoefficiens USDA 110T and ‘B. rifense’ CTAW71T were 58.11, 54.61, 53.45 and 51.89 %, respectively , respectively, which is within the range for members of the genus Bradyrhizobium.The G+C content of DNA was determined by the thermal denaturation method . The valT were assayed together with those of B. cytisi CTAW11T, B. diazoefficiens USDA 110T and ‘B. rifense’ CTAW71 in order to examine differences between the novel strain and closely related species. The strains were cultured aerobically on YMA medium at 28 °C and cells were collected during the late-exponential phase of growth. Fatty acid methyl esters were prepared and separated using the method described by T , but the percentages of these fatty acids varied .The polar lipid profiles of cells of strain RITF806Bradyrhizobium with validly published names.Phenotypic characterization of the three test strains in this study was based on the API 20NE kit (bioMérieux), according to the manufacturer’s instructions, using YM-minus-mannitol as the basal medium. Carbon-source utilization was determined using a Biolog GN2 microplate , according to the manufacturer’s instructions. Tolerance to dyes, antibiotics and sodium chloride, and other characteristics were assessed as described by Bradyrhizobium ganzhouense sp. nov.The newly isolated strains can be differentiated genotypically and phenotypically from previously described species and we therefore propose naming the new group Bradyrhizobium ganzhouense .d-fructose, α-d-glucose, d-mannitol, d-mannose, citric acid, methyl pyruvate, d-galactonic acid lactone, d-galacturonic acid, d-gluconic acid, dl-lactic acid, d-saccharic acid, succinic acid, d-alanine and glycerol as carbon sources. Does not grow on d-glucose, maltose, l-rhamnose, N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, i-erythritol, myo-inositol, α-lactose, lactulose, melibiose, raffinose, sucrose, xylitol, inosine, uridine or thymidine. Resistant to the following antibiotics (μg ml−1) erythromycin (100), kanamycin (100), neomycin sulfate (50), streptomycin (5), chloramphenicol (300), gentamicin (5) and totomycin (100). Summed feature 8 (C18 : 1ω6c/C18 : 1ω7c) and C16 : 0 are the dominant fatty acids. The polar lipid profile contains aminolipid, diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol. Can form effective nodules on its original host plant Acacia melanoxylon and other species including Acacia aneura, Acacia victoriae and Acacia implexa, but not Medicago sativa, Pisum sativum, Trifolium albus or Vigna unguiculata.Cells are Gram-negative, aerobic, non-spore-forming rods, 1.29–3.32 µm long and 0.50–0.60 µm wide. Colonies are circular, convex and translucent, 1–2 mm in diameter within 7 days of growth at 28 °C on YMA medium. Grows at pH 5–12, with optimum growth at pH 7.0. Growth occurs at 4 °C, 10 °C, 28 °C and 37 °C . Grows on YMA in the presence of 3 % NaCl. Cannot tolerate 60 °C for 10 min on YMA. No growth in Luria–Bertani broth. Positive for catalase, oxidase and urease production. Nitrate reduction, Nile blue reduction and methylthionine chloride reduction are negative. Uses Tween 80, T ( = CCBAU 101088T = JCM 19881T), was isolated from effective nodules of Acacia melanoxylon R. Br. Its DNA G+C content is 64.6 mol% (Tm).The type strain, RITF806


AbstractPageBreakCalcaridorylaimus Andrássy, 1986 was collected from the litter of broadleaf forests dominated by Castanea sativa Mill. and mixed with Quercus daleshampii Ten. and Fagus sylvatica L. on Belasitsa Mountain, south-western Bulgaria. Calcaridorylaimus castaneaesp. n. is characterised by its long body (1.4–2.1 mm), lip region practically not offset, vulva transverse, short odontostyle (14.5–16 μm) and tail  in females and 38–46 μm long spicules with small spur before their distant end in males. It is most similar to C. andrassyi Ahmad & Shaheen, 2004, but differs in having transverse vs pore-like vulva and shorter spicules (38–46 μm vs 52–57 μm). An identification key to the species of the genus Calcaridorylaimus is proposed. Phylogenetic analyses were performed on 18S and D2-D3 expansion domains of 28S rRNA genes by Neighbor-Joining, Maximum Likelihood and Bayesian Inference methods. The phylograms inferred from 18S sequences showed closest relationships of the new species with some species belonging to the genus Mesodorylaimus. However, insufficient molecular data for members of both genera do not allow the phylogenetic relationships of Calcaridorylaimus and the new species described herein to be elucidated.An unknown species belonging to the genus Calcaridorylaimus Andrássy, 1986 was recovered. The genus Calcaridorylaimus is represented by nine species worldwide: Calcaridorylaimus calcarifer Andrássy, 1986, Calcaridorylaimus promissus Andrássy, 1986, Calcaridorylaimus ruwenzorii  Andrássy, 1986, Calcaridorylaimus signatus  Andrássy, 1986, Calcaridorylaimus simillimus Andrássy, 1986, Calcaridorylaimus sirgeli Heyns & Meyer, 1995, Calcaridorylaimus arcticus Gagarin, 1997, Calcaridorylaimus andrassyi Ahmad & Shaheen, 2004 and Calcaridorylaimus beatus Andrássy, 2011. The genus is distributed mainly in the southern hemisphere: three species occur in Africa, two in South America and one each in Antarctic, Central America, and Europe, and Calcaridorylaimus promissus was recorded from Australia and Alaska, North America  an undescribed species belonging to the genus  America , 2003 . Subsequently, on 17.10.2012 new litter samples were collected by Dr Michaela Ilieva from one of these sites, sub-compartment 140b, in order to obtain fresh material for molecular studies. Nematodes were recovered from the litter using the Baermann funnel method. They were killed by heat (65 °C), fixed in TAF  from three sites on Belasitsa Mountain representing different types of broadleaf forests dominated by Genomic DNA was extracted from two female and two male worms using a standard nematode digestion protocol . Two ovePageBreakKF717497 and KF717498 for the 18S and the D2-D3 rRNA genes, respectively. A BLAST  search at NCBI  was performed using the obtained sequences as queries to confirm their nematode origin and to identify the most closely related nematode sequences. The sequences revealing a similarity up to 97% and 85% with nematodes from various Dorylaimida families were included in the phylogenetic analyses of 18S and D2-D3 regions, respectively  was used as an outgroup taxon for both 18S and D2-D3 rDNA sequence datasets . Posterior probabilities values of ≥0.80 (BI) and bootstrap values of ≥70 (NJ and ML) were considered as credible support values for nodes.The sequences of the new species have been deposited in GenBank with the accession numbers ectively . The Mulectively  via the g MEGA 5 . EudorylPageBreakhttp://zoobank.org/9BF1D302-1986-47C5-B0D8-2F5DC75BD6D2http://species-id.net/wiki/Calcaridorylaimus_castaneaeSee Female. Body slender, more or less curved ventrally. Cuticle ca 2 μm at anterior part of neck, 3 μm thick at midbody, 4.5–5.5 μm at postanal region; outer layer with fine transverse striae. Lateral chord ca 1/4 of body width. Three dorsal, two ventral and three lateral pores are observed in the spear area. Lip region practically not offset, lip region width ca 20% of its height. Lips partly fused, labial papilae slightly protruding. Body at proximal end of pharynx 3–4 times the width of the lip region diameter. Amphidial aperture 5–6 μm wide or about half the lip region width. Odontostyle 1.2–1.3 times the lip region diameter, aperture occupying 35–40% of its length. Odontophore simple 1.3–1.7 times odontostyle length. Guiding ring at 8.5–9 μm from anterior end. Nerve ring surrounding the pharynx at 36–39% of neck length from head end. Hemizonid and conspicuous excretory pore observed in the nerve ring region. Pharyngeal characters : pharynx beginning to widen at 56–60% and attaining its full width at 61–64% of neck length from anterior end. DO  smaller, ca 2 μm diam; S1N2 comparatively large and distinct, 3–4 μm diam. DN nucleolus 4–4.5 μm diam.; S2N 2–3 μm diam. (DN>S1N2>S2N). Locations (%):PageBreakPageBreakPageBreakPageBreakPageBreakPageBreakpars dilatata oviductus well developed. Sperm present in uteri and pars dilatata oviductus. Synchronous uterine eggs 1–3, measuring 72–78 × 32–44 μm. Vulva transverse. Vagina extending to 60–70% inwards: pars proximalis 9–13 μm wide, 14.5–19.5 μm long, pars refringens more or less rounded trapezoid, 10–12.5 μm wide, 4–6 μm deep; pars distalis approx. 1.5 μm , regularly spaced ventromedian supplements present . Prerectum 4.5–7.5 times the corresponding body diameter long, extending 0.7–1.8 body widths anterior to the supplement series. Tail dorsally conoid and broadly rounded. One subdorsal and one subterminal pair of caudal pores.Calcaridorylaimus castaneae sp. n. differs from all species in the genus by a combination of the following characters: long body (1.4–2.1 mm), lip region practically not offset, short odontostyle  and short female tail ; vulva transverse, 38–46 μm long spicules with spur before its distal end. The new species is most similar to Calcaridorylaimus andrassyi from which it can be differentiated by having transverse vulva vs pore-like and shorter spicules (38–46 μm vs 52–57 μm). Further, Calcaridorylaimus castaneae differs from Calcaridorylaimus ruwenzorii by having shorter odontostyle (14.5-16 μm vs 19.5-25 μm), and tail  and lower c’ (c’=2.9–4.4 vs 7) values in females. It can be differentiated from Calcaridorylaimus arcticus, Calcaridorylaimus beatus, Calcaridorylaimus calcarifer and Calcaridorylaimus signatus by having different vulva shape  and shorter spicules . From Calcaridorylaimus sirgeli it differs by having transverse vulva vs pore-like, higher c (c=14.7–23.6 vs c=9.7–11.3) and lower c’ (c’=2.9–4.4 vs c’=5.3–6.7) values. Finally, Calcaridorylaimus castaneae differs from Calcaridorylaimus promissus and Calcaridorylaimus simillimus by having longer odontostyle (14.5–16 μm vs 13 μm and 11 μm) and shorter tail (75.5–110.5 μm vs 158–178 μm and 175 μm).Calcaridorylaimus castaneae is unusual for members of Dorylaimida, however, similar structure has been mentioned only for two Longidorus species, namely Longidorus macrosoma Hooper, 1961 and Longidorus carniolensis Širca et al., 2011 . Second locality: young sweet chestnut forest (30-40 years old) near Belasitsa village (sub-compartment 104g).Belasitsa Mountain, south-western Bulgaria, litter from old broadleaf forest (100-140 years) dominated by Holotype and 80 paratype females and 47 males deposited in the nematode collection of the Institute of Biodiversity and Ecosystem Research, Sofia, Bulgaria. Other paratypes deposited as follows: four females and two males in the Nematode Collection of the Foodland Environment Research Agency, Sand Hutton, UK (former Rothamsted Nematode Collection); three females and three males in the USDA Nematode Collection, Beltsville, Maryland, USA; two females and two males in the Riverside Nematode Collection, University of California, Riverside, USA; four females, and four males in the Wageningen Nematode Collection (WANECO), Wageningen, the Netherlands; four females and three males in the Nematode Collection of the Zoology Museum of the Ghent University, Belgium.Castanea) in the forest where this nematode was found.The scientific name is derived from the generic name of dominant tree species, the sweet chestnut tree (PageBreakPageBreakKF717498 and KF717497). A BLAST search for D2-D3 region showed highest similarity (88%) to the sequence of Labronema vulvapapillatum clone 2  from France (AJ875133 and JN049666)  have been submitted to GenBank (accession numbers m France  and two N049666) .PageBreakgroup with several Mesodorylaimus spp. , Mesodorylaimus japonicus  and Mesodorylaimus cf. nigritulus ) and two unidentified nematodes from environmental samples in the 18S rDNA phylogenetic reconstructions  in the partial D2-D3 LSU reconstructions  were aligned along with our sequence. The phylograms obtained by NJ, ML and BI methods showed similar topology and differed only in the positions of poorly supported clades. The BI trees  and 8 wiructions  and 9; aructions  and 10.PageBreakPageBreakPageBreakCalcaridorylaimus was erected by Mesodorylaimus, with the males and females being practically indistinguishable. The phylograms inferred from 18S sequences showed the closest relationships of Calcaridorylaimus castaneae with some members of the latter genus; however, the insufficiency of molecular data complementary to detailed morphological studies of species belonging to both genera does not allow the elucidation of evolutionary relationships among them and the position of the new species herein described.The genus PageBreak


Amyotrophic lateral sclerosis (ALS) is characterized by selective degeneration of motor neurons. The gene encoding Cu/Zn superoxide dismutase (SOD1) is responsible for 20% of familial ALS cases. Growth hormone (GH) concentrations are low in the cerebrospinal fluid of patients with ALS; however, its association with motoneuronal death is not known. We tested the neuroprotective effects of GH on human SOD-1-expressing cultured motor neurons and SOD1G93A transgenic mice.In cultured motor neurons, cytotoxicity was induced by A23187, GNSO, or homocysteine, and the effects of GH were determined by MTT, bax, PARP cleavage pattern, Hoechst nuclear staining, MAPK, and PI3K assay. In SOD-1 transgenic mice, rotarod motor performance was evaluated. Survival analysis of motoneuronal loss was done using cresyl violet, GFAP, and Bcl-2 staining. GH prevents motorneuronal death caused by GSNO and homocysteine, but not that by A23187. It activates MAPK and PI3K. GH-treated mice showed prolonged survival with improved motor performance and weight loss. GH decreased cresyl violet positive motoneuronal loss with strong Bcl-2 and less GFAP immunoreactivity.Our results demonstrate that GH has a protective effect on mutant SOD-1-expressing motor neurons. Amyotrophic lateral sclerosis (ALS) is a degenerative disorder ,2 characUp to 10% of ALS cases are familial (fALS); the remaining 90% have no hereditary component and are known as sporadic ALS . About 2Many studies have implicated oxidative stress and an excitotoxic mechanism in the pathogenesis of ALS. Plasma homocysteine (HC), which is continuously produced from diet and increases with age, has been considered to play a role in motor neuronal death, thus resulting in ALS. HC in vascular systems produces reactive oxygen species (ROS), such as superoxide anions, and reduces transitional metals by reaction of the metal with molecular oxygen. NO (Nitric oxide) might be involved in the mechanism of action of HC. NO is scavenged for transformation into peroxynitrite by reaction with the superoxide anion generated by HC. This reactive peroxynitrite causes cell death by oxidative damage, disruption of energy metabolism, calcium homeostasis, and mitochondrial function ,10. In aGrowth hormone (GH), a high molecular weight peptide composed of 191 amino acids, is produced by the anterior lobe of the pituitary gland. GH functions either by direct action on tissues or through the insulin-like growth factor-1. It causes proliferation of many types of cells and controls differentiation in adipose or muscle tissues. It also controls the metabolism of proteins, carbohydrates, or fatty acids ,14. AlthIn the present study, we examined the protective effect of GH on motor neuronal death. We tested the effect of GH on the viability of motoneuron-neuroblastoma hybrid cells (VSC4.1) expressing mutated human SOD1 against cytotoxic stimuli. In addition, we attempted to determine whether GH treatment has a neuroprotective effect on transgenic mice with the SOD1G93A mutation. Lifespan and body weight measurements, rotarod test, and immunohistochemistry for motor neuronal loss were performed.Cell lines were cultured with various GH concentrations for 24 h (0.0004 I.U./ml to 0.4 I.U./ml). The number of living cells increased in the A4V cell line (p = 0.015) and G93A cell line (p = 0.034) as determined by the MTT assay. However, there was no statistical difference with the number of viable cells in the WT cell line.When treated with W13 (P13K inhibitor), the increasing viability of A4V with pretreated GH decreased (p = 0.039) , cell viabilities decreased but did not reach significance. Viabilities in A4V and G93A decreased by 10% and 20%, respectively, which was higher than that in WT. Pretreatment with 0.04 I.U./ml and 0.4 I.U./ml GH had no protective effect on WT, A4V, and G93A cell lines.GSNO 200 μM) treatment caused a non-significant reduction in viabilities. The reduction was 20% in A4V and 10% in G93A, and was higher than in WT. Pretreatment with 0.04 I.U./ml and 0.4 I.U./ml GH caused viabilities in WT and A4V to increase, but not significantly , cell viabilities decreased but was not significantly different compared to WT. Viabilities in A4V and G93A decreased more than that in WT . The protective effect of GH was observed in A4V, especially at 0.4 I.U./ml (p = 0.046)   and 19 weeks (p = 0.040) Figure B.To determine the protective effects of GH on lifespan, GH was injected weekly from postnatal 11 weeks till death  in GH-treated SOD1G93A transgenic mice. After treatment, lifespan was compared with saline-treated SOD1G93A transgenic mice. GH-injection resulted in a significant improvement in lifespan (P = 0.019)  Figure A, D. To A few studies on halting disease progression in SOD1-dependent ALS have been carried out. The effect of GH on the CNS appears to involve brain growth and development, and action as a neuroprotective factor -17. We tet al. -2,5-diphenyltertazolium bromide (MTT) assay was used for determination of cell viability. In brief, MTT solution (5 mg/ml in PBS) was added to cultures. Following a 3-h incubation period, the MTT solution was removed and formazan precipitate was dissolved in 200 μl dimethyl sulfoxide (DMSO).d-lysine  and were fixed with 4% paraformaldehyde for 20 min. Hoechst 33, 342 dye  was diluted at 1: 1000 with phosphate buffered saline (PBS) and incubated for 5 min at room temperature. Morphological changes in the nuclei were observed using a fluorescence microscope. To determine any evidence of apoptosis, nuclear fragmentation or chromatin condensation were examined [For staining, cells were plated on coverslips coated with poly-examined .In order to induce cytotoxicity by production of reactive oxygen species (ROS), we used homocysteine and S-nitrosoglutathione (GSNO), a nitric oxide donor, which have been previously reported. For calcium induced cytotoxicity, the ionophore A23187 was used for calcium-influx. The concentrations were used as previously described: GSNO 200 μM, homocysteine 10 mM, A23187 5 μM ,36. We ePI3K and MAPK were inhibited using wortmannin , and 10 μM P098050 , respectively. Phosphorylated Akt (pAkt) was assayed for PI3K activation and phosphorylated ERK (pERK) for MAPK activation.Total proteins were extracted with RIPA buffer, to which protease inhibitors  had been freshly added. Protein concentration was determined by the Bradford method . Protein extracts (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein separation was performed in 10% polyacrylamide with 0.05% bis-acrylamide and transferred to a nitrocellulose membrane. Anti-PARP , anti-Bax , anti-pAkt , and anti-pERK  were used as primary antibodies. Anti-tubulin antibody  was used for standardization of the protein amount. Immunoreactivity was detected by enhanced chemiluminescence .Transgenic TgN (SOD1G93A) 1GUR mice were obtained from the Jackson Laboratory . These mice express high-copy numbers of the transgenic human mutant SOD1 containing the Gly 93 to Ala (G93A) substitution . SOD1G93In ALS model mice, shaking of the limbs and suspension of the tail in the air are the initial symptoms of disease onset. At this stage, clonus, hyperreflexia, and crossed spread of spinal reflexes are also detectable in most mice. Subsequently, these mice develop gait impairments and paralysis of one (or both) hind limbs; in the final stage these mice are completely paralyzed. The end point was noted when ALS model mice could not right themselves within 30 s when placed on their sides on a flat surface; at this point they were sacrificed by cervical dislocation ,53.Using a previously described protocol, the Rotarod  test was used in SOD1G93A mice from 11 weeks of age . TraininFor immunohistochemistry, mice were deeply anesthetized and perfused transcardially with cold 0.1 M PBS, followed by cold 4% paraformaldehyde for 10 min. Spinal cords were carefully dissected to obtain 4–10 mm long segments. Lumbar and cervical cords were identified by enlargement of the spinal cord. Tissues were postfixed in 4% paraformaldehyde for 8 h, and the tissue blocks produced were cryoprotected in a series of cold sucrose solutions of increasing concentration for 24 h. Transverse sections (30-μm thicknesses) were taken through lumbar and cervical cords. Cresyl violet acetate  staining was performed using these sections and incubated using the free-floating method  with one of the following primary antibodies: monoclonal anti-glial fibrillary acidic protein, GFAP ; or Bcl-2 .in vitro studies, values were expressed as mean ± SD, inter-group analysis by treatment was performed using Kruskal-Wallis or Mann-Whitney tests. Dose-dependent decrease in viability was determined using LD50. For in vivo studies, values are expressed as mean ± SD. Kaplan-Meier survival analysis and the Log-rank test were used for comparisons of survival. Body weight and rotarod test analysis by treatment were performed using the Mann-Whitney test. Statistical significance was accepted when p-value was less than 0.05. SPSS10.0  program was used for statistical analysis.The samples were analyzed in triplicate. For


Sphingosylphosphorylcholine (SPC) elicits vasoconstriction at micromolar concentrations. At lower concentrations (≤1 µmol/L), however, it does not constrict intrapulmonary arteries (IPAs), but strongly potentiates vasoreactivity. Our aim was to determine whether this also occurs in a systemic artery and to delineate the signalling pathway.+] to induce a small vasoconstriction. SPC (1 µmol/L) dramatically potentiated this constriction in all arteries by ∼400%. The potentiation was greatly suppressed or abolished by inhibition of phospholipase C , PKCε (inhibitory peptide), Src (PP2), and NADPH oxidase (VAS2870), and also by Tempol (superoxide scavenger), but not by inhibition of Rho kinase (Y27632). Potentiation was lost in mesenteric arteries from p47phox–/–, but not NOX2−/–, mice. The intracellular superoxide generator LY83583 mimicked the effect of SPC. SPC elevated reactive oxygen species (ROS) in vascular smooth muscle cells, and this was blocked by PP2, VAS2870, and siRNA knockdown of PKCε. SPC (1 µmol/L) significantly reduced the EC50 for U46619-induced vasoconstriction, an action ablated by Tempol. In patch-clamped mesenteric artery cells, SPC (200 nmol/L) enhanced Ba2+ current through L-type Ca2+ channels, an action abolished by Tempol but mimicked by LY83583.Rat mesenteric arteries and IPAs mounted on a myograph were challenged with ∼25 mmol/L [K2+ entry and thus vasoreactivity. We speculate that this pathway is not specific for SPC, but may also contribute to vasoconstriction elicited by other G-protein coupled receptor and PLC-coupled agonists.Our results suggest that low concentrations of SPC activate a PLC-coupled and NOX1-mediated increase in ROS, with consequent enhancement of voltage-gated Ca The actions of SPC are, however, stereospecific and dependent on phospholipase C (PLC), suggesting that they are mediated via a G-protein coupled receptor.5Sphingosylphosphorylcholine (SPC) is derived from membrane sphingomyelin and is present in the plasma in submicromolar-free concentrations and as a major component of low- and high-density lipoproteins; it is also released from activated platelets.2+ entry and Rho kinase-mediated Ca2+-sensitization, with an EC50 of ∼12 µmol/L.9 However, plasma concentrations are at least 10-fold less than this, raising questions concerning the physiological relevance of vasoconstriction induced by SPC. Conversely, we have demonstrated in rat intrapulmonary arteries (IPAs) that although low concentrations (1 µmol/L or less) of SPC do not on their own elicit vasoconstriction, cause depolarization, elevate [Ca2+]i, or activate Rho kinase, they strongly potentiate vasoreactivity by enhancing Ca2+ entry induced by other stimuli.10 Although this effect was also stereospecific and dependent on PLC,10 it therefore clearly differs from the mechanisms previously shown to underlie vasoconstriction induced by higher concentrations of SPC. This implies an additional and hitherto unrecognized high affinity signalling pathway that could be of physiological relevance.SPC induces vasoconstriction in isolated arteries via activation of Ca14 and ROS have been shown to enhance Ca2+ entry through L-type voltage-gated Ca2+ channels in vascular smooth muscle and cardiac myocytes.18 ROS have also been implicated in signalling pathways initiated by other PLC-coupled vasoconstrictor agonists, including angiotensin II and endothelin.19 This led us to hypothesize that a NOX/ROS-mediated pathway might underlie the SPC-induced enhancement of IPA vasoreactivity. As the pulmonary vasculature exhibits some unique responses to changes in redox state and ROS,21 we focused here on mesenteric artery (MA) to determine whether our previous observations were specific to the pulmonary vasculature. We show that low, subcontractile concentrations of SPC potentiate vasoreactivity of both MA and IPA through the same pathway, which involves PLC- and PKCε-dependent activation of NOX1, increased production of ROS, and consequent enhancement of Ca2+ entry via L-type channels.SPC has been reported to induce generation of reactive oxygen species (ROS) in a variety of non-muscle cell types, probably via NADPH oxidase (NOX),2.2.13, 1 MgSO4, 0.44 NaH2PO4, 4 KCl, 5.5 glucose, and 1.8 CaCl2). Male mice (6–8 weeks old) lacking genes for gp91phox22 (NOX2), p47phox 23 (background for both C57BL/6) or PKCδ (background 129/SV)24, or matched wild-type (WT), were killed by a Home Office approved method, the mesentery removed and placed in cold PSS.The study conforms with UK Home Office regulations and Directive 2010/63/EU of the European Parliament. Adult male Wistar rats were killed by a lethal overdose of pentobarbital (i.p.). The lungs, mesentery, and in some cases sections of small renal or main femoral artery were excised and placed in cold physiological saline solution  at 37°C. Vessels were stretched to equivalent transmural pressures of ∼25 (IPA) or ∼90 mmHg , and pre-conditioned by repeated exposure to 80 mmol/L K+ PSS  as previously described.20 Experiments were performed after ∼30 min to allow for stabilization. Tension was recorded using Acquisition Engine software .Small IPA, MA, renal artery (200–500 µm i.d.) or femoral artery segments (1–2 mm i.d.) were dissected free of connective tissue, mounted on a myograph , and bathed in PSS gassed with 5% CO2.210 PASMCs from passages 3–4 were growth-arrested in serum-free medium for 24 h before use; each cell line was verified as smooth muscle by immunostaining for smooth muscle α-actin, calponin, and desmin .Pulmonary artery smooth muscle cells (PASMCs) were dispersed from IPA using collagenase  and papain (1 mg/mL), and cultured in DMEM containing 10% FCS as previously described.25 The 19-nucleotide target sequences  were synthesized into 64–65 mer oligonucleotides with BamHI/HindIII overhangs (Sigma-Aldrich) and cloned into the expression vector pSilencer 3.0-H1 . All clones were purified using an EndoFree Plasmid Maxi Kit  and sequenced . PASMCs were transfected using the Basic Nucleofector™ Kit for primary mammalian smooth muscle cells and a nucleofector device ; after 72 h cells were serum starved for 24 h prior to use. Transfection efficiency was >80%, as determined using pmaxGFP (green fluorescent protein-expressing vector) provided in the kit and confirmed by fluorescence microscopy. Efficiency and selectivity of knockdown was confirmed by western blot.siRNAs were designed as described previously.2.3As a qualitative, real-time estimation in intact arteries maintained under identical conditions to contraction studies, MAs were mounted on a confocal wire myograph (Danish Myo Technology) and pre-conditioned as above. Following incubation with 10 µmol/L carboxy 2′,7′-dichlorofluorescin–diacetate (C-DCFH/DA) for 45 min at 37°C, excess dye was washed off and tissue fluorescence of oxidized C-DCF (excitation 490 nm and emission 530 nm) recorded every 30 s using an inverted microscope (Zeiss UK Ltd) and microfluorimeter (Cairn Research Ltd). After a stable baseline was established (∼30 min), SPC was added to the bath.ROS generation in cultured cells was estimated using lucigenin-enhanced luminescence. PASMCs (passage 4) were cultured to confluence in 24-well plates and growth-arrested for 24 h. Medium was replaced with gassed PSS at 37°C containing 5 µmol/L lucigenin and 100 µmol/L NADPH, to which SPC and pharmacological inhibitors were added. Luminescence was measured at 37°C using a Hidex Chameleon plate reader .2.426 Ba2+ was used as a charge carrier to record currents (IBa) through voltage-gated L-type Ca2+ channels, with an extracellular solution containing (mmol/L): 10 BaCl2, 130 NaCl, 5 CsCl, 1 MgCl2, 5 HEPES, 5 glucose, pH 7.35 and a pipette solution containing (mmol/L): 120 Cs methansulfonate, 20 CsCl, 2 MgATP, 0.5 Na2GTP, 0.3 MgCl2 and 5 HEPES, pH 7.2. Cells were equilibrated with pipette solution for 3 min after whole-cell access before recording the control (time 0) current–voltage (I–V) relationship using 120 ms voltage steps between −80 and +80 mV; holding potential was −70 mV. Cells were then incubated for 5 min with either 200 nmol/L SPC, 1 µmol/L LY83583, or in the absence of drug (time control), prior to recording of the test I–V relationship. The effect of Tempol (3 mmol/L) was studied on cells preincubated for 2 min before addition of 200 nmol/L SPC.Freshly isolated MA smooth muscle cells (MASMCs) were obtained from third- to fourth-order MA by enzymatic dispersion, and recordings of whole-cell currents performed with patch clamp as described previously.2.550 and fitted maximum ; for analysis EC50 was expressed as pD2 (−log EC50). Statistical analysis was performed using ANOVA with a Holm-Sidak post hoc unless otherwise stated . Statistical significance was deemed if P < 0.05.Tension was normalized to the response to KPSS, or for potentiation experiments to the control response prior to addition of SPC. Results are expressed as means ± SEM. Concentration–response curves were fitted to individual experiments using a Hill equation to provide EC2.6U73122, Gö6983, Gö6976, PP2, and rottlerin were obtained from Calbiochem, UK; C-DCFH/DA from Invitrogen, UK, and all other reagents including PKCε translocation inhibitor peptide from Sigma-Aldrich.3.3.110 1 µmol/L SPC alone had no effect on tension in rat or mouse MA , or rat renal or femoral artery.As previously reported for rat IPA,+] to cause a small depolarization-induced rise in tension of 5.8 ± 0.6% (n = 31) of that induced by KPSS, as previously described.10 SPC (1 µmol/L) was added to the bath, and the procedure repeated at 15 min intervals in the continued presence of SPC. SPC strongly potentiated the subsequent response to depolarization, and this gradually increased with time , such that at 30 min  force was increased to 512 ± 40% of control . Baseline tension measured immediately before each depolarizing challenge was unchanged from control , consistent with the lack of effect of SPC alone, and in the absence of SPC the response to repeated depolarization was also unchanged over 45 min . SPC (1 µmol/L) caused the same degree of potentiation at 30 min in IPA  and small renal arteries  as MA, but had a smaller effect in large femoral arteries .Rat small MAs were challenged with sequential 5 min applications of PSS containing ∼25 mmol/L  in both MA and IPA . In contrast to SPC, however, the effects were not significantly inhibited by Gö6983, PP2, or VAS2870 , consistent with ROS being downstream of PKCε- and Src-mediated activation of NOX1.The above results suggest that SPC-induced potentiation of vasoreactivity is mediated via a PLC-, PKCε- and Src-dependent activation of NOX1, and consequent generation of ROS. We therefore examined whether exogenous ROS could mimic the effects of SPC using LY83583, a membrane-permeable quinolinequinone that acts within the cell to generate intracellular superoxide.3.62+ currents using whole-cell patch-clamp and Ba2+ as a charge carrier, in freshly isolated myocytes from rat MA . We utilized 200 nmol/L SPC because 1 µmol/L caused rapid loss of attachment. To account for current rundown, comparisons were made between different cells 5 min after addition of SPC or solvent (PSS). SPC (200 nmol/L) increased peak current at 5 min to 176 ± 17%  of that in control cells, and this enhancement was abolished in the presence of 3 mmol/L Tempol . As predicted, the intracellular ROS generator LY83583 (1 µmol/L) had a similar effect to that of SPC .We examined the effects of SPC on voltage-gated Ca4.2+]i or vasoconstriction alone, nevertheless strongly potentiate depolarization- and agonist-induced constriction of small IPA by enhancing Ca2+ entry.10 The precise mechanism, however, remained unclear, and it was unknown whether this phenomenon was limited to pulmonary arteries, which have unique responses to hypoxia and some other stimuli.37 The key findings of the present study are that such concentrations of SPC also strongly potentiate vasoreactivity of small MAs and renal arteries, and that for both MA and IPA this is mediated via an increase in ROS generated by NOX1 and enhancement of Ca2+ entry through L-type channels.We previously reported that concentrations of SPC ≤1 µmol/L, insufficient to elicit elevation of [Ca+] by the same extent (∼400% at 30 min) in small IPA, MA, and renal arteries, but to a lesser degree in a large femoral artery (∼60%). The latter may reflect the suggestion that SPC plays a greater role in distal compared with large proximal arteries.38 The relatively slow onset, with a maximum effect around 50–60 min , was similar in all artery types and to vasoconstriction induced by high (e.g. 10 µmol/L) concentrations of SPC.33 While this could reflect accumulation of SPC in the membrane or of a secondary mediator, it is noted that there was no increase in basal tension over at least 45 min . As we previously reported for IPA,20 preincubation with 1 µmol/L SPC also potentiated the response of MA to PGF2α and U46619, demonstrated here by a large shift to the left of the concentration–response curve .SPC (1 µmol/L) enhanced constriction induced by mild depolarization with ∼25 mmol/L [K10 , as it was abolished by the PLC inhibitor U73122 and the putative PKCδ inhibitor rottlerin (but see below), and strongly suppressed by Gö6983 , but not by Gö6976  nor the Rho kinase inhibitor Y27632. PLC has previously been established as a critical component of SPC signalling, and at higher concentrations (>5 µmol/L) SPC strongly activates Rho kinase and Ca2+ sensitization in many vascular beds,39 though the latter is clearly not involved at low concentrations.SPC-induced potentiation of vasoreactivity in rat MA exhibited the same pharmacological profile as we reported for IPA29 which like conventional PKCs are activated by PLC-derived diacylglycerol; conventional PKCs have been previously shown to play no role in the action of SPC.28 While we originally proposed PKCδ, we show here that SPC-induced potentiation of constriction was unaltered in MA from mice lacking PKCδ . However, another novel isoform, PKCε, has been implicated in SPC-induced constriction of cat oesophagus smooth muscle.28 Consistent with this, the specific PKCε inhibitory peptide strongly inhibited SPC-induced potentiation in both IPA and MA .The differential effects of Gö6983 and Gö6976 imply a novel isoform of PKC,14 and PKCε is known to activate NOX in cardiac31 and pulmonary artery myocytes.40 Both PKC and Src phosphorylate the p47phox organizer subunit of NOX1 and NOX2, essential for translocation of cytosolic subunits and activation of the oxidase complex.32 Interestingly, Src and PKCε are themselves redox-sensitive, giving rise to the possibility of positive feedback-mediated amplification.41 Consistent with a central role for NOX-generated ROS, SPC-induced potentiation of depolarization-induced constriction was strongly suppressed in both MA and IPA by the novel NOX inhibitor VAS2870,35 Src inhibitor PP2, and superoxide scavenger Tempol . Tempol also reversed the potentiation by SPC of U46619-induced vasoconstriction . Moreover, the potentiating effect of SPC was abolished in MA from p47phox −/−, but not gp91phox −/−, mice ; this strongly implicates NOX1 as the relevant isoform, as only NOX1 and NOX2 require p47phox.SPC increases ROS generation in keratinocytes, endothelial cells, and Jurkat cells , most likely via NOX,Figure 5). Furthermore, subcontractile concentrations of the intracellular superoxide generator LY83583 mimicked the actions of SPC , and we have previously shown that such concentrations also enhance agonist-induced contraction in MA and IPA.20 Inhibition of PKC or Src did not affect LY83583-induced potentiation , suggesting that in these circumstances PKCε and Src play a primarily upstream role to generation of ROS. These results strongly suggest that the effects of SPC reported here are mediated by increased generation of NOX1-derived ROS.Consistent with the above, 1 µmol/L SPC increased ROS generation both in intact MA and in cultured vascular smooth muscle cells, an action effectively abolished in the latter by Gö6983, PP2, and VAS2870, and also following siRNA knockdown of PKCε  mediated increase in Ca2+ sensitivity, although at higher concentrations both SPC and ROS do activate Rho kinase.39 However, voltage-dependent Ca2+ entry induced by depolarization or agonist was increased.10 While this might occur if SPC induced some depolarization itself, and NOX-derived ROS have been reported to inhibit Kv channels in pulmonary artery,42 SPC did not suppress KV currents in IPA, and indeed still potentiated the response in maximally depolarized arteries.10 Moreover, we have reported that intracellularly generated ROS cause a negative shift in the activation of KV current in both MA and IPA, which would tend to have a hyperpolarizing effect.20Concerning the mechanism by which low concentrations of SPC enhance vascular reactivity, our current and previous2+ channels, the α1C subunit of which contains multiple redox-sensitive cysteine residues,15 and NOX-derived ROS are reported to enhance L-type Ca2+ channel currents in both cardiac and vascular smooth muscle.19 Consistent with this, we found that 200 nmol/L SPC potentiated L-type Ca2+ channel current (Ba2+ as a charge carrier) in MA smooth muscle cells, and this was prevented by Tempol and mimicked by the superoxide generator LY83583 .ROS and redox state are known to affect the function of L-type Ca2+ entry through L-type channels, when these are activated by other means. The pathway is apparently identical in MA and IPA. This mechanism may not be limited to SPC, as a similar pathway, albeit at concentrations sufficient to cause constriction alone, has been proposed for the archetypical NOX activator angiotensin II in cerebral arteries,16 ET-1 in cardiac myocytes,19 and U46619 in pulmonary artery.42 Notably, Tempol caused a shift to the right of the control U46619 concentration–response curve in MA . While these reports may differ in terms of specific isoforms of PKC and NOX, and in pulmonary artery the possible involvement of KV channels, the underlying signalling pathway is very similar. Note, however, that all these studies utilized agonist concentrations that in themselves cause constriction and activation of parallel Ca2+ mobilization and other pathways, which might underlie reported differences.Our results are consistent with a model where subcontractile concentrations of SPC activate NOX1 through a PLC, PKCε, and Src-dependent mechanism, and the consequent increased generation of ROS enhances Ca2+ entry and Rho kinase-mediated Ca2+ sensitization.9 Notably, all of these have been reported to be activated by ROS.41 We found that Tempol suppressed SPC-induced constriction by >80% in MA , and that 10 µmol/L SPC doubled the rate of ROS generation compared with 1 µmol/L . While this implies that ROS form a key signalling component for SPC at any concentration, the fact that Gö6983, PP2, and knockdown of PKCε only partially inhibited the elevation of ROS elicited by 10 µmol/L SPC suggests that an additional pathway may be activated by concentrations >1 µmol/L, as previously suggested for other cell types.43Considering the above, we briefly examined whether ROS and NOX signalling was important for constriction elicited by higher concentrations of SPC, which has been attributed to activation of voltage-dependent and -independent Ca5.2+ entry through L-type channels. SPC has been implicated in cardiovascular disease, though the majority of in vitro studies utilized concentrations probably well in excess of physiological levels.3 The concentrations used here and previously (≤1 µmol/L)10 are closer to those reported in plasma,44 although physiologically relevant concentrations at the cell surface are difficult to determine because of autocrine and paracrine production, and extensive binding to albumin, HDLs, and LDLs.12 Nevertheless, our results suggest that physiological or pathophysiological concentrations of SPC could greatly increase vascular reactivity to other stimuli. In addition, we can speculate from the data shown in Figure  that many of the vascular actions of SPC might be mediated via increased ROS. In this respect, there are similarities between SPC and angiotensin II, which share many downstream pathways.45We demonstrate here that low concentrations of SPC, insufficient to elicit vasoconstriction alone, strongly potentiate vasoreactivity via PLC, PKCε, and Src-dependent activation of NOX1, increased generation of ROS, and consequent enhancement of Ca20 whereas higher concentrations of exogenous ROS are reported to constrict pulmonary but relax systemic arteries.40 It is probable that this relates to compartmentalization of SPC-induced ROS signalling, synonymous to that for Ca2+. Notably, angiotensin II causes highly localized sub-plasmalemmal generation of ROS and co-localized L-type channel activity in cerebral artery, suggesting clustering of receptors, NOX, and L-type channels in a micro-signalling domain.16 We speculate that a similar situation exists for SPC and an as yet unidentified high affinity SPC receptor. Higher concentrations of exogenous ROS would, however, have more promiscuous effects, for example relaxing MA (but not IPA) by opening Kv channels,20 and constricting IPA (but not MA) in part by mobilizing ryanodine-sensitive Ca2+ stores.30The question arises as to why SPC and low concentrations of ROS should have similar effects in MA and IPA,2+ entry. Similarities with other studies suggest that this could potentially be common to other GqPCR and PLC-coupled agonists, with significant implications for vascular regulation and disease.In summary, we present evidence for a novel pathway by which physiological concentrations of SPC strongly potentiate vasoreactivity, involving PLC, NOX1, and ROS-mediated enhancement of voltage-gated CaCardiovascular Research online.Supplementary material is available at Wellcome Trust (grant #087776). Funding to pay the Open Access publication charges for this article was provided by the Wellcome Trust.This work was supported by the


Marine cold-temperature environments are an invaluable source of psychrophilic microbial life for new biodiscoveries. An Arctic marine bacterial strain collection was established consisting of 1448 individual isolates originating from biota, water and sediment samples taken at a various depth in the Barents Sea, North of mainland Norway, with an all year round seawater temperature of 4 °C. The entire collection was subjected to high-throughput screening for detection of extracellular laccase activity with guaiacol as a substrate.Psychrobacter genus. From the most diverse four strains, based on 16S rRNA gene sequence analysis, all originating from the same Botryllus sp. colonial ascidian tunicate sample, genomic DNA was isolated and genome sequenced using a combined approach of whole genome shotgun and 8 kb mate-pair library sequencing on an Illumina MiSeq platform. The genomes were assembled and revealed genome sizes between 3.29 and 3.52 Mbp with an average G + C content of around 42 %, with one to seven plasmids present in the four strains. Bioinformatics based genome mining was performed to describe the metabolic potential of these four strains and to identify gene candidates potentially responsible for the observed laccase-positive phenotype. Up to two different laccase-like multicopper oxidase (LMCO) encoding gene candidates were identified in each of the four strains. Heterologous expression of P11F6-LMCO and P11G5-LMCO2 in Escherichia coli BL21 (DE3) resulted in recombinant proteins exhibiting 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and guaiacol oxidizing activity.In total, 13 laccase-positive isolates were identified, all belonging to the Psychrobacter species with laccase-positive phenotype were isolated from a collection of Arctic marine bacteria. Four of the isolates were genome sequenced. The overall genome features were similar to other publicly available Psychrobacter genome sequences except for P11G5 harboring seven plasmids. However, there were differences at the pathway level as genes associated with degradation of phenolic compounds, nicotine, phenylalanine, styrene, ethylbenzene, and ethanolamine were detected only in the Psychrobacter strains reported in this study while they were absent among the other publicly available Psychrobacter genomes. In addition, six gene candidates were identified by genome mining and shown to possess T1, T2 and T3 copper binding sites as the main signature of the three-domain laccases. P11F6-LMCO and P11G5-LMCO2 were recombinantly expressed and shown to be active when ABTS and guaiacol were used as substrates.Thirteen The online version of this article (doi:10.1186/s12864-016-2445-4) contains supplementary material, which is available to authorized users. With 70 % of the Earth’s surface covered by oceans and 90 % of their water having temperatures of maximum 5 °C , the oceLaccases  are copper-containing oxidases that catalyze the monoelectronic oxidation of various aromatic substances at the expense of molecular oxygen, producing water as the sole by-product . In combFungal laccases have been extensively studied –24. DespPsychrobacter strains originating from the Barents Sea that exhibit laccase activity and heterologous expression of two of these enzymes in Escherichia coli BL21 (DE3). These strains and/or their aromatic compounds oxidizing enzymes may have broad application potentials as cold-active biocatalysts.In this study, we describe the discovery and genome sequencing of four new Algibacter, Aliivibrio, Alteromonas, Bacillus, Bizionia, Cellulophaga, Cobetia, Colwellia, Cytophaga, Flavobacterium, Halomonas, Lacinutrix, Leeuwenhoekiella, Marinobacterium, Marinomonas, Moritella, Neptunomonas, Olleya, Photobacterium, Planococcus, Polaribacter, Pseudoalteromonas, Pseudomonas, Psychrobacter, Psychromonas, Psychroserpens, Shewanella, Sulfitobacter, Tenacibaculum, Thalassomonas, and Vibrio species.In collaboration with UiT, the Arctic University of Norway, Tromsø, Norway, biota, water and sediments were sampled between May 14th and 26th, 2009, on a research cruise of R/V Jan Mayen in North of Norway within the Arctic Circle. Sampling was done in ten different locations, spanning a region between and around the Svalbard archipelago and the Bear Island in the Barents Sea. The collection led to the establishment of a library of 1448 single bacterial isolates originating from biota (773), sediments (418), and water (257) samples. The strain library consists of, at least, 31 genera based on 16S rRNA gene sequences of 550 isolates, including The entire strain collection of 1448 individual bacterial isolates, arrayed in 96-well plates, was screened for isolates exhibiting extracellular laccase activity, and 13 of them scored positive based on the formation of brown zones around the colonies after 24 and 48 h of incubation at room temperature (20–23 °C). The color formation was observed after 24 h Fig. . HoweverPsychrobacter based on the 16S rRNA gene sequence analysis. The aligned 16S rRNA gene sequences were used to generate a phylogenetic tree by the Weighbor weighted neighbor joining method  and Psychrobacter sp. JCM 18903 (95.65 % ANI) were found, while strain P11G3 shows significant similarity to Psychrobacter sp. JCM 18900 (94.09 % ANI) and Psychrobacter sp. JCM 18901 (93.30 % ANI). This fits well with P11F6 and P11G3 strains described as closely related to P. cibarius (strains JCM 18902 and JCM 18903) and P. nivimaris (strains JCM 18900 and JCM 18901)  grown overnight at 20 °C with 800 rpm orbital agitation. Cultures were then transferred and stamped to modified marine agar  plates (14 cm) supplemented with 0.01 % guaiacol (Sigma-Aldrich) by a 96-pin replicator. The plates were covered to protect from light and incubated at room temperature (20–23 °C) for two days. The plates were checked after 24 and 48 h. The colonies with visible brown color zones around were selected for further characterization.The bacterial collection in 96-well plate format was replicated in modified marine broth medium .Phylogenetic tree is available in the TreeBASE repository, [


To evaluate prevalence, radiological reporting and clinical management of pathologic vertebral body fractures (VBFs) of unknown origin in cancer patients receiving computed tomography (CT) examinations.We investigated all CT examinations (over 1 year) of male and female patients with an underlying malignancy and an increased risk of osteoporosis (age 55–79 years) for the presence of VBFs. We evaluated midline sagittal CT-reformations of the spine for prevalence, fracture type, severity and location, the accuracy and style of radiological reporting, subsequent clinical management and documentation in hospital discharge letters.848 patients were investigated. We found 143 VBFs in 94 (11 %) patients. 6, 49, and 45 % were grade 1, grade 2, and grade 3 fractures, respectively, while 20, 66, and 14 % were wedge, biconcave and crush fractures, respectively. 32 (34 %) radiological reports correctly classified VBFs as fractures, 25 (27 %) reports recognized VBFs, but did not type them, and VBFs were not described in 37 (39 %) reports. In 3 (3 %) patients further clinical work-up of VBFs was performed, while only 8 (9 %) hospital discharge letters contained the information of the presence of pathologic VBFs of unknown origin.VBFs of unknown origin appear frequently in cancer patients, however, clinical management and documentation was found in only few cases. Moreover, especially in cancer patients consistent radiological reporting of VBFs seems important, as aetiology of VBFs could be from osteoporosis, disease progression or oncological therapy, however, reporting is still performed inconsistently. Pathologic fractures are fractures of “diseased and weakened bone” and occur without adequate trauma  with the following constraints was performed: 12-month period; patient age 55–79 years; 64-row CT examination containing the abdomen and pelvis . Patients with previously known osseous spinal metastasis were excluded. Furthermore, patients with a recent history of trauma—in order to exclude patients with traumatic VBFs, were excluded as well. Images and radiological reports from 1229 CT examinations were retrieved. From these, 381 patients did not demonstrate an underlying malignancy, and were excluded from further investigation. All remaining 848 CT examinations  were included in the evaluation.CT examinations were indicated by clinical needs in all cases. All patients were examined with a 64-row CT scanner . From the raw data, sagittal images with a slice thickness of 3 mm and an increment of 3 mm were reconstructed utilizing a sharp (bone) kernel (70f). These images were present in the initial reading and were reevaluated.Images were investigated utilizing a standard patient archiving and communicating system (PACS) workstation with dual high-resolution/high-brightness monitors. After specific training, a resident (XX) who was blinded to the initial reports analyzed the reconstructed sagittal images of all 848 patients in a bone window setting  for the presence of VBF utilizing the Semi-Quantitative (SQ) method  consensually reviewed all suspected vertebral fractures by applying the Algorithm-Based Qualitative (ABQ) , grade 2 (25–40 % height reduction), and grade 3 (>40 % height reduction), and fractures were classified as wedge, biconcave, or crush fractures.After image analysis, the original radiological reports of all patients with VBFs were examined to determine whether the fractures were reported prospectively. The radiological reports were evaluated according to the guidelines of the IOF and ESSR . Descriptive statistics of patient specifics, fracture analysis, accuracy and style of reporting in radiological reports, and documentation in hospital discharge letters were performed.289 cervico-thoraco-abdomino-pelvic, 314 thoraco-abdomino-pelvic, and 245 abdomino-pelvic CT examinations of 848 patients were included and further evaluated. In 152 (17.9 %) cases evaluated CTs detected primary tumors, and in 696 (82.1 %) CT was indicated for response evaluation and follow-up of known tumors and metastases. Collectively, 15.298 individual vertebrae were evaluated for the presence of VBFs. Detailed information concerning the fracture locations is shown in Fig. Overall, 143 (0.9 %) VBF were detected in 94 (11.1 %) patients (mean age 66.4 ± 6.2 years) in 33 (35.1 %) women and 61 (64.9 %) men. Most fractures, 52 out of 143 (36.4 %) were found at the thoracolumbar junction (T11-L2), the pathognomonic location for VBFs. 2 (1.4 %) fractures were detected in the cervical spine (C1–C7), while 18 out of 143 (12.6 %) were found in the upper thoracic-spine (T1–T6). The most often fractured vertebral bodies were L1 (21 out of 143 (14.7 %) and L5 (19 out of 143 (13.3 %). 8 out of 143 (5.6 %) were grade 1 fractures, 71 out of 143 (49.6 %) were grade 2, and 64 out of 143 (44.8 %) were grade 3 fractures. 29 out of 143 (20.3 %) of the fractured vertebrae were wedge fractures, 94 out of 143 (65.7 %) were biconcave fractures, and 20 out of 143 (14 %) were crush fractures.fracture. Instead, characterizations such as “reduced vertebral height”, “baseplate impression”, “degenerative height reduction”, “degenerative baseplate changes”, or “sintering”, were found in the description section of the radiological reports and were not mentioned in the “In 32 (34 %) out of 94 radiological reports, VBFs were classified as fractures, either in the description or the conclusion section. In an additional 25 (27 %) reports, VBFs were recognized and described in various texts in the description section; however they were not named fractures in the conclusion section of the radiological reports.Of the 94 patients featuring VBFs of unknown origin, only 3 (3 %) patients received further clinical management of VBFs , while hospital discharge letters in 8 (9 %) patients included the information of VBFs. Indeed, in all of these 8 cases, VBF were called As presented, 94 out of 848 11 %) patients with cancer undergoing routine CT examination demonstrated one or more VBFs of unknown origin. In a study on abdomino-pelvic CT examinations (cohort of 2041 patients age 19-94) Carberry et al. (Carberry et al. 1 % patieWe demonstrated that in 32 patients, the VBFs were explicitly named fractures in the radiological report and another 25 VBFs were described; however, they were not named fractures. In 8 of these 57 patients for whom VBFs were detected in the initial reading, the medical record contained this information. This leaves two interpretations: either the clinicians did not take notice of the fractures, or the information did not seem important enough to initiate further diagnostic workup. However, this increases the importance of naming every VBF a fracture in the radiological conclusion section, which most likely is the only section reviewed by the treating physician when preparing the patients’ discharge letter.It is scientific consensus that VBFs are underdiagnosed in radiographic examinations, with reported detection rates ranging from 55 % (Kim et al. One problem might be the fact that radiologists cannot diagnose osteoporosis on routine CT examinations, only osteopenia. Instead, to date for the correct diagnose of osteoporosis dedicated quantitative CT or X-ray is required Link . HoweverThere are some limitations to our research. Firstly, our standard of reference comprises of CT-morphological image criteria and a consensus read, in order to exclude obvious malignant VBFs and vertebral deformities from our study population. Furthermore, as we do not have a clinical workup of patients with VBFs, we cannot account for the number of fractures caused by osteoporosis, malignancy, or oncological therapy related, however, this was not the focus of our work. Furthermore, as L5 is not the typical location of an osteoporotic fracture and in our work 13 % of fractures were located here, these as well might be secondary due to medical treatment. Indeed, cancer patients often receive high cortisone doses or have had a neoadjuvant radiation in rectal cancer, possibly explaining the high prevalence of L5 fractures. On the other hand, the unclear nature of the pathologic VBF should entice the clinician even more so to work up and this finding. Indeed, a missed and unmanaged metastasis causing VBF might have an even worse impact on patient morbidity and mortality than a missed and unmanaged osteoporotic VBF.VBFs of unknown origin appear frequently in cancer patients, however, clinical management and documentation was found in only few cases. Moreover, especially in cancer patients consistent radiological reporting of VBFs seems important, as aetiology of VBFs could be from osteoporosis, disease progression or oncological therapy, however, reporting is still performed inconsistently.


Corynebacterium pseudotuberculosis strains. These include a strain isolated from a patient with C. pseudotuberculosis pneumonia (48252), a strain isolated from pus in goat (Ft_2193/67), a laboratory strain originating from strain Ft_2193/67 (CS_10), and the draft genome of an equine reference strain, CCUG 27541.Here we report the genome sequencess of four  Corynebacterium pseudotuberculosis is an important animal pathogen, primarily in sheep and goat but also in other ungulates like horse and cow . The library was prepared according to the Pacific Biosciences 10-kb library preparation protocol. Size selection of the final library was conducted with BluePippin (Ampure Beads for strain CCUG 27541). Sequencing was done on a Pacific Biosciences RS II instrument using P4-C2 chemistry. Two silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) cells were used for each strain. A total of 96,456 reads with an average read length of 4,677 bp were generated for strain 48252, 7,7683 reads of 4,664 bp on average for strain CS_10, 92,607 reads of 4,859 bp on average for strain Ft_2193/67, and 89,570 reads of 2,491 bp on average for strain CCUG 27541. The reads were assembled with HGAP v. 3 (Pacific Biosciences), resulting in one continuous contig for each of strains 48252, CS_10, and Ft_2193/67. The Minimus2 software (Amos package) was used to circularize the contigs from these strains. Assembly of strain CCUG 27541 resulted in five contigs. The two shortest contigs had considerably lower coverage (8 and 34) than the other three (99–120) and were excluded from further analyses. The three remaining contigs had a combined length of 2,379,417 bp and a GC content of 52.10%. The circularized contigs of strains 48252, CS_10, and Ft_2193/67 consist of 2,338,139 bp (average coverage 150), 2,338,144 bp (average coverage 130), and 2,338,300 bp (average coverage 151), respectively. All three genomes have a GC content of 52.19%.DNA was extracted using the DNeasy blood and tissue kit (Qiagen), following the protocol for Gram-positive bacteria. The DNA was purified and concentrated using Amicon Ultra-0.5 mL 30-K centrifugal filters for DNA purification and concentration (Merck Millipore). The DNA quality was assessed by agarose gel electrophoresis combined with concentration and 260/280 measurements on a NanoDrop 2000 spectrophotometer (Thermo Scientific). The DNA was sequenced and assembled at the Norwegian Sequencing Centre  (pdr.org/ ) using dCP008922, CP008923, CP008924, and JPJB00000000. The versions described in this paper are the first versions, CP008922.1, CP008923.1, CP008924.1, and JPJB01000000.The genomes have been deposited in DDBJ/EMBL/GenBank under the accession numbers


However, the role of 5-HT2cR in the development of social behavior is unexplored. Here we address this using Htr2c knockout mice, which lack 5-HT2cR. We found that these animals exhibit social behavior deficits as adults but not as juveniles. Moreover, we found that the age of onset of these deficits displays similar timing as the onset of susceptibility to spontaneous death and audiogenic-seizures, consistent with the hypothesis that imbalanced excitation and inhibition (E/I) may contribute to social behavioral deficits. Given that autism spectrum disorder (ASD) features social behavioral deficits and is often co-morbid with epilepsy, and given that 5-HT2cR physically interacts with Pten, we tested whether a second site mutation in the ASD risk gene Pten can modify these phenotypes. The age of spontaneous death is accelerated in mice double mutant for Pten and Htr2c relative to single mutants. We hypothesized that pharmacological antagonism of 5-HT2cR activity in adult animals, which does not cause seizures, might modify social behavioral deficits in Pten haploinsufficient mice. SB 242084, a 5-HT2cR selective antagonist, can reverse the social behavior deficits observed in Pten haploinsufficient mice. Together, these results elucidate a role of 5-HT2cR in the modulation of social behavior and seizure susceptibility in the context of normal development and Pten haploinsufficiency.The development of social behavior is strongly influenced by the serotonin system. Serotonin 2c receptor (5-HT Next, we wanted to test if constitutive deletion of Htr2c affects social behavior in adult mice.We assessed the development of social behavior in ty assay , 34 as j chamber . Additiot(15) = 7.90, p<0.001, t(15) = 3.28, p<0.01, Htr2c-/Y adult males showed no significant differences in chamber time during these assays (t(16) = 1.19, p = 0.25 and t(16) = 1.31, p = 0.21 respectively, Htr2c-/Y than in wild-type mice (t(22.3) = 2.47, p<0.05, Htr2c-/Y mice showed a bimodal distribution that was reflected by a higher variation in this score than in wild-type mice  = 2.13, p<0.05 and t(30) = 2.46, p<0.05 respectively, Htr2c decreased social interaction and increased locomotor activity in adult mice.Wild-type adult males spent significantly more time with the social stimulus during social approach , which has been interpreted as reflecting a role for 5-HT2cR in tonic inhibition of excitability in neuronal networks [Htr2c knockout mice display deficits in sociability. To further explore the relationship between social behavioral deficits and seizure susceptibility, we tested for AGS at P25 and P90, after social behavioral testing was complete in juvenile and adult cohorts of mice, respectively. No Htr2c-/Y mice displayed seizures at P25, confirming previous findings that juvenile Htr2c-/Y mice are not susceptible to AGSs [Htr2c-/Y mice, but no wild-type mice, exhibited tonic-clonic seizures manifested by a brief forelimb and hindlimb flexion followed by a rigid and prolonged hindlimb extension  and Pten+/- mice (n = 21) did not exhibit any spontaneous death within the timeframe examined (up to P180). An autopsy of mice that died spontaneously did not reveal any noticeable health problems such as hemorrhage, infarction, ischemia, hamartomas or tumors. Thus, as previously observed for Htr2c-/Y mice [Htr2c-/Y; Pten+/- mice may be attributed to an increase in spontaneous epileptic seizures. Using a Kaplan-Meier analysis, we found that there was a significant effect of genotype on survival (Log-Rank test: F(2) = 36, p<0.001). Pairwise comparisons revealed a significant difference on the survival of Pten+/- mice vs. Htr2c-/Y (p<0.001) and Htr2c-/Y; Pten+/- mice (p<0.001) and also between Htr2c-/Y and Htr2c-/Y; Pten+/- mice (p<0.05). We interpret these results as consistent with mutations in Pten and Htr2c interacting to influence spontaneous death, possibly via an exacerbation of the epileptic mechanism present in Htr2c knockout mice. However, because of the accelerated age of death, we were prevented from testing social behavior in these compound mutant animals.n humans –22 and vbehavior , 39, 40.+/- mice . Moreovetal area , making er assay , we aimed to P90 . This ma-/Y mice , we conj2cR interact to influence social behavior, we used SB 242084, a selective antagonist of 5-HT2cR that has been shown to increase social interaction behavior [Pten haploinsufficient and wild-type female mice as social approach deficits in female Pten+/- mice are well described [2cR antagonist SB 242084 systemically at 0.3 mg/kg, a dose that has been shown to significantly increase social interactions in rats [Pten+/- mice did not display this preference (t(15) = 5.23, p<0.001 and t(16) = 0.40, p = 0.70, Pten+/- animals [Pten+/- mice treated with SB 242084 both displayed a significant preference for the chamber containing the social stimulus mouse (t(14) = 4.32, p<0.001 and t(15) = 4.38, p<0.001, p<0.05. Effect of SB 242084 treatment: F  = 6.32, p<0.05. Interaction between genotype and SB 242084 treatment: F  = 1.97, p = 0.17 (n.s.), Pten+/- mice treated with vehicle only and Pten+/- mice treated with SB 242084 (t(31) = 2.73, p<0.01) and between wild-type mice treated with vehicle and Pten+/- mice treated with vehicle (t(31) = 2.82, p<0.01), but no significant difference between wild-type treated with vehicle and wild-type treated with SB 242084 (t(29) = 0.76, p = 0.45) and between wild-type mice treated with SB 242084 and Pten+/- mice treated with SB 242084 (t(29) = 0.56, p = 0.58). Additionally, we found that, while Pten+/- mice treated with vehicle presented a very low preference index (5%), wild-type mice treated with vehicle or SB 242084 and Pten+/- mice treated with SB 242084 all presented a high positive preference index . There was no spontaneous death or other evidence of seizures in Pten+/- mice treated with SB 242084. These results show that SB 242084 treatment suppresses the social behavior deficits exhibited by Pten+/- mice.As an alternative approach to explore whether Pten and 5-HTbehavior  and rescbehavior  without behavior  in rodenescribed , 34. We  in rats , 13, and animals , 34. WilHtr2c null mutant mice present social behavior deficits and that these deficits are restricted to adult mice and are not present in juvenile mice. It has been hypothesized that dysregulated E/I balance may contribute to social behavioral deficits in neuropsychiatric disorders such as ASD [Htr2c mutant mice [Htr2c null mutant mice present a susceptibility to AGS only in adulthood [2cR does not increase susceptibility to seizures and can increase social investigation in rodents [Htr2c knockout mice is responsible for both social behavior deficits and susceptibility to spontaneous and audiogenic seizures. Similarly, E/I imbalance might be responsible for both social behavioral deficits and epilepsy in some ASD patients, as it has been shown that 30% of ASD patients can present a co-morbidity for epilepsy [Htr2c lead to an early dysregulation of growth and connectivity that results in abnormal neural circuitry going into the critical period for social learning, with social deficits not manifesting until after the critical period closes. It has been shown, for example, that social isolation that occurs in a critical period of 2 weeks after weaning alters prefrontal cortex function and myelination and these phenotypes are not reversible after reintroduction into a social environment [2cR [Htr2c will help answer this question.We report here that adult h as ASD –29 and ch as ASD . Evidencant mice , 41, 42.dulthood , 24, 31  rodents , 11, 13,epilepsy . Alternaironment . For thiironment , 45–49 aent [2cR . Future Pten accelerates the spontaneous death rate observed in Htr2c-/Y mice [Htr2c-/Y mice [Htr2c-/Y; Pten+/- is caused by an increase in spontaneous epileptic seizures. An increased susceptibility to seizures has been reported in different models of Pten conditional deletion [PTEN mutation can also present with epilepsy [Pten+/- mice, it is possible that Htr2c mutation uncovers a susceptibility masked by the C57BL/6 background, which is normally seizure resistant [Htr2c-/Y; Pten+/- mice might seem surprising given that Pten physically interacts with 5-HT2cR to repress its activity [Pten and Htr2c mutations would have opposing phenotypic effects. Indeed, we have demonstrated that pharmacological antagonism of 5-HT2cR with SB 242084 in Pten haploinsufficient mice is capable of restoring a preference for social investigation in a three-chamber social approach assay. In interpreting these results, it is worth considering that chronic treatment using a 5-HT2cR antagonist does not increase seizure susceptibility [Pten haploinsufficient mice treated with SB 242084, indicating that gross E/I balance is normal. Thus, it is possible that mutations in Htr2c and Pten may act in different cell types to influence E/I balance in a manner that is not recapitulated by pharmacological antagonism of 5-HT2cR. For example, mutations in Htr2c might result in decreased activity of GABAergic neurons [Pten mutations result in increased activity of excitatory neurons [2cR might have different periods and mechanisms of interaction: one complementary interaction in early development that sets up a later vulnerability to seizures, and another antagonistic interaction in mature circuitry that can reverse social behavioral deficits. Additionally, SB 242084 might rescue the social behavior deficits observed in Pten haploinsufficient mice through an anxiolytic effect, for example by modulating release of dopamine in the limbic system [2cR interacts with Pten to modulate both E/I balance and the circuitry influencing social behavior. Important issues to address in future work will be to determine the degree to which modulating 5-HT2cR is significant for the ASD-therapeutic effects of drugs such as risperidone, fluoxetine and olanzapine, which target a broad range of molecules, including 5-HT2cR.We also find that a second site mutation in -/Y mice , 24. Condeletion , 51, 52,epilepsy . Althougesistant . This enactivity , thus ontibility  and we d neurons –58 while neurons , resultic system . While t