Pleurotus ostreatus is the second edible mushroom worldwide, and a model fungus for delignification applications, with the advantage of growing on woody and nonwoody feedstocks. Its sequenced genome is available, and this gave us the opportunity to perform proteomic studies to identify the enzymes overproduced in lignocellulose cultures.P. ostreatus (PC9) was grown with poplar wood or wheat straw as the sole C/N source and the extracellular proteins were analyzed, together with those from glucose medium. Using nano-liquid chromatography coupled to tandem mass spectrometry of whole-protein hydrolyzate, over five-hundred proteins were identified. Thirty-four percent were unique of the straw cultures, while only 15 and 6 % were unique of the glucose and poplar cultures, respectively . Semi-quantitative analysis showed oxidoreductases as the main protein type both in the poplar  and straw (31 %) secretomes, while carbohydrate-active enzymes (CAZys) were only slightly overproduced (14–16 %). Laccase 10 (LACC10) was the main protein in the two lignocellulose secretomes (10–14 %) and, together with LACC2, LACC9, LACC6, versatile peroxidase 1 (VP1), and manganese peroxidase 3 (MnP3), were strongly overproduced in the lignocellulose cultures. Seven CAZys were also among the top-50 proteins, but only CE16 acetylesterase was overproduced on lignocellulose. When the woody and nonwoody secretomes were compared, GH1 and GH3 β-glycosidases were more abundant on poplar and straw, respectively and, among less abundant proteins, VP2 was overproduced on straw, while VP3 was only found on poplar. The treated lignocellulosic substrates were analyzed by two-dimensional nuclear magnetic resonance (2D NMR), and a decrease of lignin relative to carbohydrate signals was observed, together with the disappearance of some minor lignin substructures, and an increase of sugar reducing ends.Monokaryotic P. ostreatus grows on woody and nonwoody lignocellulosic substrates. One laccase occupied the first position in both secretomes, and three more were overproduced together with one VP and one MnP, suggesting an important role in lignocellulose degradation. Preferential removal of lignin vs carbohydrates was shown by 2D NMR, in agreement with the above secretomic results.Oxidoreductases are strongly induced when The online version of this article (doi:10.1186/s13068-016-0462-9) contains supplementary material, which is available to authorized users. Phanerochaete chrysosporium  has been the model lignin-degrading organism for more than two decades [P. chrysosporium belongs to the group of wood-rotting basidiomycetes known as white-rot fungi  [Postia placenta [Ceriporiopsis subvermispora [http://www.genome.jgi.doe.gov/programs/fungi) of the DOE Joint Genome Institute (JGI) [ decades . Due to  decades —this fun decades . P. chry lignin) . More replacenta , as a moplacenta ; and  . Using tte (JGI) –15, althPleurotus ostreatus is the second edible mushroom worldwide, just after Agaricus bisporus [Pleurotus and other members of the Agaricales naturally grow on wood, leaf litter, and/or other nonwoody lignocellulosic substrates . Moreover, some Pleurotus species are able to remove lignin selectively from nonwoody lignocellulosic materials [C. subvermispora growing on wood. These species have been investigated for the biological production of cellulose [bisporus   being able to grow both on woody and nonwoody lignocellulosic materials. This ability is most probably due to the presence of a new arsenal of lignin-degrading enzymes, as shown after heterologously expressing and characterizing all the ligninolytic peroxidases from the three families mentioned below [P. ostreatus is available at JGI as two monokaryons, PC9 (http://www.genome.jgi.doe.gov/PleosPC9_1) and PC15 (http://www.genome.jgi.doe.gov/PleosPC15_2), obtained from a commercial dikaryon [ cinerea  and A. bbisporus , 23, two bicolor , a model bicolor , are of ed below , 27 and dikaryon . Monokardikaryon  and pres2O2-generating oxidases and laccases, in the genomes of wood-rotting basidiomycetes [Comparative genomics, ideally combined with biochemical studies, has provided important clues on lignocellulose decay by white-rot and brown-rot saprotrophic basidiomycetes, as well as on host interactions by mycorrhizogenous and pathogenic fungi. Two conclusions of these studies are: (i) the presence of genes of ligninolytic peroxidases—from the lignin peroxidase (LiP), manganese peroxidase (MnP), and/or versatile peroxidase (VP) families—in the genomes of all typical white-rot  basidiomycetes and their absence from all the brown-rot  fungal genomes, as well as from those of some poor wood rotters; and (ii) the widespread distribution of other genes contributing to the oxidative attack on lignocellulose, such as those of Homycetes , 25, 30.omycetes . Interesomycetes .P. ostreatus growing on woody (poplar wood) and nonwoody (wheat straw) lignocellulose was analyzed and compared with that from a glucose medium, with special emphasis on lignin-modifying enzymes (LMEs) and carbohydrate-active enzymes (CAZys). Secreted proteins were identified by nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) after trypsin hydrolysis, and their differential production discussed in the context of lignocellulose modification, which was analyzed using two-dimensional nuclear magnetic resonance (2D NMR) of the whole lignocellulosic samples at the gel state [In the present study, the secretome of the model white-rot agaric el state .P. ostreatus, the secretome of this white-rot (ligninolytic) fungus was analyzed by nLC-MS/MS of the total peptides from trypsin hydrolysis. With this purpose, the fungus (monokaryon PC9) was grown on a woody (poplar chips) and a nonwoody (wheat straw) lignocellulosic substrate (with distilled water as the only additive), and the diversity and relative abundance of the secreted proteins (after 21 days) compared with those found in a glucose medium (HAT). A total of 241, 391, and 206 extracellular proteins were identified in the poplar, wheat straw, and HAT fungal cultures, respectively, as summarized in Fig. In order to understand the enzymatic mechanisms of lignin and plant polysaccharide degradation by s after 2 days comThe different proteins were functionally classified into the following seven types: (i) Oxidoreductases, including LMEs; (ii) CAZys; (iii) Esterases; (iv) Proteases; (v) Phosphatases; (vi) Other function proteins; and (vii) Unknown function proteins. The overall protein composition of the different cultures was similar, in terms of the types present, except for the minor phosphatase group that was absent from the HAT cultures. When the protein numbers in each of the above types were considered, without taking into account the abundance of each of them  only showed moderate changes as discussed above . Therefore, a more detailed analysis in the next sections focused on the 50 more abundant (top-50) proteins, whose references (JGI-ID #), type, predicted function, presence/absence of predicted signal peptide, and abundances  are shown in Fig. The abundances of the 508 P. ostreatus secretomes, 16 % were shared by the poplar wood and wheat straw secretomes, and much lower numbers were shared by the poplar–HAT (4 %) and straw–HAT (7 %) secretomes  families CE16  and PL8  only detected in the poplar and wheat straw cultures; and (iii) family CBM13 with JGI-ID# 116255 present in the three secretomes.Other CAZy groups also include well-represented proteins in the P. ostreatus secretomes. Additionally, 27 % were shared by the poplar and wheat straw secretomes, and the HAT secretome only shared 2 % proteins with each of the lignocellulose secretomes , heme peroxidases, and oxidases generating hydrogen peroxide from the glucose/methanol/choline oxidase/dehydrogenase (GMC) and copper radical oxidase (CRO) superfamilies. However, only laccases and heme peroxidases of the LiP, MnP, and VP families are recognized as LMEs. The above oxidoreductases have been considered as auxiliary enzymes in CAZy families AA1 (laccases), AA2 (peroxidases), AA3 (GMC oxidases/dehydrogenases), and AA5 (CRO oxidases), respectively , but theAs shown in Fig. P. ostreatus DyP4 (JGI-ID# 117204) was one of the few LME proteins in the HAT medium (0.6 % abundance), being also detected on poplar and wheat straw  and VP1 (JGI-ID# 137757) were the most abundant proteins, included in the top-50  was detected in the three secretomes  giving a gel-like material, which was directly analyzed under liquid 2D NMR conditions in heteronuclear single-quantum correlation (HSQC) experiments. The NMR spectra of the treated poplar wood and wheat straw are shown in Fig. Structural analysis of the whole wood and straw samples, without the need for previous isolation of the lignin and polysaccharide fractions, was possible by swelling the milled material in deuterated dimethylsulfoxide (DMSO-C/δH 90–150/6–8 ppm) included the different correlations of the lignin: (i) p-hydroxyphenyl units , only detected in the wheat straw; (ii) guaiacyl units ; (iii) normal syringyl units ; and (iv) Cα-oxidized syringyl units , only detected in the poplar wood. Other aromatic/olefinic correlations corresponded to p-hydroxybenzoic acid  in the poplar wood spectra, and tricin , ferulic acid , and p-coumaric acid  in the wheat straw spectra. On the other hand, the aliphatic-oxygenated region of the spectra (δC/δH 50–120/3–5 ppm) included the correlations of lignin side chains forming different substructures, such as: (i) β-O-4′ ethers (A dark blue); (ii) phenylcoumarans ; and (iii) resinols . This region also includes the carbohydrate correlations of hemicellulose, since cellulose is silent under the present conditions. Among them, the anomeric carbon (C1) signals of normal and acetylated xylose , arabinose (Ar), glucose (Gl), and uronic acid (U) units are indicated in cyan (including α- and β-xylose reducing ends), together with those of C2/C3-acetylated xylose units, while all the other carbohydrate correlations were not assigned on the spectra (gray). Finally, the prominent correlation of lignin methoxyls is also observed  in this region.The aromatic/unsaturated region of the spectra (δP. ostreatus on poplar wood or wheat straw (resulting in 7–10 % weight loss with respect to the controls) some modification of the remaining lignocellulosic material was produced, as shown by 2D NMR of the treated samples and controls. The first observation from this comparison is the increased ratio between carbohydrate  and lignin  in poplar wood (from 1.2 to 1.6) and, especially, in wheat straw (from 3.0 to 4.9) revealing a preferential degradation of lignin. The differential decrease of lignin and carbohydrate signals agrees with the strongest decrease of (Klason) lignin in wheat straw . Monosaccharide analysis in the Klason hydrolyzates only revealed slight changes of the glucose/mannose/xylose/arabinose/galactose percentages in the treated wood (71.9/13.9/7.7/4.9/1.6) and straw (66.0/11.6/11.2/9.9/1.3) with respect to the corresponding controls . Simultaneously with the relative decrease of lignin, the amount of p-hydroxybenzoic acid per 100 lignin units increased in the treated poplar wood.During the 21-day growth of p-hydroxycinnamic acids present in wheat straw were differentially degraded, and the ferulic content decreased (from 12 to 6 % of the lignin content) while the p-coumaric content increased (from 2 to 5 % of the lignin content). Finally, the flavonoid tricin seems to be specially recalcitrant since its content (referred to lignin) was twofold higher after the fungal treatment (passing from 8 to 22 %). Some changes in the xylan signals were also observed on both wheat straw and poplar wood, including the presence of stronger anomeric correlations (α/β X1(R)) corresponding to reducing ends.The modest delignification of poplar wood was accompanied by a low structural modification of the remaining lignin (and other wood components). In this way, the H:G:S:S′ ratio passed from 0:48:50:2 in the control to 0:46:54:1 in the treated wood, and the percentage of syringol and phenylcoumaran substructures per 100 lignin units passed from 6 to 4 % and from 2 to 0 %, respectively, while that of β-O-4′ ethers remains practically unchanged. However, the higher decrease of the lignin signals in wheat straw was accompanied by stronger modifications of: (i) the residual lignin H:G:S:S′ ratio, which passed from 3:57:40:0 in the control to 5:62:33:0 in the treated wheat straw; and (ii) the percentage of resinol substructures per 100 lignin units passed from 1.6 to 0 %, while that of β-O-4′ ethers was less significantly modified (no phenylcoumarans were found in wheat straw). Interestingly, the two Studies on the proteome of wood-rotting basidiomycetes have increased during the last years in parallel with the increasing number of sequenced genomes, which enabled protein identification by tandem mass spectrometry using genomic databases (such as JGI Mycocosm). Since wood (and lignin polymer) decay is necessarily an extracellular process , secreteP. chrysosporium was the first basidiomycete whose secretome was studied in carbon- and nitrogen-limited glucose media, and using/adding more complex carbon sources in/to liquid media, such as: (i) microcrystalline cellulose [P. chrysosporium secretome was also analyzed during solid-state fermentation (SSF) of red oak wood [ellulose –38; (ii)ellulose , 40;  teellulose ; and (v)ellulose   [Ganoderma lucidum during SSF of sugarcane bagasse [Trametes trogii growing on poplar wood blocks [Irpex lacteus during wheat straw SSF [with P. chrysosporium and P. ostreatus); (vi) C. subvermispora growing in liquid medium with microcrystalline cellulose and milled aspen (compared with P. chrysosporium) [Phlebiopsis gigantea growing on aspen and pine wood wafers [Pycnoporus cinnabarinus in (ligno)cellulose supplemented maltose liquid cultures, and as a SSF mixed secretome (from cultures on five different plant substrates) [Other white-rot fungi whose secretomes have been analyzed are: (i) cultures , 49; (iisporium) ; (iii) G bagasse ; (iv) Trd blocks ; (v) Irptraw SSF  (comparesporium) , 54;  .P. placenta in liquid medium with cellulose or aspen/pine wood (compared with P. chrysosporium) [Serpula lacrymans growing on pine wood [C. cinerea growing in glucose-peptone-yeast extract medium [Schizophyllum commune)—have been reported using milled-aspen liquid medium, with special emphasis on CAZy proteins [The secretomes of other lignocellulose-decaying basidiomycetes were also analyzed, including: (i) the brown-rotters sporium) , 39, 55 sporium) , and Serine wood ; and  [P. sapidus secretome [P. chrysosporium growing on wood from three different poplar genotypes [A total of 508 different proteins were identified in the secretome of ne media , 168 prot straw) , and onlecretome . A higheenotypes .P. ostreatus strongly varied, with only 20 % of the proteins being common to the three cultures analyzed. Interestingly, a similar additional percentage (19 %) was shared by the straw and poplar cultures, while the glucose culture only shared 2 and 4 % proteins with the poplar and wheat–straw cultures, respectively, revealing a strong and common effect of lignocellulose on the secreted proteins. More than half of the proteins were present only in one of the cultures, including 34 % on wheat straw, 15 % on glucose, and (only) 6 % on poplar wood. This reveals that colonization of wheat straw requires a high number of unique proteins in addition to those shared with the poplar culture (and the 20 % common to the three growth conditions). Moreover, strong differential production was observed for some of the shared proteins, as discussed below.The secretome of The above differences were analyzed considering the relative numbers and abundances of the main protein types. Most of them corresponded to the groups of oxidoreductases, CAZys  or unknown function proteins, with the three types showing similar diversities (protein numbers) in the poplar and straw secretomes. However, in the HAT medium the diversity of CAZys was higher, although no (ligno)cellulosic substrate was present.The differences are more remarkable when the abundances of each of the proteins were considered. In this way, it was shown that oxidoreductases (including LMEs), are by large the most abundant proteins in the two lignocellulose cultures, and less abundant in the glucose medium, where proteases and unknown proteins had larger, and CAZy slightly lower, abundances than in the lignocellulose cultures. Moreover, noteworthy differences in the different oxidoreductase “superfamilies” were observed, with peroxidases and especially multicopper oxidases (laccases) being abundant in the lignocellulose cultures but nearly absent from the glucose medium, where other oxidoreductases (including different oxidases) were more abundant. The unknown proteins, whose abundance was emphasized in early studies on wood-rotting fungal secretomes , still rP. ostreatus secretomes, but only one of them (CE16 acetylesterase) was clearly overproduced on lignocellulose (with respect to the glucose medium). When the woody and nonwoody lignocellulosic secretomes were compared, GH1 and GH3 (two β-glycosidases) were significantly more abundant in the poplar and wheat straw cultures, respectively. Among less abundant proteins, GH51 and GH35 were also more represented in wheat straw. Interestingly, the first CAZy in the top-50 proteins is glucoamylase GH15, being secreted in the three culture media. All the above CAZy families have been reported in the secretomes of other white-rot fungi growing under liquid and SSF conditions, several of them  being overproduced in the presence of lignocellulosic substrates [Seven CAZy families  were among the top-50 proteins in the bstrates , 50, 53.P. ostreatus secretomes, often represented by different proteins. However, the most abundant CAZys detected  belong to the so-called “accessory” CAZy families, with GH51 endoglucanase/endoxylanase being the exception (1 % abundance in the wheat straw secretome). The above contrasts with other studies where endo-cellulases/xylanases  are among the main proteins secreted by white-rot basidiomycetes  when growing in wood-containing media [Pleurotus species [P. ostreatus under the present growth conditions, although 18 LPMO and 1 CDH genes were annotated in the (PC9) genome. This contrast with the important role attributed to these enzymes, acting synergistically in cellulose degradation by some fungal species [Most families of “bulk carbohydrate” CAZys acting on polysaccharide backbones  were found in the ng media , 40, 54. species . On the  species .P. ostreatus secretome (GH18 was the CAZy family with the highest protein number) could be related to the need of recycling nutrients  in 3-week-old cultures. Hyphal lysis would be also related to the high levels of proteases involved in nitrogen recycling, as reported in the P. chrysosporium secretome [In addition to the above CAZys involved in degradation of plant polysaccharides, at least 28 CAZy proteins putatively contributing to the autolysis of fungal cell wall were identified. Seventy-five percent of them belong to families GH16 and GH18  but members of the GH13, GH30 and GH72 families were also present. Chitinases (GH18) and β-glucanases (GH16) have been identified in the secretomes of other basidiomycetes , 43, 61.ecretome .P. ostreatus genome includes at least ten laccase genes [2+ to Mn3+, as MnPs and VPs do [P. ostreatus when growing on lignocellulosic substrates, as shown in the present secretomic study. The above results agree with a transcriptomic study of the same fungus [P. ostreatus cultures grown on lignocellulosic substrates [The se genes , 25, togse genes . After tse genes . In parad VPs do . At lease fungus , as wellbstrates –68.P. ostreatus.LACC10 is the main protein in the two lignocellulose secretomes, and LACC2, LACC9, and LACC6 are also among the top-50 proteins, together with VP1, MnP3, DyP4, and other seven oxidoreductases. These four laccases had been previously cloned, and reported as POX1 (LACC9), POX2 (LACC10), POXA1B (LACC6), and POXA3 (LACC2) –72. HowePleurotus GMC [2O2 supply to the P. ostreatus peroxidases.The above laccases, VP1 and MnP3 were significantly more abundant in the lignocellulose cultures, while oxidases of two different superfamilies—galactose oxidases from the CRO superfamily , and sevotus GMC , was a motus GMC  occupyinC. subvermispora, G. lucidum, I. lacteus, P. ostreatus, S. lacrymans, and T. trogii [P. ostreatus cultures [P. chrysosporium represents a remarkable exception to the above enzyme presence in secretomes, due to the absence of laccase genes in its genome [P. chrysosporium [P. chrysosporium LiP and MnP isoenzymes has been reported in (carbon or nitrogen limited) glucose and (ligno)cellulose-containing media [P. carnosa suggested the presence of LiP and MnP in cellulose and wood-containing media [C. subvermispora [Pleurotus species, together with several MnPs [Laccases and MnPs have been reported in the secretomes of a series of wood-rotting basidiomycetes, such as . trogii –53, 57. cultures , 83. Theosporium , 43, theng media , 39, andng media . Moreoveng media , althougrmispora , a fungurmispora . In a sirmispora , had beeral MnPs , 53, in P.ostreatus overproduction of LMEs  when growing in lignocellulose-containing media. Although laccases and peroxidases have been reported in secretomic studies of several white-rot basidiomycetes, as discussed above, the overproduction levels were in most cases much more modest than those found here for P. ostreatus. One exception could be P. cinnabarinus that secretes one laccase (JGI-ID# 8672) as the main protein in some lignocellulose-based liquid and SSF cultures [P. cinnabarinus study is the detection of MnP proteins only in the lignocellulosic secretomes supporting their contribution to lignin decay, maybe through peroxidation reactions [The most significant finding of the present secretomic study is the cultures . Anothereactions .Interestingly, the above oxidoreductase overproduction could be correlated with the chemical modification of the lignocellulosic substrates shown by 2D NMR at the gel state, a new methodology helping lignocellulose pretreatment studies , 86. Thep-coumaric and p-hydroxybenzoic acids forming ester linkages on the Cγ-hydroxyl of wheat straw and poplar lignin units, respectively [P. ostreatus oxidoreductases with respect to lignin units (and carbohydrates), while p-coumaric acid, p-hydroxybenzoic acid, and tricin would be more recalcitrant toward the fungal attack. The differences in cinnamic acid removal may be related to the higher methoxylation degree of ferulic compared to p-coumaric acid enabling its oxidation by the secretome laccases (and improving peroxidase degradation) [The HSQC spectra also showed four aromatic compounds that are naturally incorporated to the lignin polymer: (i–ii) tricin and ferulic acid forming ether linkages on wheat straw lignin  , 90; andectively , 92. Theadation) . FinallyP. ostreatus growing both on poplar wood and wheat straw was characterized by a strong overproduction of LMEs with respect to glucose medium. These overproduced oxidoreductases included four laccases (LACC10 being the most abundant among the 434 different extracellular proteins identified in the lignocellulose cultures), one VP and one MnP. In contrast, CAZy proteins only showed slightly higher production in the lignocellulose cultures . The above results agreed with the preferential removal of lignin from the two lignocellulosic substrates shown by the ratio between the lignin (aromatic) signals and the carbohydrate (anomeric) signals in the 2D NMR spectra of the whole treated materials at the gel state, which was accompanied by structural modification of the remaining lignin and carbohydrates.Although some differences were observed between the two substrates, the secretome of P. ostreatus PC9 (CECT20311) was used in the present study. This strain was isolated (together with monokaryon PC15) from dikaryotic P. ostreatus N001 (CECT20600) [http://www.genome.jgi.doe.gov/PleosPC9_1/PleosPC9_1.home.html).Monokaryotic CT20600) . Its genP. ostreatus cultures in glucose medium and on two different lignocellulosic substrates. Glucose cultures (triplicate) were grown in 1-L shaken (200 rpm) flasks with 200 mL (surface to volume ratio of 0.7 cm−1) of HAT medium [2PO4, 0.5 g MgSO4.7 H2O, 1 g casamino acids, 1 g yeast extract, 0.368 g ammonium tartrate, and 1 L of distilled water (sterilized at 120 °C for 20 min). Inocula consisted of 15 mL of homogenized actively growing mycelium from M7GY [Triticum aestivum) straw or extractives-containing debarked poplar  small chips (particle size <4 mm in both cases) soaked with 70 mL of distilled water in 1-L flasks (surface to volume ratio of 1.9 cm−1) sterilized at 120 °C for 20 min, incubated under stationary conditions. Inocula consisted of 15 mL of homogenized mycelium from M7GY cultures. All the above cultures were maintained at 25 °C.Secretomic studies were performed on T medium  containirom M7GY  liquid c−1 trypsin in 50 mm ammonium bicarbonate, overnight at 30 °C. Peptides were extracted at 37 °C using 100 % ACN, and then 0.5 % trifluoroacetic acid, dried, cleaned using ZipTip with 0.6 μL C18 resin (Millipore), and reconstituted in 5 μL of 0.1 % formic acid in 2 % ACN.Cultures in the above media were grown for 21 days, triplicates were combined, filtered under vacuum, and the filtrates used for proteomic analyses, while the solid fraction (from the lignocellulose cultures) was used for the chemical analyses described in the next section. Total extracellular proteins in the filtrates were freeze-dried, resuspended in 10 mM tartrate (pH 5), impurities removed by a short polyacrylamide gel electrophoresis run, and stained by Colloidal Blue Kit (Invitrogen). The protein band was cut and destained using 50 mM ammonium bicarbonate in 50 % acetonitrile (ACN), reduced with 10 mM dithiothreitol for 30 min at 56 °C, alkylated with 55 mM iodoacetamide in the dark for 30 min at 24 °C, and digested with 12.5 ng·µL−1. Mass spectra were acquired in the positive ion mode and data dependent manner selecting the 20 most intense ions for fragmentation using CID (collision induced dissociation). MS spectra (m/z 300–1600) were acquired in the Orbitrap with a target value of 1,000,000 at a resolution of 30,000 (at m/z 400) and MS2 spectra were acquired in the linear ion trap with a target value of 10,000 and normalized collision energy of 35 %. Precursor ion charge state screening and monoisotopic precursor selection were enabled. Singly charged ions and unassigned charge states were rejected. Dynamic exclusion was enabled with a repeat count of one and exclusion duration of 30 s.Tryptic peptides were analyzed in an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific) coupled to a nanoEasy high-performance liquid chromatography equipment (Proxeon). Peptides were first trapped onto a C18-A1 ASY-Column 2 cm precolumn (Thermo Scientific), and then eluted onto a Biosphere C18 column  (NanoSeparations) using a 130 min gradient from 0–45 % buffer-B  at a flow rate of 250 nL.minP. ostreatus PC9 genomic database, downloaded from JGI (http://www.genome.jgi.doe.gov/PleosPC9_1/PleosPC9_1.download.html), using Sequest search engine through Proteome Discoverer (version 1.4). As for the search parameters, precursor and fragment mass tolerance were set to 10 ppm and 0.8 Da, respectively. Carbamidomethylation of cysteines was set as a fixed modification and oxidation of methionines was set as a dynamic modification. Two missed cleavages were allowed. Identified peptides were validated using Percolator algorithm with a q-value threshold of 0.01. The presence/absence of a signal peptide was predicted with SignalP 4.1 [Acquired spectra were searched against the nalP 4.1 .P. ostreatus, and from uninoculated controls kept (for 21 days) under the same conditions, were dried in an aeration oven at 65 °C until stable weight (to estimate weight losses during the treatments). The dried material was grounded in an IKA A10 cutting mill, and finely milled using a Fritsch Pulverisette six planetary mill at 400 rev·min−1 for 5 h (with 10 min breaks after every 10 min of milling) using a 500-mL agate jar and agate ball bearings (20 × 20 mm). Lignin content (as Klason lignin) was estimated as the residue after sulfuric acid hydrolysis of the samples according to Tappi test method T222 om-88 [The solid fraction from poplar wood and wheat straw treated with 22 om-88 . Neutral22 om-88 .d6 and HSQC spectra were acquired at the gel state [13C-1H correlation experiment was an adiabatic HSQC experiment . Spectra were acquired from 10 to 0 ppm in F2 (1H) with 1000 data points for an acquisition time of 100 ms, an interscan delay (D1) of 1 s, 200 to 0 ppm in F1 (13C) with 256 increments (F1 acquisition time 8 ms) of 32 scans. The 1JCH used was 145 Hz. Processing used typical matched Gaussian apodization in 1H and a squared cosine bell in 13C. The central DMSO peak was used as an internal reference (δC/δH 39.5/2.49 ppm). The aromatic 13C-1H correlation signals of the different lignin units were used for estimation of composition in p-hydroxyphenyl (H), guaiacyl (G), syringyl (S) and Cα-oxidized syringyl (S´) units, and the p-hydroxybenzoic acid (PB), p-coumaric acid (PCA), ferulic acid (FA), and tricin (T) contents were referred to total lignin (estimated as H + G+S + S´). The aliphatic 13Cα-1Hα correlation signals of the β-O-4′ ether (A), phenylcoumaran (B), and resinol (C) side chains were used to estimate the relative abundances of the above substructures per aromatic unit. The intensity corrections introduced by the adiabatic pulse program permit us to refer the latter integrals to the previously obtained number of lignin units. Assignment of lignin (and hemicellulose) signals was based on previous wheat straw and hardwood NMR studies [For NMR analysis, 100 mg of milled samples were swollen in DMSO-el state , 86, 98. studies , 99–102.


Molecular studies of the SRD5A2 revealed a new heterozygous gene variant within the coding region that results in phenotypic expression. A c.92C>T transition changing serine to phenylalanine at codon 31 of exon 1 (p.Ser31Phe) was identified in a patient with 46,XY disorder of sexual development who displayed glandular hypospadias with micropenis and bilateral cryptorchidism. The restoration of the p.Ser31Phe mutation by site-directed mutagenesis and transient expression assays using cultured HEK-293 cells showed that this novel substitution does not abolish but does deregulate the catalytic efficiency of the enzyme. Thus, the maximum velocity (Vmax) value was higher for the mutant enzyme (22.5 ± 6.9 nmol DHT mg protein−1 h−1) than for the wild-type enzyme (9.8 ± 2.0 nmol DHT mg protein−1 h−1). Increased in vitro activity of the p.Ser31Phe mutant suggested an activating effect. This case provides evidence that heterozygous missense mutations in SRD5A2 may induce the abnormal development of male external genitalia.Inactivating mutations of the 5 It is found predominantly in the stromal cells of internal and external reproductive organs. During embryogenesis, this isozyme plays a central role in the differentiation of the male phenotype by catalyzing the conversion of testosterone (T) to 5α-dihydrotestosterone (DHT), an androgen that is 10–15 times more potent than testosterone. DHT acts to drive the formation of the male urethra and prostate at the urogenital sinus and induces swelling and folding at the genital tubercle to form the penis and scrotum  and a phallus of 2.0 cm in length presenting glandular hypospadias. A pelvic ultrasound showed no Müllerian structures. The karyotype was 46,XY. By the age of 9 years 5 months, the penis had grown to 2.5 cm and the testicles had descended into the scrotal sac. When testicular reserve was assessed using the hCG stimulation test, testosterone concentration had changed from 0.45 to 2.0 ng/mL. Treatment was recommended for phallic growth with a positive response. His sex hormone profile was as follows: FSH 2.5 IU/L , LH 2.4 IU/L , basal testosterone levels 19.2 nmol/L , and DHT 0.48 nmol/L . His levels of androstenedione and estradiol were 2.0 nmol/L  and 17.5 pg/mL , respectively. At the age of 18 years, the patient's penis measured almost 6 cm in length, and the testes were 4.5 × 2.5 cm, with Tanner stages completed. He did not present breast enlargement or facial fuzz. After the signing of informed consent forms, blood samples were obtained from the patient and his mother for genomic DNA extraction; the father was not available to participate in the molecular studies. The study protocol was approved by the Institutional Ethical Committee for Investigation in Humans (INCMNSZ).SRD5A2  were assessed by exon-specific PCR, Single-Strand Conformation Polymorphism, and sequence analysis, using previously described specific primers and conditions  was used as a template to synthesize constructs. The mutant vectors were created as previously described , HSD17B3 , and NR5A1  was performed using primers designed based on database sequences.Coding sequence abnormalities in SRD5A2, revealed variations in exon 1 but not in exons 2–5. As shown in Figure Km) for testosterone was higher for the mutant enzyme (2.8 ± 0.5 μmol/L) than for the wild-type enzyme (0.7 ± 0.1 μmol/L). Likewise, the maximum velocity (Vmax) value was higher for the mutant enzyme (22.5 ± 6.9 nmol DHT mg protein−1 h−1) than for the wild-type enzyme (9.8 ± 2.0 nmol DHT mg protein−1 h−1). Based on in vitro metabolic assay data, this mutation appears to increase enzyme activity compared to the wild-type protein.Single-Strand Conformation Polymorphism and sequencing analyses of the complete coding region, including the exon–intron boundaries of Vmax/Km ratio (8.0 nmol DHT h−1 mg−1 (μmol/L)−1 T−1) than the wild-type form (14.0 nmol DHT h−1 mg−1(μmol/L)−1 T−1), which points toward an enzyme catalytically less efficient. Because the patient was originally referred for hypospadias, it was considered important to exclude genetic alterations in other candidate genes, including the androgen receptor, steroidogenic factor-1, and 17β-hydroxysteroid dehydrogenase 3. Molecular screening studies excluded mutations in the NR3C4, NR5A1, and HSD17B3 genes.However, the S31F variant displays a lower SRD5A2 gene is broad, varying from normal female external genital appearance to micropenis, isolated hypospadias, or micropenis associated with hypospadias of varying severity.The phenotypic spectrum associated with 46,XY DSD due to autosomal recessive mutations in the SRD5A2 have been reported, including 70 missense/nonsense mutations, two gross deletions, 13 small deletions, and six splicing mutations (http://www.hgmd.cf.ac.uk/ac/gene.php?gene=SRD5A2). Most of these variants are pathogenic mutations that affect binding to the cofactor Nicotinamide Adenine Dinucleotide Phosphate or the substrate, resulting in 46,XY DSD due to a deficiency of 5α-reductase. With the exception of two single-nucleotide polymorphisms (A49T and V89L), the remaining 60 single-base missense mutations have deleterious effects on enzyme activity, mainly through a loss-of-function effect  predicted that p.Ser31Phe might be damaging (score 0.874), PON-P (http://bioinf.uta.fi/PON-P/) and SIFT (http://sift.jcvi.org/) predicted a low pathogenicity for this same mutation. However, the results from expression assays in transiently transfected HEK-cultured cells showed abnormal kinetic characteristics for the p.Ser31Phe mutant, which are consistent with an atypical enzyme activity . Most patients carrying mutations in these genes are identified as heterozygous for missense mutations that impair PROKR2 signaling (Dode and Rondard NR5A1 (SF1) gene are considered a frequent cause of 46,XY DSD. It has been estimated that 5–15% of newborn male patients with genital ambiguity, underandrogenization, partial gonadal disgenesis, and absence of Müllerian derivatives are heterozygotes for mutations in NR5A1/SF1  that increases enzyme activity; however, this mutation was detected in a compound-heterozygous patient harboring a second nonsense mutation (p.P212X) in the other allele ; however, the biochemical consequences of these gene variants were not assessed , these were identified from microdissected prostate adenocarcinoma samples and were found to be somatic mutations . In summary, this report supports the concept that heterozygous missense mutations in SRD5A2 may induce masculinization defects throughout a noninactivating effect. Likely, the p.Ser31Phe mutation elicited a deregulation of the enzyme activity that led to inadequate levels of DHT to sustain the proper development of external genitalia. Such mechanisms may account for other cases of 46,XY DSD with only one affected SRD5A2 allele.In this study, we characterize a molecular defect in a case of 46,XY DSD in which the causative mutation was found to be a novel heterozygous p.Ser31Phe substitution. This mutation appears to increase rather than decrease enzyme activity. To date, approximately 96 different genetic variants of ted Chan  yielded


Betanodaviruses are the causative agents of viral nervous necrosis and affect a broad range of fish species worldwide. Their bi-segmented genome is composed of the RNA1 and the RNA2 molecules encoding the viral polymerase and the coat protein, respectively. In southern Europe the presence of the RGNNV and the SJNNV genotypes, and the RGNNV/SJNNV and RGNNV/SJNNV reassortants has been documented. Several studies have reported a correlation between water temperature and disease onset. To explore the replication efficiency of betanodaviruses with different genomes in relation to temperature and to understand the role of genetic reassortment on viral phenotype, RGNNV, SJNNV, RGNNV/SJNNV and RGNNV/SJNNV field isolates were fully sequenced, and growth curves generated in vitro at four different temperatures  were developed for each isolate. The data obtained, corroborated by statistical analysis, demonstrated that viral titres of diverse betanodavirus genotypes varied significantly in relation to the incubation temperature of the culture. In particular, at 30 °C betanodaviruses under investigation presented different phenotypes, and viruses containing the RNA1 of the RGNNV genotype showed the best replication efficiency. Laboratory results demonstrated that viruses clustering within the same genotype based on the polymerase gene, possess similar growth kinetics in response to temperature, thus highlighting the key role of RNA1 in controlling viral replication at different environmental conditions. The results generated might have practical implications for the inference of viral phenotype according to genetic features and may contribute to a better understanding of betanodavirus ecology. The increasing amount of genetic information obtained from viral genomes sequencing, aids research into the exploration of the genotype-phenotype relationships to determine the genetic traits responsible for different phenotypic features, which in turn may have practical implications for disease recognition and control. Nevertheless, the phenotype of a virus is not only dependent on its “intrinsic” genetic features  but it is also governed by “extrinsic” variables -4. AmongBetanodavirus are the causative agents of a highly infectious fish disease known as viral nervous necrosis (VNN), also known as viral encephalopathy and retinopathy (VER). The genome of betanodaviruses is composed of two single stranded positive sense RNA molecules. The RNA1 segment encodes the RNA-dependant RNA polymerase (RdRp), or protein A, and gives rise to the RNA3 sub-genomic transcript which is translated into proteins B1 and B2, whilst the RNA2 segment encodes the coat protein (CP) [Scophthalmus maximus named as turbot nervous necrosis virus (TNNV) is awaiting for official classification [Viruses within the genus ein (CP) -8. The pein (CP) . A putatfication . Iwamotofication  demonstrfication . More refication ,14. Notefication . Cold wafication -21. The fication ,22-25. Cfication ,24,26-33fication ,29,32. TIn order to identify the genetic regions involved in temperature dependency of piscine nodaviruses, the infectious RNA transcription system established by Iwamoto et al.  has receDicentrarchus labrax). However, strain 484.2.2009 isolated from a Senegalese sole  was also included in the selection, because to date no SJNNV infection has been reported in sea bass.On the basis of previous phylogenetic analysis of partial RNA1 and RNA2 sequences ,36, fourg for 15 min at 4 °C, and subsequently subjected to further investigations.Betanodavirus isolates were propagated in E-11 cell monolayers  incubatFor each virus, total RNA was extracted from 100 μL of cell culture supernatant using the NucleoSpin® RNA II , and subsequently treated with Tobacco Acid Pyrophosphatase (TAP)  for 5’-cap removal. The 3’ and 5’ ends were ligated with the T4 RNA Ligase 1  in a 25 μL reaction mix. Briefly, 15 μL of de-capped RNA were incubated for 5 min at 65 °C with 20U rRNasin RNase Inhibitor , 1X T4 RNA Ligase Reaction Buffer, 10% dimethyl sulfoxide (DMSO) and water, and subsequently cooled on ice for 2 min. The reaction mix was then incubated for 60 min at 37 °C with 20U of rRNasin RNase Inhibitor (Promega) and 20U of T4 RNA Ligase 1 (New England BioLabs). Enzymes were inactivated by incubating the reaction mix for 10 min at 65 °C. Circular RNA was subjected to reverse transcription (RT) for cDNA synthesis by using the SuperScript® III Reverse Transcriptase  according to the manufacturer’s instructions and adding 10% DMSO to the reaction mix. PCR was subsequently carried out in a final volume of 25 μL containing 2 μL of template cDNA, 1X Cloned Pfu DNA polymerase reaction buffer, 240 μM dNTPs, 1.25U Pfu Turbo DNA polymerase  and 0.2 μM specific forward and reverse primer hybridizing respectively upstream the 3’ end and downstream the 5’ end. Primer sequences are available upon request. The cDNA was denaturated at 95 °C for 2 min, and 40 cycles of the following conditions were applied: denaturation at 95 °C for 45 s, annealing at 50 °C for 1 min, elongation at 72 °C for 2 min. The amplification was completed with 2 min elongation at 72 °C. PCR products were subjected to electrophoresis in agarose gel, and were subsequently purified using the QIAquick Gel Extraction Kit . The sequences of the 3’-5’ junctions were generated using the BigDye Terminator v3.1 cycle sequencing kit (Life Technologies). The products of sequencing reactions were cleaned up using the Performa DTR Ultra 96-Well kit  and sequenced in a 16-capillary ABI PRISM 3130×l Genetic Analyzer (Life Technologies). Sequencing data were assembled and edited with the SeqScape® software v2.5 (Life Technologies).In order to obtain complete genetic data related to the four selected betanodavirus strains, the nucleotide sequences corresponding to the full length RNA1 and RNA2 were determined for each virus. Previously de-capped RNA was subjected to RT by using the High Capacity cDNA Reverse Transcription Kit (Life Technologies) following the manufacturer’s instructions. The identification of the 3’ and 5’ terminal sequences allowed to design of specific primer sets for the whole genome amplification. PCR and sequencing were carried out as described above. Primer sequences can be provided on request.The obtained sequences were aligned and compared to reference betanodavirus sequences publicly available in GenBank using the MEGA 4 package . For botinocula were brought to the same volume with L-15 medium without growth factor (FCS). Inoculated monolayers were then incubated at 15, 20, 25 and 30 °C and checked regularly for CPE. A volume of 700 μL was sampled from each flask at 0 (T0), 20 (T1), 30 (T2), 45 (T3), 55 (T4), 69 (T5), 79 (T6), 93 (T7), 117 (T8), 141 (T9) and 165 (T10) hours post infection (hpi), and subsequently replaced with an equal amount of medium. Collected cell culture supernatants were subjected to viral titration in E-11 monolayers by endpoint dilutions assays. Viral titres were calculated according to the Spearman-Karber formula [50/mL were calculated among replicates, and were used for developing growth curves for each incubation temperature.E-11 cells were grown in 5 mL culture flasks with Leibovitz medium (L-15) (Sigma-Aldrich), supplemented with 10% FCS, L-Glutamine (2 mM) and antibiotics . Cell monolayers were infected in three replicates with each of the isolates at a multiplicity of infection (MOI) of 1.0. After 1 h adsorption at 25 °C, all the  formula . FinallyP-values < 0.10 were considered significant. Graphical Student residuals analysis  and tests for residuals normality (Shapiro Wilks and Kolmogorov Smirnov tests) were used to verify the goodness of the proposed models [The linear mixed model (LMM) for longitudinal data  was usedd models . SAS sofd models ,43.The RNA1 genetic segment differed in length among the four betanodavirus strains. The 5’-untranslated regions (UTRs) were 78 nt long, whereas the 3’-UTRs varied from 75 to 77 nt. Strains 283.2009 and 367.2.2005 showed a 2949 nt ORF, corresponding to 983 aa, while the RNA1 coding region of samples 484.2.2009 and 389/I96 was 3 nt longer, determining an amino acid insertion in the RdRp at position 888. The RNA2 5’-UTRs and 3’-UTRs varied from 26 to 27 nt and from 370 to 390 nt, respectively. The complete ORF for strains 283.2009 and 389/I96 was 1017 nt, encoding a coat protein of 339 amino acids. Viruses 484.2.2009 and 367.2.2005 presented an insertion of 6 nt within the T4 variable region , correspNucleotide and amino acid similarities calculated for the RNA1 and the viral polymerase are 96.4% and 98% between strains 283.2009 and 367.2.2005, and 97.2% and 98.3% between strains 484.2.2009 and 389/I96. The RNA1 nucleotide sequence of the RGNNV and the RGNNV/SJNNV viruses was 97.6 and 96.7% identical to that of strain SGWak97 (corresponding to 99.3 and 97.6% amino acid identity). The polymerase gene of the SJNNV and of the SJNNV/RGNNV viruses was 97.1 and 97.8% identical to that of strain SJNag93 (corresponding to 97.8 and 98.1% amino acid identity). Pairwise similarity was calculated also for the RNA1 nucleotide region spanning from position 84 to position 1419 and putatively responsible for betanodavirus temperature sensitivity , which c7) onwards, an alteration of all the infected monolayers was observable, where cells appeared dark and contracted and tended to detach from the surface of the flask. However, no specific lesions related to betanodavirus infection (i.e. vacuolization of cells) were detected and none of the four viruses determined the complete disruption of cell monolayers at this temperature. At 20 °C, no CPE was noticed at the early stages of the infection. However, at 69 hpi (T5) all the betanodavirus strains under investigation showed typical cellular vacuoles which evolved into extended foci of rounded, granular and vacuolated cells after 93 hpi (T7). All the viral strains determined the complete disruption of cell monolayers by 117 hpi (T8). At 25 °C, the RGNNV-type, the SJNNV-type and the two reassortants showed the same phenotype: after 69 hpi (T5) the presence of diffuse CPE was observed, resulting in the disruption of the monolayers at 93 hpi (T7). Interestingly, clear phenotypic differences among the four betanodavirus strains were noticed at 30 °C. In particular, the RGNNV strain and the RGNNV/SJNNV reassortant determined an early appearance of CPE just after 45 hpi (T3), which became more severe at 69 hpi (T5). The complete disruption of cell monolayer was observable after 93 hpi (T7). Noteworthy, the SJNNV strain and the SJNNV/RGNNV reassortant showed a completely different phenotype. In particular, the SJNNV/RGNNV strain determined the appearance of diffuse vacuoles after 69 hpi (T5), while no CPE was observable for the SJNNV isolate. By 93 hpi (T7) both the SJNNV strain and the SJNNV/RGNNV reassortant induced the emergence of multiple foci of vacuolated cells. CPE severity worsened over time, but none of these strains determined the complete disruption of the cell monolayers by the end of the experiment.Inoculated cells were inspected regularly for the appearance of CPE Table . At 15 °5.1 TCID50/mL; SJNNV: 105.7 TCID50/mL; RGNNV/SJNNV: 105.3 TCID50/mL; SJNNV/RGNNV: 106.1 TCID50/mL). At 20 °C, all the viruses displayed comparable kinetics. However, this temperature showed a higher compatibility with an improved replication fitness, determining a regular increase of viral titre over time, particularly for strains SJNNV and SJNNV/RGNNV. The higher titres were obtained at 117 hpi . At 25 °C, the RGNNV strain and the two reassortants showed an acute growth trend characterized by a rapid and efficient replication, yielding high titres (107.9 TCID50/mL) after 93 hpi. However, this condition was suboptimal for the SJNNV strain, which showed a slow growth and had a lower titre (106.3 TCID50/mL after 93 hpi). Interestingly, at 30 °C the differences in replication efficiency among strains became striking. RGNNV and RGNNV/SJNNV rapidly multiplied during the first hours of incubation, reaching the peak of replication  only after 55 hpi. The acute phase was followed by a progressive decrease in viral titres, until the cell monolayer was completely disrupted. On the contrary, the SJNNV and the SJNNV/RGNNV betanodaviruses showed a chronic growth trend, characterized by a slow and poor replication. The higher titres were obtained at 117 hpi .The average titres determined in culture supernatant samples collected at different time points were used to develop graphs describing the growth kinetics of the RGNNV and the SJNNV genotypes and their natural reassortants at different incubation temperatures Figure . At 15 °9 (141 hpi) and T10 (165 hpi) at 20 °C and T8 (117 hpi), T9 (141 hpi) and T10 (165 hpi) at 25 °C are missing for all viruses, as well as data associated with T8 (117 hpi), T9 (141 hpi) and T10 (165 hpi) at 30 °C for viruses RGNNV and RGNNV/SJNNV. This is why statistical analysis was performed using a reduced dataset, so as to have the same number of exposure times  for each temperature and genotype.Given the diverse phenotypes and growth kinetics of viral strains at different incubation temperatures, data related to the observation points TThe first tested model, where replicates were considered as fixed effects, demonstrated the repeatability of the experiment, highlighting that differences in titres measurements among different replicates are not significant.The best LMM selected for longitudinal data presented genotype, exposure time, temperature and their interaction as fixed effects while the replicate was considered as a random effect, with a AR1) structure of VAR/COV matrix (ρ = 0.18). Graphical Student residuals analysis, Shapiro Wilks and Kolmogorov Smirnov tests of posterior analysis, confirmed the goodness of the proposed model .In order to evaluate whether the statistical observations might have been biased by the use of a restricted dataset, additional analyses were performed considering all the observation points up to 165 hpi for strains RGNNV, SJNNV, RGNNV/SJNNV and SJNNV/RGNNV at 15 °C, and for viruses SJNNV and SJNNV/RGNNV at 30 °C. The model developed for 15 °C showed no significant differences in the replication fitness of strains RGNNV, SJNNV, RGNNV/SJNNV and SJNNV/RGNNV. At 30 °C, the difference in terms of replication efficiency observed between strains SJNNV and SJNNV/RGNNV in the time slot TGenerally speaking, strains RGNNV and RGNNV/SJNNV showed similar growth trends at 15, 20, 25 and 30 °C. Similarly, the SJNNV and the SJNNV/RGNNV viruses exhibited comparable replication fitness at all the temperatures considered. Differences in viral titres between pairs RGNNV-RGNNV/SJNNV and SJNNV-SJNNV/RGNNV became more evident at 30 °C.Betanodavirus natural infections can occur at different water temperatures, depending on the genotype. The association between the onset of VNN and environmental conditions has been documented in several papers ,46. A nu7-RGNNV vs Glu7-SJNNV; Met223-RGNNV/Leu223-RGNNV vs Lys223-SJNNV; Thr241-RGNNV vs Leu241-SJNNV; Pro251-RGNNV vs Gln251-SJNNV. Positive-strand RNA viruses commonly associate their polymerase and viral RNA to the membranes of cellular organelles to replicate their genomes [nodaviridae family, namely AHNV, Flock house virus (FHV), GGNNV, Nodamura virus, Wuhan Nodavirus (WhNV), have shown to replicate in the mitochondria [Interesting observations resulted from the sequencing analysis carried out in this study. Within the amino acid region 1–445 which controls betanodavirus temperature dependency , four tr genomes . Severalchondria ,51,53-55Results on viral replication obtained in this study were fully corroborated by extensive statistical analysis, and substantiate previous findings reported by Hata et al. , achieveRecently, Vendramin et al.  have comEpinephelus septemfasciatus) with a live vaccine strain by keeping the water temperature at 17 °C. This vaccination strategy, despite the obvious limitations for application in open farms or sea cages, might be used inside hatcheries or, alternatively, during transportation of fish to reduce the volume of vaccine needed [Data gained in the present study might have practical implications, as they could help infer the viral phenotype on the basis of the genetic data. Furthermore, the in vitro characterization of the viral phenotype appears to be a suitable methodology for the prediction of betanodavirus pathogenicity under controlled environmental conditions, and might have important applications in fish farming and vaccinology. Indeed, Nishizawa et al.  applied e needed . AdditioVNN: viral nervous necrosis; VER: viral encephalopathy and retinopathy; RdRp: RNA-dependant RNA polymerase; CP: coat protein; SJNNV: Striped jack nervous necrosis virus; TPNNV: Tiger puffer nervous necrosis virus; BFNNV: Barfin flounder nervous necrosis virus; RGNNV: Red-spotted grouper nervous necrosis virus; TNNV: Turbot nervous necrosis virus; SSN-1: Striped snakehead cell line; FCS: Foetal calf serum; CPE: Cytopathic effect; TAP: Tobacco acid pyrophosphatase; DMSO: Dimethyl sulfoxide; RNA: Ribonucleic acid; RT: Reverse transcription; cDNA: Complementary deoxyribonucleic acid; PCR: Polymerase chain reaction; dNTPs: Deoxinucleotides; ML: Maximum likelihood; GTR: General time-reversible; MOI: Multiplicity of infection; TCID: Tissue culture infectious dose; LMM: Linear mixed model; VAR/COV: Variance and covariance matrix; CS: Compound symmetry; CSH: Heterogeneous compound symmetry; UN: Unstructured, AR(1): first order autoregressive; AR(1): First order autoregressive; UTRs: Untranslated regions; ORF: Open reading frame; TMD: Transmembrane domain; GGNV: Greasy grouper nervous necrosis virus; AHNV: Atlantic halibut nodavirus; FHV: Flock house virus; WhNV: Wuhan nodavirus.The authors declare that they have no competing interests.GC conceived the study and coordinated the work described. VP, AT, CT were involved in the experimental design. EC, VP, AM performed the experiments and interpreted the results. MM carried out the statistical analysis. VP wrote the manuscript. GC, AT, CT were involved in the interpretation of the results and critically read the manuscript. All authors read and approved the final manuscript.Graph Student residual analysis: residuals versus predicted plot, residuals distribution and residuals Q-Q plot. Residuals without a particular trend, with normal distribution and good alignment over line indicate that the model is correct. The hypothesis of normal distribution of residuals is further tested using the Shapiro-Wilk and Kolmogorov-Smirnov test. Value of p > 0.10 indicates that the residual has a normal distribution.Click here for file


Synoviocytes from 12 osteoarthritic (“oaTSB”) and 6 normal joints (“nTSB”) were cultured as tensioned bioscaffolds and compared for their ability to synthesize fibrocartilage sheets. Gene expression of collagens type I and II were higher and expression of interleukin-6 was lower in oaTSB versus nTSB. Compared with nTSB, oaTSB had more glycosaminoglycan and alpha smooth muscle staining and less collagen I and II staining on histologic analysis, whereas collagen and glycosaminoglycan quantities were similar. In conclusion, osteoarthritic joint—origin synoviocytes can produce extracellular matrix components of meniscal fibrocartilage at similar levels to normal joint—origin synoviocytes, which makes them a potential cell source for canine meniscal tissue engineering.Meniscal tears are a common cause of stifle lameness in dogs. Use of autologous synoviocytes from the affected stifle is an attractive cell source for tissue engineering replacement fibrocartilage. However, the diseased state of these cells may impede  Meniscal injury is a common cause of lameness and pain of the dog. Due to the virtually absent healing response in the majority of the meniscus, injured meniscal tissue is commonly removed to relieve the clinical signs of lameness, joint locking, and painful popping. Unfortunately, partial meniscectomy hastens the development of secondary arthritis  and thusin vitro culture and extracellular matrix (ECM) formation. Because cells cannot be synthesized de novo, they must be harvested autologously, or obtained from living or deceased tissue donors. When determining ideal cell sources for tissue engineering, location of the source tissue, quantity of donor tissue available for harvest, and ability to harvest the cells in a minimally invasive fashion must be considered. Autologous cells are particularly attractive because they have a low potential for infectious disease transmission , CD29 (β- integrin), CD44  and negative for markers of hematopoetic progenitors (CD34) and leukocyte antigens  osteoarthritis as per Institutional Animal Care and Use Committee approval. Dogs were treated for degeneration of the cranial cruciate ligament and medial meniscal injury via exploratory arthroscopy, partial meniscectomy if indicated, and tibial plateau leveling osteotomy. Synovial villi were arthroscopically harvested during routine partial synovectomy using a tissue shaver  as previously described . SynoviaNormal synovium was also harvested from six dogs which were euthanatized via sodium pentobarbital overdose for reasons unrelated to the study, as per the Institutional Animal Care and Use Committee Protocol and in accordance with the American Veterinary Medical Association Humane Euthanasia Guidelines. Dogs were assessed by a Diplomate of the American College of Veterinary Surgeons—Small Animal to not have any orthopedic disease based on medical history, pre-mortem physical examination, and post-mortem gross joint evaluation. Post-mortem, a lateral arthrotomy and patellar tendon transection was performed on each stifle joint. The parapatellar, suprapatellar, lateral, and medial wall synovium were dissected off the joint capsule using a #15 bard parker blade. Synovium was transported as described above. In the laboratory, synovium harvested from normal joints was additionally was minced into 2 × 3 mm pieces using sterile technique.2, 95% humidity, with sterile media change performed every 24 h.Osteoarthritic joint—origin synovial villi and normal joint—origin synovium tissue fragments were completely digested with sterile Type 1A clostridial collagenase 10 mg/mL in RPMI 1640 solution (Invitrogen) over 2–6 h at 37 °C. Tissue was deemed to be completely digested when no ECM could be visualized microscopically at 20 × objective magnification. Cells were cultured in monolayer for four passages to isolate Type B fibroblast-like synoviocytes  and Type2 flasks and allowed to become hyperconfluent cell sheets, defined as cells overlapping each other in greater than 100% confluency , , ; and glyn (ASM), , includin (ASM), . Differeviocytes .2, 95% humidity. Cells were then visualized in at least five regions of the bioscaffolds,  using a laser microscope . The number of viable (green) and non-viable (red) cells per each field counted by hand. Due to the complex three-dimensional nature of the bioscaffolds, these cell counts provided an estimate of cell viability.One TSB per dog was washed three times in sterile phosphate buffered saline and immersed in 4 µM ethidium homodimer and 6 µM acetomethoxy calcein  solution  for 20 min at 37.6 °C, 5% COTwo TSB per dog were fixed in 10% buffered formalin, paraffin embedded, and tissue blocks cut in 4 µm sections for histologic and immunohistologic analysis. All slides were labelled with randomly generated acquisition numbers and analyzed in a blinded fashion. Sections were stained with Hematoxylin and Eosin (“H&E”), Masson’s Trichrome, and Toluidine Blue. Cell morphology and general ECM architecture was assessed using H&E; organization and intensity of collagen staining was described using Masson’s Trichrome, and intensity of GAG staining was assessed using Toluidine Blue.Immunohistochemistry was performed as previously described  for typeOne TSB per dog was lyophilized and a dry weight obtained. Samples were digested in 1.0 ml Papain Solution (2 mM Dithiothreitol and 300 µg/ml Papain) at 60 °C in a water bath for 24 h. This papain digest solution was used to obtain double stranded DNA (dsDNA), GAG and collagen content of the bioscaffolds.Double stranded DNA quantification assay  was performed per manufacturer’s instructions; double stranded DNA extracted from bovine thymus was used to create standards of 1,000, 100, 10, and 1 ng/mL. Standard and sample fluorescence was read by a fluoromoter  at 485 nm excitation/ 528 nm emission, and dsDNA concentration was determined based on the standard curve.Glycosaminoglycan content was determined by the di-methyl-methylene blue sulfated glycosaminoglycan assay  using a One TSB per dog was snap frozen in liquid nitrogen and stored at −80 °C. Total RNA was isolated using the phenol-chloroform extraction  with sliThe RNA quality and quantity was determined using capillary electrophoresis , and RNA integrity numbers  were determined .First-strand cDNA synthesis was performed from 400 ng total RNA  and Oligo-(dT) 20 primers. To control for possible genetic DNA contamination, non reverse-transcribed samples were also processed.® Primers and Probes; Applied Biosystems Inc., Foster City, CA) were obtained for each of the genes of interest: IL-1β, IL-6, TNF-α, SOX-9 (an embryonic chondrogenic transcription factor), collagen type I α1, collagen type II α1, aggrecan, and the reference gene GAPDH (see Pre-designed primers and probes (Taq-ManAPDH see . All assCT−ΔΔ = [(CT gene of interest − CT housekeeping gene GAPDH)nTSB − (CT gene of interest − CT housekeeping gene GAPDH)oaTSB].Quantitative gene expression was determined in triplicates using the comparative CT method . The genP < 0.05. Data were analyzed with a statistical software program Graph Pad Prism, San Diego, CA.A D’Agostino & Pearson omnibus normality test was performed on all data to test for normality. Cell harvest data was non-parametric data and was analyzed with a Wilcoxon matched-pairs signed rank test, and data reported as median and interquartile range. Significance was declared at t-test, assuming unequal variances. The effect of osteoarthritis on the histologic scoring of TSB extracellular matrix formation was analyzed using a non-parametric Mann–Whitney U-test; ranking of the histologic scores was performed using a Kruskal-wallis analysis on ranks followed by a Fisher’s exact test. Significance was declared at P < 0.05. Data were analyzed using Statistical Analysis System, version 9.3 .The effect of osteoarthritis  on gene expression and ECM composition was analyzed using a 2-tailed Student’s 6 ± 3.7 × 105 cells per joint, and cells were 99.5% ± 0.002 viable at harvest.The mean age of dogs with stifle osteoarthritis was 4.7 years (range: 2–8 years). Breeds represented included: Golden Retriever (1), American Staffordshire Terrier (2), Labrador Retriever (3), Australian Shepherd (1), Rottweiler (2), Boston Bull Terrier (1), Goldendoodle (1), and mixed breed (1), with 7 neutered males, 4 spayed females, and one intact female dog. As observed by a Diplomate of the American College of Veterinary Surgeons–Small Animal, all dogs had marked villous synovial hyperplasia and osteophytosis, and grade 1–2 Outerbridge cartilage lesions of the medial femoral condyle and tibial plateau . Cell yi7 ± 2.6 × 106 per joint and cells were 99.5% ± 0.01 viable. As the entire stifle joint synovial membrane could be harvested post mortem, a greater volume of tissue and thus greater cell numbers were obtained from the normal joints versus arthroscopic harvest of the osteoarthritic joints (P = 0.01).Mean age of dogs with normal stifles was 4.3 years (range: 3–6 years); breeds represented included: Red Tick Hounds (4), Labrador Retriever (1), and American Staffordshire cross (1), with 3 female intact dogs, 2 male intact dogs, and one neutered male. Cell yield per joint was 1.4 × 102 flasks in order to have enough TSB for tissue analyses. This, however, resulted in greater cell seeding numbers for nTSB versus oa TSB. Thus, normal joint—origin synoviocytes were seeded at 1.49 × 107 cells per flask, wherease 6.52 × 106 osteoarthritic joint—origin cells were seeded per flask. At 4th passage, normal joint—origin cells were 99.0 ± 0.4% viable compared with 98.8 ± 0.4% viability of osteoarthritic joint—origin cells (P = 0.85). Culture duration from tissue harvest to hyperconfluent cell membrane formation and synthesis of TSB was 37.6 days and similar for both cell origins (range 20–49 days).At 4th passage, cells were transferred into eight 150 cmDuring the first week of tensioned bioscaffold culture, the culture media phenol red pH indicator changed to yellow by the time the 24 h media change was required, indicating marked increase in media acidity. In addition, during the first 7–10 days of culture, approximately 2–3 bioscaffolds per normal and osteoarthritic joint unraveled or slipped off their wire hoops (no group differences observed), and were not analyzed in this study. The typical appearance of intact nTSB and oaTSB is pictured in P = 0.008).At harvest, tensioned bioscaffolds from normal dogs had a dry weight of 39.3 mg (range 27.5–50.4 mg), which was more than for oaTSB . Cell viability was not associated with peripheral versus central location on the TSB. Laser microscopy revealed cells with fusiform, fibroblastic morphology, oriented parallel with the vector of tension, as well as the presence of acellular, circular regions in the bioscaffolds. Hematoxylin and eosin staining revealed highly cellular bisocaffolds, with layers of fibroblastic cells organized in parallel, as sheets or bands, or variably arranged in whorls, with eosinophilic ECM . SubjectP = 0.01).Percent dsDNA content was used to quantify tissue cellularity. Despite an initial higher seeding cell count at 4th passage, dsDNA accounted for 0.11 ± .02% of nTSB dry weight, versus 0.21 ± 0.03% of oaTSB dry weight  and type II collagen  and a lower gene expression of interleukin-6  versus nTSB. No significant changes were observed for relative expression of SOX-9 (P = 0.72), aggrecan (P = 0.84), and tumor necrosis factor-α  were found in any bisocaffolds .in vitro canine synoviocyte fibrochondrogenesis in monolayer culture  and oaTSB (16%) did not reach that of the healthy meniscus, at 60–70% of dry weight (Other osteoarthritic cell types, such as chondrocytes, have reduced cell proliferation compared to normal cells in monolayer culture . In conty weight , althougy weight .in vitro chondrogenic activity (α gene, indicating an inflammatory response in in vitro culture (Consistent with prior studies , all oaTactivity  by contractivity . Synoviuactivity . Both nT culture , indepenβ-1 (β-1 present in FBS (Decreased IL-6 in oaTSB may have reflected better mechanical homeostasis  in the cβ-1 . Endogent in FBS  may explt in FBS . Given tWhen cultured as TSB in high concentrations of FBS, osteoarthritic joint—origin synoviocytes can produce ECM components of meniscal fibrocartilage at similar levels to normal joint—origin synoviocytes. Potential reasons for this include increased collagen and decreased IL-6 gene expression and the greater GAG and ASM staining in oaTSB compared with nTSB Osteoarthritic joint—origin synoviocytes are a viable cell source toward meniscal tissue engineering. Further investigation of culture environments to optimize cell viability and ECM formation and strength are justified due to the promising data reported here.10.7717/peerj.581/supp-1Supplemental Information 1Data sheet showing cell viability, ECM content, gene expression, and histologic scores for tensioned bioscaffolds originating from cell harvested from normal and osteoarthritic joints.Click here for additional data file.10.7717/peerj.581/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.581/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.581/supp-4Appendix S1Assays used for RT-PCRClick here for additional data file.


The aim of the study was to characterize synovial cells from OA synovium with low-grade and moderate-grade synovitis and to define the role of synovial macrophages in cell culture.Synovial tissue explants were analyzed for the expression of typical markers of synovial fibroblasts (SF), synovial macrophages (SM) and endothelial cells. Synovial cells at passage 1 (p.1) and 5 (p.5) were analyzed for different phenotypical markers by flow cytometric analysis, inflammatory factors by multiplex immunoassay, anabolic and degradative factors by qRT-PCR. P.1 and p.5 synovial cells as different cell models were co-cultured with adipose stem cells (ASC) to define SM effects.Synovial tissue showed a higher percentage of CD68 marker in moderate compared with low-grade synovitis. Isolated synovial cells at p.1 were positive to typical markers of SM  and SF , whereas p.5 synovial cells were positive only to SF markers and showed a higher percentage of CD55 and CD106. At p.1 synovial cells released a significantly higher amount of all inflammatory  and some anabolic (IL10) factors than those of p.5. Moreover, p.1 synovial cells also expressed a higher amount of some degradative factors  than p.5 synovial cells. Co-culture experiments showed that the amount of SM in p.1 synovial cells differently induced or down-modulated some of the inflammatory  and degradative factors .We found that p.1 (mix of SM and SF) and p.5 (only SF) synovial cells represent two cell models that effectively reproduce the low- or moderate-grade synovitis environment. The presence of SM in culture specifically induces the modulation of the different factors analyzed, confirming that SM are key effector cells.The online version of this article (doi:10.1186/s13075-016-0983-4) contains supplementary material, which is available to authorized users. Osteoarthritis (OA) is defined as a disease of the whole joint because it affects not only the cartilage but the subchondral bone and the synovial tissue that undergo structural and metabolic modifications , 2. DiffSynovial inflammation is a process characterized by synovial thickening (hypertrophy and hyperplasia) and cell infiltration (lymphocytes and macrophages) , 9. HistNormal synovial layers in OA are composed of synovial fibroblasts (SF) and inflammatory leukocytes (lymphocytes and macrophages) . SF are Up to now there has been no in-depth characterization of the synovium and isolated synovial cells in OA. Recent papers , 21 haveTo gain new insight into this topic we first analyzed synovial tissue biopsies for the expression of typical markers of SF, SM and endothelial cells and then we followed their expression in isolated synovial cells both at passage 1 (mix of SF and SM), and passage 5 (SF). Subsequently, we performed in-depth analysis of isolated cells for different phenotypical markers, inflammatory, anabolic and degradative factors. Finally, as inflammation induces adipose stem cells (ASC) to exert anti-inflammatory effects , we used2, disease duration 7 ± 4.8 years) and Kellgren/Lawrence grade 3/4 [Synovial tissues were obtained from 26 patients with OA  at room temperature for 2 h and embedded in paraffin, and serial tissue sections (4 μm thick) from each specimen were prepared and routinely stained with hematoxylin-eosin. The histopathological features of each synovial tissue specimen were evaluated according to the synovitis inflammation scoring system described by Krenn , which r2 area.Serial sections were incubated overnight at 4 °C with monoclonal anti-human-CD55 , −CD68 , −Factor VIII (10 μg/ml Dako), −CCL3/MIP1α  and -S100A8 (4.5 μg/ml R&D) diluted in Tris-buffered saline (TBS) containing 0.1 % bovine serum albumin (BSA). Samples were then rinsed in TBS and sequentially incubated at room temperature for 20 minutes with multilinker biotinylated secondary antibody  and alkaline phosphatase-conjugated streptavidin . The reactions were developed using fast red substrate , counterstained with hematoxylin, and mounted in glycerol gel. Negative controls were performed using isotype control (Dako Cytomation). Semiquantitative analysis of immunohistochemically stained slides were performed on 20 microscopic fields (×200 magnification) for each section. The analysis was performed using Red/Green/Blue (RGB) with Software NIS-Elements and Eclipse 90i microscope (Nikon Instruments Europe BV). Briefly, we acquired the total number of blue-stained nuclei and the total number of positive-stained red cells. The data were expressed as percentage of positive cells for CD55 and CD68, respectively. For Factor VIII analysis we counted the number of positive vessels in 20 microscopic fields. The data were expressed as the mean number of positive vessels/5 mm. [2.Synovial cells were isolated following a standardized procedure as previously described.  and wereSynovial cells at both passages 1 (p.1) and 5 (p.5) were characterized by flow cytometry using the following markers expressed by SF , CD73, CD90, and CD105 , CD106 , SM , CD68 , CD80 , and CD163 ), and by endothelial  and mononuclear cells . Briefly, after harvesting cells upon detachment, they were washed twice with PBS, centrifuged, and washed in a flow cytometry buffer (PBS supplemented with 2 % BSA and 0.1 % sodium azide).5 cells were then incubated with primary antibodies at 4 °C for 30 minutes, washed twice with a flow cytometry buffer, and incubated with polyclonal rabbit anti-mouse immunoglobulins/fluorescein isothiocyanate (FITC) conjugate (Dako Cytomation) at 4 °C for 30 minutes. After two final washes, the cells were analyzed using a fluorescence-activated cell sorting (FACS) CantoII Cytometer (Becton Dickinson). For isotype control, non-specific mouse IgG was substituted for the primary antibody.Aliquots of 1 × 10Synovial cells at both passages 1 and 5  were seeded in the lower chamber of a 6-well plate and co-cultured with clinical grade ASC  for 7 days (medium was changed at day 2) in complete DMEM using a defined cell ratio (1:1) that assures no cell proliferation, as we previously reported . ASC werThe concentrations of IL1β, IL4, IL6, CXCL8/IL8, IL10, CCL2/MCP-1, CCL3/MIP1α, CCL5/RANTES, TGFβ, and TNFα were simultaneously evaluated in the supernatants of ASC, synovial cells at both passages (1 and 5) in mono- and co-cultures, using multiplex bead-based sandwich immunoassay kits  following the manufacturer’s instructions. Briefly, we added 50 μl to each well of the diluted standards (fourfold dilution series), controls, and samples in triplicate and added 50 μl of coupled beads, and the plate was incubated at room temperature for 30 minutes. The plate was then washed three times with 100 μl of wash buffer and incubated with 25 μl of detection antibodies for 30 minutes. Finally, the plate was washed three times and incubated with 50 μl of streptavidin-PE for 30 minutes and measured in a reader .Total RNA was extracted from human ASC, synovial cells in mono- and co-cultures, using RNA PURE reagent  according to the manufacturer’s instructions, and then was treated with DNase I . Reverse transcription was performed using SuperScript VILO (Life Technology) reverse transcriptase and random hexamers, following the manufacturer’s protocol.-∆Ct, and expressed as a percentage of the reference gene, as this was expressed in the same amount in all conditions tested.Forward and reverse oligonucleotides for PCR amplification of ADAMTS4, ADAMTS5, MMP13, S100A8, and S100A9 are described in Table U test was used to analyze unpaired two-group data and the Wilcoxon test was used to analyze paired two-group data. Groups with small samples were evaluated using the exact method. Values were expressed as the median and interquartile range. CSS Statistica Statistical Software  was used for analysis and values of p < 0.05 were considered significant.Statistical analysis was performed using non-parametric tests because the data did not have a normal distribution (Kolmogorov-Smirnov test). Friedman’s analysis and Dunn’s post hoc test was used to analyze more than two groups of paired data, the Mann–Whitney 2 area) in low-grade than in moderate-grade synovitis (222 ± 79/5 mm2 area). Then, for in-depth analysis of the main synovial cell populations present in the synovial tissue, we analyzed CD68 and CD55 to establish the percentage of synovial macrophages and synovial fibroblasts, in both low- and moderate-grade synovitis. As shown in Fig. Synovial tissue explants from 26 patients with OA were first scored on hematoxylin-eosin-stained slides, as reported by Krenn  and we fThe cells outgrowing from cultured-synovium tissue fragments Fig.  were theThese cells at both passages (p.1 and p.5), were then characterized by flow cytometry for markers expressed by SF , SM , endothelial cells (CD31), and mononuclear cells . As shown in Fig. We subsequently evaluated inflammatory factors  and anabolic factors  released by p.1 and p.5 OA synovial cells. As shown in Fig. Then we analyzed different factors  involved in the degradation of joint tissue. As shown in Fig. The presence of SM in p.1 synovial cells significantly increased the release of inflammatory, anabolic and degradative factors, thus creating a significantly different milieu from p.5 synovial cells. Therefore, as p.1 and p.5 synovial cells represent two different cell culture models, we tested whether they could differently affect another cell type in co-culture. We chose adipose stem cells (ASC) as the cell model because they are reportedly activated by an inflammatory environment . We analMoreover, to confirm that the effects observed were directly dependent on the amount of SM in synovial cells p.1 we also evaluated all the inflammatory and degradative factors in p.1 synovial cells isolated from low-grade synovial explants, which, as shown in Fig. Furthermore, to confirm that CCL3/MIP1α and S100A8 were specific markers of SM, we also immunostained serial sections of synovial tissue from patients with moderate synovitis using the positive control macrophage marker CD68. As shown in Fig. Synovitis is a typical feature in a high percentage of patients with OA, even in the early phase of the disease . HyperplOur data show that synovium from patients with OA and moderate synovitis had approximately 27 % CD68 (SM) and 70 % CD55 (SF) positive cells, which are well-standardized markers for SM and SF, respectively. The synovial cells outgrowing in culture were evaluated at both p.1 and p.5. SF (p.5) were not positive to typical endothelial (CD31), hematopoietic  and macrophage-like markers  but they expressed more CD55 and CD106, which are considered relative specific markers of SF . MoreoveWe found that p.1 synovial cells (mix of SF and SM) release significantly more IL6, CXCL8/IL8, CCL2/MCP-1, CCL3/MIP1α, CCL5/RANTES, and IL10 than those of p.5 (SF), thus suggesting that the absence of SM significantly reduces inflammation and does not induce the anabolic factor IL10. It has been shown that CCL2/MCP1 enhances CD106 ; our datBondeson et al.  reportedSynovial cells p.1 and p.5 represent two different in vitro cell models characterized by the presence and absence of SM, respectively, which are directly responsible for the low- and high-level inflammatory/degradative milieu. Different studies , 29 haveIt has been shown that CCL3/MIP1α has an important role in the recruitment of infiltrating leukocytes in the arthritic joint. Moreover, the CCL3-null mouse arthritis model is associated with a reduction of infiltrating cells and normal appearance of the synovium and cartilage, and the absence of pannus or bone resorption, thus confirming the important role of this chemokine in OA . This evOur data show that ASC in co-culture with p.1 synoviocytes from moderate-grade OA decreased the typical inducible factors ADAMTS5, S100A8, and S100A9, and did not affect ADAMTS4 and MMP13. Conversely, in co-culture with p.5 synovial cells they only induced the expression of MMP13, thus suggesting that in the absence of SM, SF appear to acquire a characteristic more typical of SM, such as increased expression of MMP13. Interestingly, we also found that S100A8 and S100A9, the main catabolic factors produced by activated macrophages, were not detected in p.5 synovial cells co-cultured with ASC, thus confirming their specific expression on SM. These data were also confirmed on moderate-grade OA synovial tissue, where we found that S100A8 and CCL3/MIP1α were co-expressed with CD68, the typical macrophage marker.Moreover, we found that S100A8 and S100A9 inhibition in p.1 synovial cells was also associated with IL6 and CXCL8/IL8 inhibition, which, as already reported , are strIn summary, our data from in vitro analysis show the importance of using the correct in vitro cell models to recreate a milieu that closely resembles OA synovial tissue as the target tissue organ. The availability of in vitro cell models  with large or small numbers of SM effectively reflects the different degrees of OA, which are characterized by different degrees of synovial inflammation, giving the opportunity of testing cells, anti-inflammatory drugs, or factors in a well-defined milieu.


With our procedure, alkaline phosphatase catalyzes the dephosphorylation of l-ascorbic-2-phosphate, which then serves as a reducing agent for salts of iron, gadolinium, and holmium, forming magnetic precipitates of Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5. The nanoparticles were found to be paramagnetic at 300 K and antiferromagnetic under 25 K. Although weakly magnetic at 300 K, the room-temperature magnetization of the nanoparticles found here is considerably greater than that of analogous chemically-synthesized LnxFeyOz  samples reported previously. At 5 K, the nanoparticles showed a significantly higher saturation magnetization of 45 and 30 emu/g for Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5, respectively. Our approach of enzymatically synthesizing magnetic labels reduces the cost and avoids diffusional mass-transfer limitations associated with pre-synthesized magnetic reporter particles, while retaining the advantages of magnetic sensing.We report the first  Magnetic nanoparticles (MNPs), commonly used in sample capture, clean-up, and concentration, are also now evaluated as labels for sensitive biomolecule detection  since th in 1998 , and by  in 1998 . This ap in 1998 ,5,6 and  in 1998 ,8,9,10 h in 1998 , MagniSe in 1998 , and Mag in 1998 .p-nitrophenyl phosphate is dephosphorylated by alkaline phosphatase (AP) to form a soluble yellow product (p-nitrophenol) that is readily detected at 405 nm using a spectrophotometer  rely on modification of a substrate to form a detectable product that absorbs light, fluoresces, or luminesces. For example, otometer . The subhosphate , p-aminohosphate , and 3-ihosphate ) that acl-ascorbic-2-phosphate, which then reduces iron, gadolinium, and holmium chlorides to yield paramagnetic MNPs at room temperature. Our strategy offers a novel approach to magnetic sensing in which the magnetic reporter can be enzymatically synthesized in situ. In contrast to conventional sandwich ELISA that uses an optical read-out, in our method  or the iron sulfide greigite (Fe3S4). The synthesis of these magnetic particles is encoded by at least 28 different genes, [In nature, magnetotactic bacteria  possessit genes,  and trant genes, . In contet al. found that ZnO nanoparticles lacking a doping metal exhibit weak or no magnetic properties, but when Fe was used as a dopant, the resulting Znx1−FexO product showed noticeable levels of magnetization that increased as Fe was increased from 0% to 10% [l-ascorbic-2-phosphate to l-ascorbic acid and found that the latter could serve as a reducing agent for iron, gadolinium, and holmium salts. The resulting precipitates were magnetic. In our novel enzymatic process, gadolinium and holmium are incorporated into the products as dopants, producing measurable magnetic properties as compared to the non-magnetic iron oxide precipitate formed in the absence of these dopants. The synthesis conditions and characterization methods are described in detail in the The introduction of dopants during the chemical synthesis of MNPs has been previously demonstrated ,28,29. J43±18Gd2±0O55±18 and Fe3±1Ho11±2O85±3, while the enzymatically-synthesized nanoparticles were composed of Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5.Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of the chemically- and enzymatically-synthesized MNPs are shown in 45±14Gd5±2O50±15 and 90 mg Fe42±4Ho6±4O52±5 for each of the MNP syntheses . Based on the EDX data, the weight % ratio of Fe:Gd and Fe:Ho was (59 ± 14):(20 ± 2) and (57 ± 9):(23 ± 12), respectively. The initial masses of Fe, Gd, Ho were 40 mg, 20 mg, and 20 mg, respectively. Taking a mass balance with respect to iron, gadolinium, and holmium, recovery was estimated at 118% ± 28% and 80% ± 8% for Fe and Gd in the Fe–Gd–O precipitate and 128% ± 20% and 100% ± 54% for Fe and Ho in Fe–Ho–O, where the non-homogeneous composition likely led to significant deviations in the measured recovery.Elemental mass balances on the synthetic process were estimated from the following data. An aliquot  of AP protein gave magnetic precipitates of 80 mg FeUsing SEM/EDX and TEM/EDX, we were able to analyze the composition further for each nanoparticle. As noted above and elsewhere, the compositions of the nanoparticles varied. x, y, and z for FexGdyOz and FexHoyOz produced by the two procedures  might be a reflection of a variation in composition for the individual particles as observed in the TEM/EDX data discussed below. The TEM diffraction data showed that in each sample, some of the nanoparticles were crystalline and some were amorphous (data not shown). The diffraction patterns gathered by the TEM showed a crystalline selected area electron diffraction (SAED) that pointed to the presence of FeO in some, and matched CaO in other nanostructures; the rest of the nanoparticles in each sample revealed amorphous SAED patterns. The varied diffraction patterns observed in each sample indicated that the samples were heterogeneous with respect to composition, which led us to study the composition of these nanoparticles further. To accomplish this task, we isolated about fifteen particles of each of the chemically- and enzymatically-synthesized Fe-Gd-O and the enzymatically-synthesized Fe-Ho-O, and five particles of chemically-synthesized Fe-Ho-O. x, y, z scatter plot. The plot shows that most of the chemically-synthesized nanoparticles cluster around single-element oxides, and there are only a few nanoparticles that contain all three elements . Additionally, none of the chemically-synthesized nanoparticles contained all three components . However, a small population of the enzymatically-synthesized nanoparticles contained all three elements, suggesting increased synthetic potential of the enzymatic approach.The deviation of l-ascorbic acid failed to yield magnetic precipitates. However, we characterized the non-magnetic precipitate using XRD and compared it to the magnetic precipitate, as shown in The nanoparticles were further characterized by vibrating sample magnetometry (VSM), and the magnetization curves at 300 K for chemically-synthesized and enzymatically-synthesized nanoparticles are shown in 45±14Gd5±2O50±15 and 30 emu/g for Fe42±4Ho6±4O52±5 at 5 K. Both chemically and enzymatically synthesized MNPs were observed to be paramagnetic at 300 K and antiferromagnetic under 25 K. Although Gd and Ho possess a higher number of unpaired f electrons as compared to the unpaired d electrons in Fe, enhancement of the magnetic properties by the coupling of these electrons was observed only at low temperature.On doping with the rare earth elements Gd and Ho, the resulting enzymatically-synthesized nanoparticles were found to be weakly magnetic (~5 emu/g) at 300 K, but with a comparatively higher saturation magnetization of 45 emu/g for Fe3 (Ln = rare earth), phase-selective or homogeneous composition is difficult to achieve during chemical syntheses; the hydrothermal and co-precipitation synthesis of GdFeO3 gave an amorphous precipitate, and the combustion route yielded a crystalline powder [3 [3 and HoFeO3, which, however, exhibited weak magnetizations of 0.03 and 0.3 emu/g, respectively [xFeyOz  samples reported previously [The saturation magnetization of the samples might be reduced by the significant presence of non-magnetic precipitates of single-element oxides  with only a small percentage of the MNPs of Fe–Gd–O or Fe–Ho–O present. As previously reported in the case of LnFeOe powder ,31. In aowder [3 . Furtherectively . In all eviously ,31,32.−13 emu); consequently, to detect an analyte using our method, we only need 20 fg MNPs of 5 emu/g. Even with an overall 10% efficiency, this signal would translate to a 10,000-fold improvement in potential limit of detection over conventional ELISA. The synthesized magnetic nanomaterials are not monodisperse. However, polydispersity (population of reporter particles of varied size), is not a substantial barrier to analytical performance. Silver intensification and BCIP/NBT staining are successful examples [Our in-house nanoscale GMR sensor can detect one MNP with 60–70 emu/g  from a Milli-Q water system was used in the synthesis and washing steps.l-ascorbic acid. Using this reduction procedure, we obtained precipitates under various experimental conditions of pH (from 6 to 10), temperature , and magnetic field conditions during synthesis (presence or absence of a strong bar magnet), but the precipitates from these compounds proved to be non-magnetic. We also observed that although gadolinium and holmium are common components of a permanent magnet, reduction of these rare-earth elements from their chloride salts alone failed to yield magnetic precipitates.In initial efforts to obtain Fe–Gd–O and Fe–Ho–O precipitates through enzymatic means, we first explored the chemical reduction of ferric chloride, ferric nitrate, cobalt nitrate, nickel sulfate, platinic acid, and copper sulfate. We evaluated the initial matrices and ensured that the precursor concentration was not toxic to the alkaline phosphatase enzyme. In each experiment, 0.1 to 2 mmol of the salt were dissolved in 5 mL Millipore water and evaluated to determine whether a magnetic precipitate was formed upon the addition of l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (p-aa) by alkaline phosphatase (AP) (“enzymatic synthesis” approach). Samples of AP were obtained from Sigma . One unit activity of AP is defined to hydrolyze 1 μmole of substrate (4-nitrophenyl phosphate) per minute at pH 9.8 at 37 °C. Zeba desalting columns  were used to remove more than 95% of the salts (5 mM MgCl2 and 0.2 mM ZnCl2) present in the AP solution. The enzyme was then resuspended in 100 µL diethanolamine buffer (pH 9.8) containing 5 mM MgNO3 and 0.25 mM ZnNO3 to give a final concentration of 20 units AP/mL. In a 50-mL centrifuge tube, 0.16 g (0.60 mmol) of FeCl3·6H2O and 0.05 g (0.1 mmol) GdCl3·6H2O were dissolved in 5 mL of Millipore water. For chemical or enzymatic synthesis of FexGdyOz, 0.1 g (0.6 mmol) of aa or 0.1 g (0.3 mmol) of p-aa, respectively, were added to the salt solution. In the case of enzymatic synthesis, 15 μL of 60 units/mL AP enzyme were added to the centrifuge tube containing the metal salts. For the synthesis of FexHoyOz, we used a similar procedure with 0.16 g (0.60 mmol) of FeCl3·6H2O, 0.05 g (0.1 mmol) of HoCl3.6H2O, 0.1 g (0.6 mmol) of aa  or 0.1 g (0.3 mmol) of p-aa (enzymatic synthesis), and 15 μL of 20 units/mL AP enzyme (enzymatic synthesis). The reactions were carried out at 20 °C.Ascorbic acid (aa) was used either as purchased  or was produced enzymatically via dephosphorylation of Nanoparticles were characterized by transmission electron microscopy  and equipped with energy dispersive spectrometer , scanning electron microscopy , Oberkochen, Germany), vibrating sample magnetometry , and X-ray diffraction , Karlsruhe, Germany). For the TEM analyses, we deposited the nanoparticles suspended in ethanol on a holey carbon film coating a 300-mesh copper grid and allowed them to dry. For the SEM analyses, we deposited them on a silicon wafer and allowed them to dry. We used EDX, XRD, and SAED  to confirm the composition and phases of the nanoparticles. For the latter studies, a concentrated sample of nanoparticles in ethanol was deposited on a piranha-cleaned glass slide, and XRD was carried out using Cu Kα radiation (λ = 1.540562 Å) in the 2θ range from 0° to 90°.The magnetic properties  of a known mass of sample were measured using VSM. Saturation magnetization and coercivity were obtained from the hysteresis loop analysis at 300 K and at 5 K. Measurements were recorded with uniform spacing in log field by sweeping the field 100 Oe/s with a maximum applied field up to ±90 kOe. Zero-field-cooling (ZFC) and field-cooling (FC) magnetization curves were measured in the temperature range of 1.9–300 K using a field of 100 Oe. Data were obtained by first cooling the sample from 300 to 1.9 K without applying any magnetic field. To obtain the ZFC curve, a small field of 100 Oe was applied after reaching 1.9 K, and the magnetization was measured at 0.5 K intervals while heating the sample to 300 K with a heating rate of 2 K/min. The FC curve was obtained by cooling the sample from 300 to 1.9 K while keeping the same applied field.SEM/EDX and TEM/EDX were used to obtain the composition of the chemically- and enzymatically-synthesized Fe–Gd–O and Fe–Ho–O precipitates. Each SEM/EDX spectrum is an average of at least five samplings, and the average composition with standard deviation was calculated using at least three spectra for each sample. An example of the spectrum obtained for each precipitate is given in the Results and Discussion section.3 ) synthesized by other routes. This first in vitro enzymatic synthesis of magnetic nanoparticles opens a novel approach to magnetic sensing in which the magnetic reporter is enzymatically synthesized in situ, thus circumventing any mass-transfer limitations. The enzymatically-synthesized nanoparticles, paramagnetic at 300 K and antiferromagnetic below 25 K, exhibited a strong saturation magnetization, up to 45 emu/g at 5 K. Future optimization of the reaction conditions can potentially lead to a homogeneous composition that reduces (or even eliminates) the presence of precursors or by-product components. Nevertheless, with further optimization of the process parameters, the current precipitated particles can be readily integrated with GMR sensors, such as the one with a reported sensitivity of 10−13 emu [In summary, we have demonstrated a novel alternative to optical/electrochemical reporters by enzymatically synthesizing MNPs with higher saturation magnetization than similar nanoparticles (LnFeO0−13 emu ,34. The 0−13 emu  offers a