Erysiphe necator, is one of the most devastating diseases of grapevine. To better understand grapevine-PM interaction and provide candidate resources for grapevine breeding, a suppression subtractive hybridization (SSH) cDNA library was constructed from E. necator-infected leaves of a resistant Chinese wild Vitis quinquangularis clone “Shang-24”. A total of 492 high quality expressed sequence tags (ESTs) were obtained and assembled into 266 unigenes. Gene ontology (GO) analysis indicated that 188 unigenes could be assigned with at least one GO term in the biological process category, and 176 in the molecular function category. Sequence analysis showed that a large number of these genes were homologous to those involved in defense responses. Genes involved in metabolism, photosynthesis, transport and signal transduction were also enriched in the library. Expression analysis of 13 selected genes by qRT-PCR revealed that most were induced more quickly and intensely in the resistant material “Shang-24” than in the sensitive V. pseudoreticulata clone “Hunan-1” by E. necator infection. The ESTs reported here provide new clues to understand the disease-resistance mechanism in Chinese wild grapevine species and may enable us to investigate E. necator-responsive genes involved in PM resistance in grapevine germplasm.Powdery mildew (PM), caused by fungus  Vitis vinifera L.) is the most widely-grown fruit crop worldwide and used for wine, table grape, and dry fruit production. Powdery mildew (PM) caused by Erysiphe necator (Schw.) Burr., is one of the major fungal diseases in grapes, resulting in huge losses of berry quality and grape production. Due to the high cost and toxicity associated with fungicide application, developing resistant cultivars using wild species could be an efficient, economical, and environmentally friendly strategy to reduce the threat of the disease. Therefore, understanding the mechanisms of PM resistance, identifying and functionally characterizing key genes in the resistant germplasm may provide candidate genes and valuable information on transgenic engineering for improving plant resistance to PM and withstanding harsh conditions.Grapevine  is an effective method that can be used to maximize the identification of genes involved in host responses to pathogen infection and disease development. This technique has been successfully used to isolate functional genes that are responsive to pathogen infection in plants –10. In tV. quinquangularis clone “Shang-24” and V. pseudoreticulata clone “Hunan-1”, we conducted a microscopy study of conidiospore germination and hyphal development during a 5-day time period. Microscopic images of 24, 48 and 120 hpi are presented in To investigate the developmental time-course of the pathogen in Chinese wild V. quinquangularis and V. pseudoreticulata leaves. In V. quinquangularis leaves, most epidermal cells invaded by the conidiospores led to a hypersensitive response and the attachment by the conidiospores induced H2O2 accumulation, as indicated by brown staining due to DAB polymerization  nr database using the BLASTX program. A total of 205 (77.1%) unigenes including 77 contigs and 128 singletons showed significant similarities to known functional gene sequences in the database, 25 had matches against genes of unknown function or hypothetic proteins. The remaining 36 sequences (15 contigs and 21 singletons) did not match to any known gene sequences in the database.E. necator-inoculated grapevine leaves were further assigned gene ontology (GO) terms. Based on the GO annotations, unigenes were classified into high-level plant specific GO slims within the two ontology categories, namely, molecular function and biological process. A total of 176 unigenes were assigned to 22 GO slims within the molecular function category and 188 unigenes to 41 GO slims within the biological process category  unigenes were related to metabolism, 15 (5.6%) encoding proteins and enzymes putatively involved in photosynthesis and energy, the majority of which encoded ribulose-1, 5-bisphosphate carboxylase/oxygenase activase, 7 (2.6%) unigenes were related to protein synthesis and fate, and genes involved in signal transduction, transport, and transcription accounted for 3.0%, 4.5%, and 1.5%, respectively.Although a large amount of unctions . The larE. necator at different time points, qRT-PCR was performed with 11 genes represented by one EST and two genes by two or more ESTs on E. necator- and mock-inoculated samples at eight time points . These 13 genes in different functional groups were selected for the qRT-PCR analysis because of defense-related roles of their homologs in hypersensitivity, ubiquitin/proteasome pathway and phenylpropanoid synthesis reported previously, and because of their novel sequences. Nine of them corresponded to three functional categories: metabolism (3 genes), protein synthesis and fate (3 genes) and response to defense (3 genes)  , and fouHunan-1” .Three genes involved in defense , which encode a thaumatin-like protein, a senescence-associated protein, and a class IV chitinase, respectively, shared similar expression patterns in the resistant plant. They did not start increasing until 12 hpi, and all of them reached their maximum expression levels at 24 hpi, then decreased gradually from 48 to 120 hpi; while in the susceptible material, the expression levels of these three genes were consistently close to the control levels throughout the entire time course .E. necator from 6 to 72 hpi in the resistant material “Shang-24”, while in “Hunan-1”, their transcripts consistently remained at control levels throughout the entire time course , coding for a coronatine insensitive 1 (COI1), an F-box family protein, and a jasmonate ZIM-domain protein, respectively, were activated strongly by e course .S-adenosylmethionine synthetase, chalcone-flavanone isomerase, and phenylalanine ammonia-lyase, respectively  showed homology to ectively . They alculation .E. necator is shown in E. necator, while in “Hunan-1”, the reaction was delayed until 72 hpi. Gene UN103 showed a completely different expression pattern with other genes in our experiment: induced at 6 hpi and reached its maximum expression level at 12 hpi in “Hunan-1” though also slightly increased in “Shang-24”  unigenes appeared to share significant homologies with genes induced by pathogen attack in V. vinifera, A. thaliana, N. tabacum and O. sativa, etc.As a valuable tool in functional genomics, SSH has been applied to maximize the identification of genes involved in host responses to pathogen infection and disease development. In this study, a PCR-based cDNA subtractive strategy was used to isolate genes related to PM-resistance from a PM-resistant clone of Chinese wild ransport . ESTs faE. necator infection (Puccinia striiformis interaction  in lacto glycerol) was pipetted onto leaf surfaces immediately before they were photographed.For microscopy, the bleached leaf segment was mounted on a microscope slide without a coverslip and observed using a microscope  with a “no coverslip” 100× objective and 20× objective lens. For micrographs, a drop of aniline blue  following the manufacturer’s instructions. Differentially expressed cDNA fragments digested with RsaI after a two-round PCR selection were cloned into the pGEM-T easy Vector  and transformed into E. coli DH5α . The clones were cultured overnight on an LB media plate containing ampicillin and X-Gal/IPTG at 37 °C.For SSH library construction, total RNA of g et al.  with minTaq DNA polymerase  and 1 μL of bacterial culture. The PCR was performed as follows: an initial denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1.5 min, and a final extension at 72 °C for 5 min. The PCR products were electrophoresed on 1.0% agarose gels to confirm the amplification. A subset of positive clones whose PCR products were longer than 200 bp were selected for preparation of plasmid DNA using the Plasmid DNA extraction kit from U-gene .cDNA inserts from white colonies were PCR-amplified using SP6 and T7 primers. The PCR reactions contained a total volume of 25 μL, including 16 μL sterile water, 2.5 μL 10× buffer, 1.0 μL of each primer (10 μM each), 0.5 μL dNTP mix (2.5 μM each), 0.5 units of The selected positive clones were sequenced with the universal M13 sequencing primer. The raw chromatogram files were base-called with Phred . Vector,The ESTs were assembled into unigenes using iAssembler  with minE. necator-inoculated “Shang-24” and “Hunan-1” plants at 0, 6, 12, 24, 48, 72, 96 and 120 hpi as well as from their corresponding mock-inoculated grapevines, was treated with DNase I to remove DNA contamination before cDNA synthesis. The cDNA was synthesized from 1.0 μg total RNA using 500 ng of random hexamers and the M-MLV reverse transcriptase (Promega). cDNAs, derived from inoculated healthy V. quinquangularis clone “Shang-24” leaves harvested at 0, 6, 12, 24, 48, 72, 96 and 120 hpi, were used as templates for qRT-PCR. Gene-specific primers for the 13 selected defense-related genes were designed using the Primer Premier 5.0 software. All the primers are listed in For qRT-PCR, total RNA extracted from leaves of ® Premix Ex Taq™ II (TaKaRa Biotechnology), 1.0 μL of cDNA template (6× diluted cDNA from leaf samples), 0.8 μL of each primer (10 pmol·L−1), and 7.4 μL ddH2O with the following cycling parameters: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. Dissociation curves were generated to ascertain only a single product that was produced in each case. Relative gene expression was determined from standard curves produced using serial cDNA dilutions.qRT-PCR reactions were performed in triplicates using iCycler Detection system . Grape Actin1 (GenBank accession no. AY680701) was used as the internal control and for data normalization. A 168 bp fragment of Actin1 gene was amplified with a pair of primers . Reactions were performed in a total volume of 20 μL containing 10.0 μL SYBRV. quinquangularis accession “Shang-24”. The EST data reported herein provides new insights into the grapevine-PM interaction and the candidate defense-related genes involved in the process. Analyzing gene expression patterns in response to pathogen infection provides a basis for their functional studies.The results obtained from the present study are the first to provide a global view of gene expression profiles in response to PM pathogen attack in a PM-resistant Chinese wild


P<0.05). For identifying hepatic necroinflammation (G≥2), after adjusting other factors, the odds ratio (OR) of HexCer (d18∶1/22∶0) reached 1.01 . Furthermore, the area under the curve (AUC) of HexCer (d18∶1/22∶0) was 0.7 (P = 0.01) and approached that of ALT (AUC = 0.78). However, in CHC patients with normal ALT, HexCer (d18∶1/22∶0) was an independent factor  to identify the hepatic necroinflammation (G≥2). HexCer (d18∶1/22∶0) not only showed the largest AUC , but also exhibited the highest specificity of all indicators. These results indicate that plasma HexCer (d18∶1/22∶0) is a potential indicator to distinguish hepatic necroinflammation in CHC patients. For CHC with normal ALT, the ability of HexCer (d18∶1/22∶0) to distinguish hepatic necroinflammation might be superior to conventional serum indicators.Accurate estimation of hepatic necroinflammation caused by chronic hepatitis C (CHC) is crucial for prediction of prognosis and design of therapeutic strategy, which is particularly true for CHC patients with normal alanine aminotransferase (ALT) level. Recent studies have shown that sphingolipids have a close relationship with hepatitis C virus infection. The present study aimed to identify plasma sphingolipids related to hepatic necroinflammation. We included 120 treatment-naïve CHC patients and 64/120 had normal ALT levels (<40 U/L). CHC patients who underwent liver biopsies were subjected to Scheuer scoring analysis for scope of hepatic inflammation. Plasma sphingolipids were detected by high-performance liquid chromatography tandem mass spectrometry. Our results showed 44 plasma sphingolipids were detected altogether. Of all detected sphingolipids, hexosylceramide (HexCer) (d18∶1/22∶0) and HexCer (d18∶1/24∶0) showed a significant difference among G0/G1, G2, and G3/G4 ( Hepatitis C is a major public health problem, affecting over 2% of the global population who are at risk of cirrhosis or hepatocellular carcinoma de novo synthesis of sphingolipids in vitroSphingolipids are enriched in lipid rafts of biological membranes and provide a distribution platform for different signal processes including proliferation, differentiation and apoptosis We have previously established a mature high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) method to quantify sphingolipids in other diseases Therefore, in the present study, based on our previous improved HPLC-MS/MS methods and liver biopsy, we aimed to evaluate the diagnostic ability of plasma sphingolipids to identify hepatic necroinflammation (G≥2) among patients with CHC and the subgroup of patients with normal ALT levels, respectively.A group of 122 CHC patients with long-term follow-up, from Dingxi City, Gansu Province, China, were enrolled from July 2010 to June 2011. All the patients had a history of paid plasma donations with blood cell re-transfusions between 1992 and 1995, and did not receive antiviral treatment before being recruited into the present study. CHC diagnosis was made in accordance with established criteria Fasting blood samples were collected on the day of the liver biopsy. Liver function and coagulation function tests were performed in all patients. Each patient gave signed informed consent at the beginning of the study. The study protocol was conducted in accordance with the provisions of the Declaration of Helsinki, 1975 and approved by the Institutional Review Board of Beijing YouAn Hospital, Capital Medical University, Beijing, China.Liver biopsies were obtained from CHC patients under ultrasound guidance. Specimens were fixed in formalin and embedded in paraffin. Specimens longer than 1.5 cm, containing at least six complete portal areas, were reviewed by two senior pathologists who were unaware of the patients’ clinical data. The Scheuer scoring system was used to assess hepatic necroinflammatory activity, and G≥2 was considered to show the presence of necrosis g. The samples were stored at –80°C. All lipid standards were from Avanti Polar Lipids . Ultra Resi-analyzed grade methanol and HPLC grade methyl-tert-butyl ether (MTBE) were purchased from Mallinckrodt Baker . Formic acid of analytical grade was obtained from TEDIA . Ammonium formate (purity >99.99%) was purchased from Sigma–Aldrich . Ultrapure water was prepared using a Milli-Q purification system . HPLC-MS/MS was performed on an Agilent 6410B Triple Quad mass spectrometer  comprising a triple quadrupole MS analyzer with an electrospray ionization interface and an Agilent 1200 RRLC system. Sphingolipidomic assays were performed at the Institute of Materia Medica, Peking Union Medical College  as previously described Blood was collected into cold lithium heparin used as an anticoagulant and immediately centrifuged at 4°C at 8000 × t test or Mann–Whitney test. Additionally, the independent-samples t test or Mann–Whitney test was used to compare G<2 against G≥2. Categoric variables were analyzed by Pearson χ2 test. Correlation analysis between plasma sphingolipids and inflammation grades was performed by Spearman’s rank correlation. Moving forward, the (LR) multivariate logistic regression analysis was performed, and the P values of entry and removal were respectively set to 0.05 and 0.1. The diagnostic value of each indicator with significant difference in univariate analysis was assessed by the area under the receiver operating characteristic (ROC) curve. Statistical analysis was performed using SPSS version 19.0 , while a two-sided P value <0.05 was considered statistically significant.Data were expressed as mean ± standard deviation (SD). In univariate analysis, depending on the data distribution according to the Kolmogorov–Smirnov test, continuous variables among multiple groups were analyzed by one-way analysis of variance or the nonparametric Kruskal–Wallis test. Comparisons between pairs of groups subsequently were analyzed by the least significant difference P<0.001), aspartate aminotransferase (AST) (P<0.001), γ-glutamyl transpeptidase (GGT) (P<0.001), total bile acid (TBA) (P<0.05), and prothrombin activity (PTA) (P<0.001) showed a significant difference among G0/G1, G2, and G3/G4 (The characteristics of the 120 patients who underwent a liver biopsy are summarized in nd G3/G4 .P<0.05). hexosylceramide (HexCer) (d18∶1/22∶0), which differed significantly between G0/G1 and G2 (P = 0.02), also showed a significant difference between G0/G1 and G3/G4 (P = 0.01). Similarly, another sphingolipid HexCer (d18∶1/24∶0) showed a significant difference in both G2 versus G0/G1 (P = 0.02) and G3/G4 versus G0/G1 (P = 0.02) (P = 0.02). HexCer (d18∶1/24∶0) also showed a correlation with inflammation grade .Plasma sphingolipids in the 120 patients were analyzed by HPLC-MS/MS, and 44 sphingolipids were identified and quantified . Two pla = 0.02) . To analP<0.05) in univariate analysis. Routine serological indicators such as ALT, AST, albumin, globulin, GGT, TBA, cholinesterase, and PTA also showed significant differences between G<2 and G≥2 (P<0.05). To determine independent factors for the absence or presence of necroinflammation (G<2 versus. G≥2), the multivariate logistic regression analysis was performed. Sex, ALT, AST, GGT, cholinesterase, PTA, Cer (d18∶1/18∶1), HexCer (d18∶1/16∶0), HexCer (d18∶1/22∶0), HexCer (d18∶1/24∶1), and HexCer (d18∶1/24∶0) were initially included in multivariate analysis. The final results showed that the odds ratio (OR) for HexCer (d18∶1/22∶0) reached 1.01 [95% confidence interval (CI): 1.0–1.02]. In addition, the OR of AST and PTA was 1.07 (95% CI: 1.01–1.13) and 0.89 (95% CI: 0.81–0.98), respectively (Hepatic necroinflammatory activity is one of the most important predictors of fibrosis progression in CHC patients ectively .P = 0.01). Although it was lower than that of ALT , the specificity and positive likelihood ratio of HexCer (d18∶1/22∶0)  were higher than the routine serological indicators, including ALT. The AUC value of the other plasma sphingolipids HexCer (d18∶1/24∶0), HexCer (d18∶1/24∶1) ranged from 0.69 to 0.70 ; both of which were larger than that of globulin, cholinesterase, albumin, and TBA  of HexCer (d18∶1/22∶0) reached 0.7 ( and TBA .P<0.05). HexCer (d18∶1/22∶0), HexCer (d18∶1/24∶1), and HexCer (d18∶1/24∶0) showed significant differences between G<2 and G≥2 (P<0.05) among all the plasma sphingolipids. During the logistic regression analysis, sex, AST, total bilirubin, GGT, PTA, HexCer (d18∶1/22∶0), HexCer (d18∶1/24∶1), and HexCer (d18∶1/24∶0) were included. The final multivariate results revealed that HexCer (d18∶1/22∶0) was independent of other factors and its OR value was 1.02 (95% CI: 1.01–1.03).In order to evaluate whether sphingolipids could differentiate the hepatic necroinflammation (G≥2) in the subgroup of CHC patients with normal ALT levels, we also performed a univariate and multivariate analysis . RoutineP = 0.001) exhibited the highest specificity (1.0) when the optimal cutoff value was set to 281.37 pmol/mL. The AUC of HexCer (d18∶1/24∶1) followed closely, which reached 0.75  and was the same as AST. The AUCs of total bilirubin, PTA, and GGT fluctuated from 0.69 to 0.71  with the largest AUC  might be a surrogate marker for assessing hepatic necroinflammation in patients with CHC, especially, for those with normal ALT level.in vivo study has reported that α-galactosylceramide induces pronounced cytokine responses that could contribute to hepatic inflammation, by activating natural killer T cells Currently, although increasing evidence confirms the close relationship between sphingolipids and liver diseases Furthermore, we focused on the potential of plasma sphingolipids to identify the presence of hepatic necroinflammation (G≥2) in CHC. Another interesting finding in our study was that, although the AUC value of these sphingolipids to identify hepatic necroinflammation (G≥2) was not larger than that of routine serological indicators in the total patient population, others such as negative likelihood ratio and positive likelihood ratio of some sphingolipids were better indicators than routine serological indicators. This indicated that plasma sphingolipids, especially HexCer (d18∶1/22∶0), which was independently linked to hepatic necroinflammation with the largest AUC of all sphingolipids, had a certain superiority in identifying hepatic necroinflammation in CHC patients.P = 0.001) and the highest specificity (1.0) of all the indicators including plasma sphingolipids and routine serological indicators. This suggests that HexCer (d18∶1/22∶0) might become a potential marker for identifying hepatic necroinflammation in CHC patients with normal ALT.We also verified the superiority of plasma sphingolipids compared with routine serological indicators to reflect the presence of necroinflammation (G≥2) in CHC patients with normal ALT levels. At present, elevated ALT level, as an indirect surrogate marker for hepatic necrosis, still has poor ability to identify hepatic inflammation in CHC patients Although HexCer (d18∶1/24∶0) showed that its AUC approached that of HexCer (d18∶1/22∶0) in ROC analysis, HexCer (d18∶1/24∶0) was not an independent factor to identify hepatic necroinflammation in CHC in multivariate analysis. However, we speculated that the association with small samples and poor statistical power could not be ruled out. We confirmed the superior diagnostic value of HexCer (d18∶1/22∶0) to identify hepatic necroinflammation in patients with CHC and normal ALT level.There were some limitations in our study. We analyzed the relationship between the plasma sphingolipids and hepatic necroinflammation based on a long-term (∼20 years) follow-up in a cohort of interferon-naïve CHC patients, and the actual number of cases was not large, and the different inflammation groups were not matched exactly for sex. However, by adjusting for sex, the multivariate analysis showed that sex had minimal effects on plasma sphingolipid level.In summary, based on liver biopsies and HPLC-MS/MS, we have found for the first time that plasma sphingolipid HexCer (d18∶1/22∶0) has a positive diagnostic value to distinguish hepatic necroinflammation in CHC, especially in patients with normal ALT levels. Meanwhile, our discovery of plasma sphingolipids has opened new directions of research to identify novel noninvasive markers for progression of CHC.Table S1Plasma sphingolipids profile of untreated chronic hepatitis C patients with G0/G1, G2, G3/G4. Data are expressed as mean ± standard deviation, P values were calculated by one-way analysis of variance or the nonparametric Kruskal-Wallis test.(DOC)Click here for additional data file.Table S2Performance of indicators for identification of liver necroinflammation (G≥2) in ROC analysis. The indicators whose P values of AUC were less than 0.05 are listed in the Table.(DOC)Click here for additional data file.


Talaromyces secrete large amounts of red pigments. Literature has linked this character to species such as Talaromyces purpurogenus, T. albobiverticillius, T. marneffei, and T. minioluteus often under earlier Penicillium names. Isolates identified as T. purpurogenus have been reported to be interesting industrially and they can produce extracellular enzymes and red pigments, but they can also produce mycotoxins such as rubratoxin A and B and luteoskyrin. Production of mycotoxins limits the use of isolates of a particular species in biotechnology. Talaromyces atroroseus sp. nov., described in this study, produces the azaphilone biosynthetic families mitorubrins and Monascus pigments without any production of mycotoxins. Within the red pigment producing clade, T. atroroseus resolved in a distinct clade separate from all the other species in multigene phylogenies , which confirm its unique nature. Talaromyces atroroseus resembles T. purpurogenus and T. albobiverticillius in producing red diffusible pigments, but differs from the latter two species by the production of glauconic acid, purpuride and ZG–1494α and by the dull to dark green, thick walled ellipsoidal conidia produced. The type strain of Talaromyces atroroseus is CBS 133442 Some species of  Monascus species are known to produce six major azaphilone pigments being the yellow monascin and ankaflavin; the orange monascorubrin and rubropunctatin and the red monascorubramine and rubropunctamine, in addition to more than 20 related pigments + and [M+Na]+ using Agilent MassHunter Qualitative Analysis B.06.00 software and a database of potential extrolites in T. atroroseus with support from UV-VIS spectra. The list of compounds searched for including the extrolite standards can be found in The extrolite extractions from http://www.mycobank.org/MB. The online version of this work is archived and available from the following digital repositories. PubMed Central, LOCKSS.1. The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature of algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Name Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix Talaromyces atroroseus Yilmaz, Frisvad, Houbraken & Samson 2013 sp. nov. [urn:lsid:mycobank.org: 804901]2. Repository of Talaromyces atroroseus sp. nov. and its close relatives were studied using multigene phylogenies, bason on ITS, RPB1 and β-tubulin sequences. The aligned datasets were 482, 888 and 374 bp long, respectively. The new species resolved in a clade together with other red pigment producing species such as T. albobiverticillius, and T. minioluteus. Talaromyces purpurogenus resolved in a distantly related clade  and NRRL 2120 (= CBS 133452). Pitt Holotype: CBS 133442 in Centraalbureau voor Schimmelcultures is designated as the holotype of Talaromyces atroroseus. It was isolated from indoor house dust, Stellenbosch, South Africa by C. Visagie in 2010. Cultures ex type: CBS 133442 = IBT 32470 = DTO 178A4 = KAS 3778 Etymology: Named after the dark rosy diffusing azaphilone pigment mixture produced.Diagnosis: Dark green ellipsoidal rough-walled conidia and a dark red diffusing pigment, strains of the species produce the unique combination of secondary metabolites: glauconic acid, ZG–1494α, purpuride, red Monascus pigments, mitorubrins, and purpactins in fresh isolates.CYA 25 °C 7d: Colonies are 30–40 mm in diameter, low, plane; margins narrow (1–2 mm), entire, low; mycelia white; texture velvety; sporulation dense, conidia en masse dark to dull green; exudate absent; soluble pigment red; reverse coloration dark cherry red.MEA 25 °C 7d: Colonies 35–40 mm in diameter, low, plane, having a pinkish colour because of exudates diffusing into mycelia; margins narrow (1–2 mm), entire, low; mycelia white; texture velvety overlaying floccose; sporulation moderately dense, conidia en masse bluish green; exudate red droplets especially close to margin; soluble pigment absent, after prolonged incubation red pigments produced; reverse coloration dark red.YES 25 °C 7d: Colonies are 33–45 mm in diameter, raised at centre, sulcate; margins wide (2–3 mm), entire, low; mycelia white; texture velvety; sporulation dense, conidia en masse dark to dull green; exudates small red droplets; soluble pigment red in some isolates; reverse coloration brownish red.CYAS 25 °C 7d: Commonly no growth, some strains up to 5 mm in colony diameter.CREA 25 °C 7d: Colonies 9–13 mm in diameter, weak acid production close to colony periphery, some strains acid absent; reverse dark red.OA 25 °C 7d: Colonies 30–35 mm in diameter, low, plane; margins wide (2–3 mm), entire, low; white mycelia; texture velvety; sporulation dense; conidia en masse dull to dark green, almost appears blackish green; exudates absent; soluble pigment absent; reverse coloration commonly greenish yellow to green, red in some isolates.DG18 25 °C 7d: Colonies 27–30 mm in diameter, low, plane; margins wide (2 mm), entire, low; mycelia white; texture velvety, floccose mycelia present at centre; sporulation dense, conidia en masse greyish green, at margins bluish green; exudates absent; soluble pigment absent; reverse colour is beige.Conidiophores mostly biverticillate, subterminal branches produced, have a greenish to brownish pigmentation; Stipes smooth walled, 90–150 × 2.5–3 µm; Branches 2–3 when present, 15–50 × 2–3 µm; Metulae in verticils of 3 to 5 per stipe, 8–15 × 3.0–4.0 µm; Phialides acerose, 3 to 6 per metula, 9.5–12.5 × 2.5–3 µm; Conidia rough walled, ellipsoidal, 2–3.5 × 1.5–2.5 µm.Talaromyces albobiverticillius  Samson, Yilmaz, Frisvad & Seifert, Studies in Mycology 70: 174, 2011. MycoBank MB560683 , entire, low; mycelia white and yellow; texture floccose to velvety; sporulation sparse, in some isolates moderately dense; conidia en masse when sparse white, otherwise greyish green; exudates red small droplets; soluble pigmentation red; reverse coloration dark cherry red.MEA 25 °C 7d: Colonies 24–28 mm in diameter, low, crateriform, in some isolates sulcate; margins wide (2–3 mm), entire, low; mycelia white and yellow; texture velvety with overlaying floccose in the centre; sporulation sparse, in some isolates moderately dense; conidia en masse when sparse white, otherwise greyish green; exudates clear and red droplets; soluble red pigment absent; reverse coloration dark red.YES 25 °C 7d: Colonies 23–25 mm in diameter, raised at centre, sulcate; margins wide (2–3 mm), entire, low; mycelia white and yellow; texture velvety; sporulation sparse, in some isolates moderately dense; conidia en masse when sparse white, otherwise greyish green; exudates small orange to red droplets; soluble pigment red in some strains; reverse coloration red to pale brown.CYAS 25 °C 7d: No growth.CREA 25 °C 7d: Colonies 4–8 mm in diameter, no acid produced.OA 25 °C 7d: Colonies 25–28 mm in diameter, low, plane; margins wide (3–4 mm), entire, low; mycelia white; texture velvety; sporulation sparse to moderately dense; conidia en masse when sparse white, otherwise greyish green; exudates absent; soluble pigment absent; reverse coloration red in the centre and the rest greenish yellow to green.DG18 25 °C 7d: Colonies 15–35 mm in diameter, low, plane; margins narrow (1–2 mm), entire, low; mycelia white; texture velvety; sporulation sparse; sparse to moderately dense; conidia en masse when sparse white, otherwise greyish green; exudates clear to red droplets; soluble pigment red in some isolates absent; reverse coloration brownish red, in some isolates beige.Conidiophores strictly biverticillate, subterminal branches absent; stipes smooth walled, 200–380 × 2.5–3.5 µm; metulae in verticals of 3–6, 8–12 × 1.5–4.5 μm; phialides acerose, 3–7 per metula, 8–13.5 × 2–3 μm; conidia smooth to finely roughened, spheroid to subglobose, in some isolates fusiform, 2–3.5 (4) × 1.5–2.5 μm.Talaromyces atroroseus is a new species that produce large amounts of red pigments that can be potentially used for colouring foods, as it does not produce any known mycotoxins. Certain strains of T. albobiverticillius may also be used for these purposes.Table S1Table S1 contains the extrolites searched for by ultra high performance-liquid chromatography-diode array detection-high resolution mass spectrometric detection (UHPLC-DAD-HRMS) the fungal extracts analysed. The table also includes data on the available standards used in the study.(DOCX)Click here for additional data file.


Porcine circovirus type 2 (PCV2), is nowadays associated with a number of diseases known as porcine circovirus-associated diseases (PCVAD), especially postweaning multisystemic wasting syndrome (PMWS). The epidemiological investigation of PCV2 infection was usually conducted by PCR, nested PCR, PCR-RFLP, TaqMan-based assay and nucleotide sequencing. However, there is still no rapid, sensitive and practical method for detecting PCV2 genotypes. As a novel nucleic acid amplification method, the loop-mediated isothermal amplification method (LAMP) has been used to detect a variety of pathogenic microorganisms.Herein, a LAMP method is developed to detect the genotypes of PCV2. The diagnostic sensitivity of LAMP is 1 copy/reaction for differentiating genotypes PCV2a and PCV2b. The reaction process was completed at 65°C for 1 hour in a water bath. Cross-reactivity assay shows that this method is specific for PCV2a and PCV2b and no reactive for PCV2c and other swine-origin viruses . Identity between LAMP and nested PCR was 92.3% on 52 field clinical samples.LAMP method provides a rapid, sensitive, reliable way to detect PCV2a and PCV2b, and a better means for the large scale investigation of PCV2a and PCV2b infection. Porcine circovirus type 2 (PCV2), a small, nonenveloped, single stranded DNA virus with a rather small circular genome , is cons9 in less than an hour under isothermal conditions [LAMP method with high DNA specificity was developed in 2000 as a novel nucleic acid amplification method . The usenditions . Therefonditions -25, inclnditions ,26. HoweThe concentrations of the forward inner primers (FIP) and backward inner primers (BIP), deoxynucleoside triphosphates (dNTPs), betaine and the reaction temperature were optimized when the LAMP assay was developed. The optimal LAMP reaction mixture (25 μL) was composed of 0.8 μM (each) of inner primers, 0.2 μM (each) of outer primers, 0.2 mM of dNTPs, 0.5 M of betaine, 10 × ThermoPolbuffer 2.5 μL, 8U of Bst DNA polymerase, and 1 μL of template DNA. The mixture was incubated at 65°C for 1 hour, inactivated at 80°C for 10 min, and characterised a bolder ladder like bands on 2% (W/V) agarose gel under UV light after EB staining (data not shown). The similar results can also be observed by naked eyes after adding SYBR Green I dye (data not shown).Data in Figure 3 copies/reaction for both of PCV2a and PCV2b  and the LAMP method had higher sensitivity than the nested PCR method.Generally, the nucleotide sequencing has been used to detect PCV2a and PCV2b , other aThe specific genotype primers for differential PCR amplification with high sensitivity and high specificity are not easy to design, because the genetic identity between PCV2a and PCV2b is very high (>95%) . HoweverTake together, we can find that the LAMP method has better specificity, higher sensitivity and efficiency, and simpler equipment requirements than the nested PCR. Furthermore, the detection of the amplification product is more flexible. Therefore, the LAMP method developed in this study provides a better means for the molecular epidemiological investigation of PCV2 genotypes.4)2SO4, 2 mM MgSO4, 0.1% Triton X-100) were purchased from New England Biolabs . Betaine was purchased from Sigma-Aldrich Co. . SYBR Green I dye was obtained from Solar Biotechnology .Porcine circovirus type 1 (PCV1), porcine circovirus genotype 2a (PCV2a), porcine circovirus genotype 2b (PCV2b), classical swine fever virus (CSFV), porcine respiratory and reproductive syndrome virus (PRRSV), bovine viral diarrhea virus (BVDV) were kept in our laboratory. Transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and rotavirus (RV) came from purchased commercial triple attenuated vaccine. Bst DNA polymerase and 10 × ThermoPolbuffer . RNA isolation of CSFV, PRRSV, BVDV, TGEV, PEDV and RV was carried out using Trizol Reagent  according to manufacturer’s specification. The extracted RNA was used as template after reverse transcription with random primer according to the instructions of the RevertAid™ First Strand cDNA Synthesis Kit . All templates were stored at -20°C until used.http://primerexplorer.jp/eLAMP3.0.0/index.html). Inner primers contain sequences of the sense and antisense strands of the target DNA linked by a TTTT spacer (Hightlighted in bolder pattern in Table According to the reference sequences of PCV2a (GenBank:AF055382) and PCV2b (GenBank:AF055394) , two setThe LAMP reaction mixture contains inner primers (FIP/BIP), outer primers (F3/B3), dNTPs, betaine, buffer, Bst DNA polymerase and the template DNA. To optimize the LAMP reaction, final concentration of FIP/BIP at 0.4 μM, 0.6 μM, 0.8 μM, dNTPs at 0.2 mM, 0.4 mM, 0.6 mM and betaine at 0.5 M, 1 M, 1.5 M, were selected to test the best reaction condition in a total of 25 μL reaction volume. In addition, the reaction temperature was selected to test the optimized LAMP reaction condition. The LAMP reaction was carried out in a water bath for 1 h, and then heated at 80°C for 10 min to terminate the reaction. The amplified products (5 μL) were electrophoresed on 2.0% (W/V) agarose gel and visualized under UV light after EB staining.The nested PCR method for detecting PCV2a and PCV2b was developed by Lyoo in 2008 , and it To detect the specificity of the LAMP method, PCV2a, PCV2b, PCV1, CSFV, PRRSV, BVDV, TGEV, PEDV and RV templates were amplified with LAMP primers for PCV2a and PCV2b respectively. As compared, all these templates were detected by conventional PCR with their special primers to ensure the template for individual virus is testable.7 copies/μL to 2.5 × 101 copies/μL.To detect the sensitivity of the LAMP method, the concentration of the extracted DNA of PCV2a and PCV2b was quantified with ND-1000 spectrophotometer , and serial ten-fold dilution method was used to dilute it from 2.5 × 10Four PCV2a and PCV2b postive templates were chosen to test the stability of the LAMP method, respectively. Each template was repeated three times under identical conditions. The amplified products (5 μL) were analyzed by agarose gel electrophoresis.The authors declare that they have no competing interests.XQ, TL, GZ and JZ designed this experiment. XQ, JC carried out the experiments. XQ, GZ, ML and JZ wrote the manuscript. YJ and GX provided clinical samples. JZ has given final revision and approval of the version to be published. All authors read and approved the final manuscript.


Indeed, FTY720 (non-selective S1PX receptor agonist) produces modest hypertension in patients  as well as acute bradycardia independent of changes in blood pressure. However, the precise receptor subtypes responsible is controversial, likely dependent upon the cardiovascular response in question , and perhaps even species-dependent since functional differences in rodent, rabbit, and human have been suggested. Thus, we characterized the S1P receptor subtype specificity for each compound in vitro and, in vivo, the cardiovascular effects of FTY720 and the more selective S1P1,5 agonist, BAF312, were tested during acute i.v. infusion in anesthetized rats and after oral administration for 10 days in telemetry-instrumented conscious rats. Acute i.v. infusion of FTY720  or BAF312  elicited acute bradycardia in anesthetized rats demonstrating an S1P1 mediated mechanism-of-action. However, while FTY720  elicited dose-dependent hypertension after multiple days of oral administration in rat at clinically relevant plasma concentrations , BAF312  had no significant effect on blood pressure at any dose tested suggesting that hypertension produced by FTY720 is mediated S1P3 receptors. In summary, in vitro selectivity results in combination with studies performed in anesthetized and conscious rats administered two clinically tested S1P agonists, FTY720 or BAF312, suggest that S1P1 receptors mediate bradycardia while hypertension is mediated by S1P3 receptor activation.Sphingosine-1-phospate (S1P) and S1P receptor agonists elicit mechanism-based effects on cardiovascular function  K(ACh) current in pacemaker cells 1 and/or S1P3) responsible for these effects is controversial, dependent upon the cardiovascular effect in question , and perhaps even dependent on species since functional differences in rodent, rabbit, and human have been suggested Sphingosine-1-phospate (S1P) and S1P receptor agonists elicit mechanism-based effects on cardiovascular function in animal models 1,2,3) are present in the heart and vasculature 1 exceeds that of S1P2 and S1P3 in the cardiac myocyte but S1P3 predominates in cardiac fibroblasts At least three of the S1P receptor subtypes , known to contribute to the cardiac resting membrane potential. Modulation of the current elicits changes in APD in multiple species in vivo. While S1P-mediated bradycardia is thought to be due to IK(ACh) stimulation in SA nodal cells, independent of a central mechanism, the negative inotropic effects of S1P are thought to be a result of KAch activation in ventricular myocytes , Tango™ EDG3-bla U2OS cells (human S1P3), Tango™ EDG6-bla U2OS cells (human S1P4) and Tango™ EDG8-bla U2OS cells (human S1P5) cell lines were cultured and assayed according to manufacturers instructions with one modification; agonists incubated on the cells for 18 hours prior to addition of the GenBlazer substrate. Final DMSO concentration in the assay is 1%.t-test comparing treatment change from baseline (baseline was defined as the period from −30 to 0 minutes) vs. vehicle controls change from baseline at each timepoint without adjustment for multiple comparisons. In a separate group of anesthetized rats in which left ventricular (LV) contractility was also recorded with a Millar pressure transducer introduced into the left ventricle, BAF312 was infused in the same dosing regimen as the studies described above  and effects on LV dP/dtmax were recorded.The cardiovascular effects of FTY720 and BAF312 were tested in anesthetized rats (180–200 g). In brief, male Sprague Dawley rats (Charles River) were anesthetized with Inactin (110 mg/kg i.p.) and a tracheotomy performed. The femoral artery and veins were catheterized for measurement of mean arterial pressure and heart rate, collection of blood samples at 20-min intervals (0.2 mL/sample), and for compound infusion/hydration as previously described Mean arterial pressure was assessed in conscious, freely moving male Sprague Dawley rats  instrumented with telemetry transmitters (DSI-Ponemah) and housed in metabolism cages as previously described 1/5 agonism in vitro (B arrestin) with EC50s of 2.5/0.8 nM respectively and >10,000 fold selectivity over S1P3 and 100 fold selectivity over S1P4. FTY720-P was run as a control and agonism on S1P1,3,4,5 with EC50 values of 0.4/54/26/1.8 nM were observed consistent with the selectivity profile previously reported BAF312 was confirmed to have the reported selectivity  and heart rate decreased to −71±26, −139±37, and −173±26 beats/min below baseline values .FTY720 was first profiled in anesthetized rats. In anesthetized rats plasma concentrations of parent and phosphorylated FTY720 at the end of each dosing period were 54±9, 198±13, and 375±46 nM (parent) and 285±87, 683±214, and 908±264 nM (phosphorylated), respectively; at the end of the 60-min post-treatment period values decreased to 121±5 nM (parent) and 211±17 nM (phosphorylated). At the end of each infusion period mean arterial pressure decreased to −9±3, −22±4, and −24±4 mmHg below baseline , A and h. Immediately upon infusion BAF312 elicited an acute decrease in heart rate; 10-min into the first infusion period heart rate decreased to −51±8 beats/min (vehicle = −6±4 beats/min) and values remained statistically reduced for 30-min . However, despite an increase in plasma concentrations of BAF312 throughout the infusion period heart rate returned toward baseline and was not different from vehicle controls during the latter half of the 60-min infusion. In a separate group of animals instrumented with a Millar catheter into the left ventricle, BAF312 at the same doses elicited only a modest, but statistically-significant, decrease on LV dP/dtmax at the end of the 1.5 mg/kg i.v. infusion . In the same study heart rate was acutely reduced (vs. vehicle) only in the 10 mg/kg group on day 1 . Thereafter, changes in heart rate were not different from vehicle control rats. The acute hemodynamic effects of a single oral dose of BAF312  were also quantified. Compared to vehicle controls, the compound had no statistically significant effect on either mean arterial pressure  or heart rate  at all doses tested up to 30 mg/kg.Prior to longer-term hypertension studies with FTY720 an acute, single oral-dose study was performed to define the lower and upper limits of a pharmacologically-active dose with respect to acute hemodynamic effects. Thus, FTY720  was administered p.o. in telemetry instrumented rats as a single dose and hemodynamic values recorded for 72 hours post-treatment due to the prolonged half-life of the compound. Plasma concentrations of the parent at Tectively . Values ectively . Values ectively . Values ectively . Values ectively . Values . On day 1, plasma concentrations of FTY720 at Tmax in satellite rats (n = 3/group) were 13±2, 53±9, and 120±17 nM, respectively. Plasma concentrations of the phosphorylated metabolite at Tmax were 103±24, 275±55, and 773±55 nM, respectively. Terminal plasma concentrations of FTY720 at Tmax were 63±37, 81±4, and 229±21 nM, respectively, and concentrations of the phosphorylated metabolite at Tmax were 546±358, 800±77, and 2913±92 nM, respectively. Concentrations of FTY720 and FTY720-P in heart at the end of the study were also determined, mean FTY720 tissue concentrations were 379, 490, and 1848 ng/g, respectively, in the 0.5, 1.5, and 5.0 mg/kg/d dose groups and corresponding concentrations of (S)-FTY720-P were 513, 757, and 3799 ng/g.We sought to ensure that effects of FTY720 on blood pressure could be tested in the absence of significant changes in heart rate and thus the doses selected were below that shown to elicit acute bradycardia after a single dose (observed only in the 10 mg/kg/d group in the single-dose study). In the repeat-dosing study with FTY720 , plasma concentrations were also determined . On day 1, plasma concentrations of BAF312 at Tmax (4 hrs) in satellite rats (n = 3/group) were 91±7, 685±113, and 6395±390 nM, respectively. Terminal plasma concentrations at Tmax were 81±8, 735±73, and 8328±650 nM, respectively. Cardiac tissue concentrations of BAF312 were not determined.Plasma concentrations in the repeat-dosing study with BAF312  were determined . No differences in baseline mean arterial pressure between the groups were detected in the BAF312 arm of the study. BAF312 elicited no significant increase in mean arterial pressure during the study; average changes in 24-mean values during 14-days of treatment in the 0.3, 3.0, and 30 mg/kg/d dose groups were 3.0±0.5, 1.1±0.5, and 0.1±0.5 mmHg, respectively . In the FTY720 arm of the study, the compound elicited no acute or sustained bradycardia at any dose tested . Unexpectedly, values in the 1.5 mg/kg treatment group tended to be higher than those in all of the comparator groups, including higher doses of the compound (1.5 and 5.0 mg/kg) and those in the vehicle control group); 24-hr mean changes in heart rate in the vehicle, 0.5, 1.5, and 5.0 mg/kg dose groups were −2±1, 11±2, 1±1, and 1±3 beats/min, respectively. In the BAF312 treatment arm and throughout the 14-day study, no bradycardia was observed in any dose group tested ; 24-hr mean change in heart rate in the 3, 10, and 30 mg/kg dose groups were −2±2, −2±3, and −2±4 beats/min, respectively.In the repeat-dosing study no differences in baseline mean arterial pressure were detected at any timepoint between the treatment groups. FTY720 (n = 8/group) elicited dose-dependent increases in mean arterial pressure at all doses tested . Relative to baseline, the average mean arterial pressure values over the treatment period increased 8.4±0.4, 12.8±0.4, and 16.2±0.8 mmHg, respectively, (vehicle = 3.7±0.5 mmHg) and values reached statistical significance in all treated groups . No diff. No diff. No diff1 receptor activation, likely through activation of IK(Ach) in pacemaker cells 3 receptor subtype is principally responsible for sustained hypertension as observed in the present study and in human clinical trials In the present study using differentially-selective, and clinically-tested and efficacious S1P receptor agonists, we demonstrate that acute bradycardia is mediated by S1P1/5 receptors, elicited immediate decreases in heart rate during the 0.5 mg/kg (low-dose) infusion. Thereafter, decreases in heart rate were attenuated despite continued infusion and escalating plasma concentrations of the compound. A similar effect was noted for acute decreases in dP/dt in the presence of BAF312 whereby values were not maintained in the 5.0 mg/kg dose group . The reduced effect on heart rate and dP/dt at high doses was likely due to ligand-receptor internalization in the presence of high concentrations of BAF312 1 agonists, such as SEW2871, also elicit S1P1 receptor internalization In anesthetized rats, both FTY720 and BAF312 produced bradycardia. While heart rate was dose-dependently decreased in the presence of FTY720, infusion of BAF312, with selectivity only for S1PAt high enough oral doses of FTY720 (10 mg/kg/d) in rats, acute bradycardia was observed. Indeed, when the effect of the compound was monitored for 72 hours after a single oral dose, heart rate was decreased on day 1 but not thereafter despite prolonged plasma concentrations of the compound and despite sustained increases in blood pressure. The effect of FTY720 during repeated administration was tested at doses below that shown to elicit acute bradycardia. In those studies no bradycardia was observed and in fact values were increased in the 0.3 mg/kg dose group although the reason for this increase is not clear and the same effect was not observed at higher doses of the compound. More importantly, in those studies we demonstrate that FTY720 elicits dose-dependent hypertension after repeated administration at doses below those necessary to elicit bradycardia and consistent with hypertension observed clinically with FTY720 3 receptors, but not in the presence of BAF312 that lacks S1P3 activity, is consistent with clinical observations in patients 3 receptor subtype in the development of hypertension both in rats and in patients administered FTY720. Interestingly, data from a subset of telemetry animals in the present study suggest that rats administered FTY720 had a reduction in urinary sodium excretion (unpublished preliminary observation), a finding consistent with a study by Tawadrous et al. 3 activity of the compound is not clear.Sustained hypertension in telemetry rats in the presence of FTY720 that possesses activity at S1POf note, while FTY720 elicited hypertension in telemetry rats after repeated administration, acute increases in blood pressure were not observed during an escalating i.v. infusion in anesthetized rats. In fact, blood pressure fell dramatically in that model due to the immediate and marked decrease in heart rate, and presumably cardiac output, as opposed to any direct effect on the systemic vasculature. In addition, the drop in blood pressure during i.v. infusion was exacerbated by the limited ability of the anesthetized rat to elicit a sympathetic reflexive response secondary to vasodilation 1 receptor activation, some pre-clinical studies have suggested that bradycardia may be elicited by S1P3 receptor stimulation and this has led to a high degree of controversy in the field and speculated differences between rodent and man. However, many of these hypotheses were proposed within SAR-driven publications with limited biological data or context. Saha et al. 18, with reported selectivity for human S1P1 over S1P3, administered as an oral dose to telemetered conscious rats had no effect on heart rate, thereby implying that S1P3 activity is responsible for bradycardia in rodents. However, in that study heart rate was assessed only in a repeat-dosing paradigm and it has been demonstrated that bradycardia observed after stimulation of these receptors is transient in both animals and patients 3 affinity, higher doses were required to elicit similar changes in heart rate in anesthetized rats vs. those with greater S1P3 affinity. Without disclosure of the hemodynamic data, nor the pharmacokinetic properties of the compounds tested in the model, it is not possible to adequately corroborate these claims. A separate study 3-mediated bradycardia in rats after synthesis of differentially-selective S1P agonists due to correlation in heart rate and S1P3 IC50 values however, values correlated just as well, if not better, with activity at S1P1 receptors.Although results from the present study using two differentially selective and clinically-tested S1P agonists suggest that bradycardia in rats is mediated by S1P3 may have a role in bradycardia, Shimizu et al 1 agonist, KRP-203 , when administered as an i.v. bolus to anesthetized guinea pigs had no effect on heart rate although the highest tested dose, 0.03 mg/kg/d, was below that demonstrated to be efficacious in a disease-relevant model (0.1–1.0 mg/kg/d). However, in a separate study KRP-203 was administered to Wistar rats and while the compound had no effect on heart rate at 0.03 mg/kg, it elicited acute bradycardia (to ∼150 beats/min below baseline) at 0.1 and 0.3 mg/kg. These results clearly implicate an S1P1-dependent mechanism 3 antagonist, TY-52156, could partially inhibit FTY720-induced bradycardia in vivo, the effect of FTY720 in the presence of TY-52156 was still markedly attenuated vs. vehicle controls (40–50 beats/min) suggesting another S1P receptor subtype, likely S1P1, was responsible for the majority of the bradycardia observed in the study.Lending to the hypothesis that S1PA, with 10-fold S1P1 selectivity over S1P3, elicited decreases in heart rate in conscious rats during i.v. infusion. However, in mice reductions in heart rate were observed only in wild type mice administered A or S1P, but not in S1P3 KO animals, thereby implicating an S1P3 dependent mechanism. While these data are convincing in genetically-modified mice, it may be a phenomenon limited to mouse since results of the present study in rat, and clinical studies in humans both implicate the S1P1 receptor subtype as the primary driver of acute bradycardia in vivo. Moreover, results of the present study and data reported in humans implicate the S1P3 receptor subtype is the primary mediator of hypertension and suggest that S1P receptor agonists devoid of activity at S1P3 would not be expected to elicit hypertension clinically.Finally, Forrest et al. Limitations of the Study: one cannot rule out the possibility that agonist activity at S1P5 is responsible for bradycardia since both FTY720 and BAF312 maintain activity at this receptor subtype. However, this possibility seems less likely since the S1P5 receptor has not been shown to play a role in coupling to sino-atrial nodal pacemaker activity. In addition, one cannot rule out the possibility that FTY720 agonist activity at S1P4 is responsible for hypertension observed in the present study since it has been suggested that this receptor subtype plays a role in vasoconstriction in the pulmonary circulation Figure S1max in anesthetized rats.Effect of BAF312  on left ventricular dP/dt Values are shown at the end of the 0.5, 1.5 and 5.0 mg/kg doses and at an equivalent timepoint in the vehicle control group. Values were statistically reduced at the end of the 1.5 mg/kg i.v. infusion, but not the 5.0 mg/kg i.v. infusion, an effect likely due to ligand-receptor internalization in the presence of high concentrations of BAF312.(TIF)Click here for additional data file.Figure S2Brain, muscle, and plasma concentrations of FTY720 (A) and FTY720-P (B) after a single oral dose of FTY720 (3.0 or 10 mg/kg). A blood sample or tissues were harvested from satellite rats at 4, 8, 24, and 72-hrs post-dose. No compound was detectable in the CSF.(TIF)Click here for additional data file.


In a previous preliminary study, we developed the polymerase-endonuclease amplification reaction (PEAR) for amplification and large-scale preparation of natural antisense ODNs. Here we extended the method in preparation of a widely used modified oligonucleotide with 5′-O-(1-Thiotriphosphate) modifications. Using electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection, the purity of the PEAR product was measured as high as 100.0%. Using PEAR a large amount of a specific oligonucleotide can be produced starting from a small amount of synthetic seeds. It is suggested that PEAR can be a useful tool for large-scale production of modified oligonucleotides.Antisense oligonucleotides (ASODNs) have been widely used as an important tool for regulating gene expression, and developed into therapeutics. Natural ODNs are susceptible to nuclease degradation, nucleic acid analogues, however, have less side effects, stronger stability and more potent activities. Large-scale  In addition, synthetic ODNs are often contaminated with highly homologous failure sequences. A few studies have been reported that modified nucleic acid analogues were produced by enzymatic reactions, such as primer extension At present, oligonucleotides are produced mostly by chemical synthesis. Large-scale synthesis of a certain oligonucleotide by We conducted PEAR reactions by using one or two dNTPαSs  , to subsslipping and cleaving mechanism”. An exponential PEAR consist a slipping reaction that extends the number of repeats in the PEAR product increases linearly, and a subsequent cleaving reaction that drives the number of molecules increases exponentially. When a thermostable DNA polymerase was present, but PspGI was absent  nucleotides by primer extension, Phusion DNA polymerase was able to incorporate up to three, eight and five LNA nucleotides successively, respectively for LNA-A, LNA-T and mixed LNA-A+LNA-T nucleotides We employed a well-established LC/MS technique Among the five ODN strands, A, B and C are all in full length, while the other two ones, D and E, were truncated by one or two 3′-terminal bases. The sequencing results shows that in the PEAR products most of the repeats were perfect, therefore, the truncations of the 3′-terminal Gs were not caused by incomplete reaction to extend the 3′-terminus to full length, but occurred after the products had been cleaved into monomers. Because truncations happened only in modified strands, but not in unmodified strands, they must be caused by the 3′ to 5′ exonuclease activity of the Phusion High Fidelity DNA polymerase, which was triggered by the modified bases. However, the truncations were ceased when a modified base was encountered, owing to thio-modified bases are resistant to exonuclease.To obtain full length oligonucleotides, we conducted PEAR reactions in which both A and G were modified (*A*G). As shown in In addition, as shown in de novo synthesis, PEAR has several advantages such as lower equipment cost, easier purification process, higher product purity, the ability to avoid failure sequences, and the use of hazardous chemicals. Therefore, PEAR can be a useful tool for large-scale production of modified oligonucleotides, as it allows the production of a large quantity of modified oligonucleotides starting from a small amount of seeds using a simple thermocycler, the only equipment required.Based on the “slipping and cleaving mechanism”, PEAR amplifies a specific target oligonucleotide through polymerase elongation and endonuclease cleaving under control of thermal cycling. Compared to traditional Usually, ASODNs are used in the form of single-stranded in vivo, since some nascent bases are required for triggering cellular RNase H to recognize and degrade the target RNA transcripts. In addition, in CpG ODNs, one or more unmodified CpG dinucleotides are required to stimulate the innate immune responses.In the present scenario only dATPαS, dGTPαS and dCTPαS, but not dTTPαS, could be incorporated into a desired oligonucleotide. It is, however, often sufficient to resist nuclease degradation by modifying only 3 to 6 bases in the ends of an oligonucleotide. Chimeric ODNs comprises both natural and modified bases are often more favorable for use in vivo stability. It has been reported that LNA ODNs can be produced enzymatically Thiotriphosphate oligonucleotides, as well as 2′-MOE and LNA modifications, have been widely used in biomedical studies due to their higher potency and New England Biolabs, Inc. The recognition site (R) of PspGI is CCWGG, where W = A or T. Synthetic ODNs, including a target (X) and a probe (P), are synthesized by Integrated DNA Technologies, Inc. and purified by HPLC. The sequence of X is: TGT AAA CAT CCT CGA CTG GAA G, which is derived from human microRNA hsa-miR-30a. The structure of P is X'R'X'R'X', where X' and R' is complementary respectively to X and R. The sequence of P is: CTCC AGG CTT CCA GTC GAG GAT GTT TAC ACC AGG CTT CCA GTC GAG GAT GTT TAC AT CCA GTC GAG GAT GTT TAC A, where the recognition site of PspGI is underlined. Four 2′-deoxyribonucleotides-5′-O-(1-Thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS and dTTPαS were purchased from Trilink BioTechnologies, Inc.Phusion high fidelity DNA polymerase, highly thermostable restriction enzyme PspGI and dNTPs are purchased from 4)2SO4, 2.5 mM MgCl2, 0.02% BSA, 0.08 U/µL Phusion DNA polymerase, 0.4 U/µL PspGI, 0.1 µM target and 1.0 µM probe (or seeds PEAR product). In desired reactions, one or two natural dNTPs were completely replaced with the corresponding dNTPαS . The reactions were initiated at 95°C for 1 min, followed by 35 cycles of denaturing at 95°C for 15 sec, annealing at 55°C for 35 sec, elongation and cleaving at 75°C for 3 to 5 min. If desired, PspGI digestion of the product is conducted under 75°C for 1 to 16 h by adding 0.1 volume 10X NEBuffer 4, 0.4U/µL PspGI, and ddH2O to 2X volume. PEAR products were examined by non-denaturing polyacrylamide gel electrophoresis (PAGE) in 15% gels at 5V/cm, stained with ethidium bromide and detected by an UV illuminator. Yields of products were estimated by absorbance measurements OD260 of diluted samples.PEAR reactions were run in a 96-well Applied Biosystems 9700 Thermal Cycler, each in a 100 µL volume reaction mixture containing 200 µM each dNTP, 15 mM Tris-HCl, 30 mM KCl, 5 mM  analysis was performed by Novatia, LLC using their High-Throughput Characterization System (HTCS) PEAR products were fully digested by the addition of 1 volume of cleavage mixture containing 1X NEBuffer 4, and 1.0 U/µL of PspGI. Cleavage reactions were incubated for 8 hours at 75°C. Before and/or after PspGI digestion, the products were ethanol precipitated, washed twice with 75% ethanol, dried and resuspended in ddHE. coli DH5α competent cells were purchased from TaKaRa Co. Ltd., and operated following the manufacturer’s instructions. To allow efficient TA cloning, PEAR products were pretreated with Taq DNA polymerase in the presence of dNTPs to fill in the sticky ends, and to add an additional adenine nucleotide at the 3′-ends. After ligation, transformation, plating and overnight culture, E. coli colonies were picked randomly, plasmids were extracted, double-digested with EcoRI and HindIII and screened using PAGE electrophoresis to identify inserted fragments. Fifty clones containing insertions were sequenced using Sanger method. Mutation rate was computed by dividing the number of mutations by the total number of nucleotides.TA cloning vector pMD18-T and File S1Figure S1,PAGE electrophoresis of PspGI digestions of PEAR products. (A) Digestion of natural PEAR products;(B) Digestion of PEAR products incorporating PS-dATP; (C) Digestion of PEAR products incorporating PS-dGTP. Figure S2, The nucleotide sequence of the recognition site of PspGI. Figure S3, Sanger sequencing of PEAR products.  Figure S4, The LC/UV Chromatogram and Deconvoluted Mass Spectrum of the *A*G PEAR product incorporating dATPαS+ dGTPαS fully digested by PspGI. Components: (A) RT = 7.45 min: MW = 6742.0; (B) RT = 8.00 min; (C) RT = 8.38 min; (D) RT = 8.76 min; (E) RT = 9.05 min; See Table S2 for detailed characterization of components. Table S1,Characterization components of the *A PEAR product by LC/UV/MS analysis. Table S2, Characterization components of the *A*G PEAR product by LC/UV/MS analysis.(DOC)Click here for additional data file.


P≤0.05) with the risk of overall breast cancer in the same direction as previously reported: rs10069690 (5p15/TERT), rs999737 (14q24/RAD51L1), rs13387042 (2q35/TNP1), rs1219648 (10q26/FGFR2), rs8170 (19p13/BABAM1), rs17817449 (16q12/FTO), and rs13329835 (16q23/DYL2). A marginally significant association (P<0.10) was found for three additional SNPs: rs1045485 (2q33/CASP8), rs4849887 (2q14/INHBB), and rs4808801 (19p13/ELL). Three additional SNPs, including rs1011970 (9p21/CDKN2A/2B), rs941764 (14q32/CCDC88C), and rs17529111 (6q14/FAM46A), showed a significant association in analyses conducted by breast cancer subtype. The risk of breast cancer was elevated with an increasing number of risk variants, as measured by quintile of the genetic risk score, from 1.00 (reference), to 1.75 (1.30–2.37), 1.56 (1.15–2.11), 2.02 (1.50–2.74) and 2.63 (1.96–3.52), respectively, (P = 7.8×10–10). Results from this study highlight the need for large genetic studies in AAs to identify risk variants impacting this population.Genome-wide association studies (GWAS), conducted mostly in European or Asian descendants, have identified approximately 67 genetic susceptibility loci for breast cancer. Given the large differences in genetic architecture between the African-ancestry genome and genomes of Asians and Europeans, it is important to investigate these loci in African-ancestry populations. We evaluated index SNPs in all 67 breast cancer susceptibility loci identified to date in our study including up to 3,300 African-American women , recruited in the Southern Community Cohort Study (SCCS) and the Nashville Breast Health Study (NBHS). Seven SNPs were statistically significant ( Breast cancer is one of the most common malignancies diagnosed among women worldwide, including women of African descendent. African American (AA) women experience a disproportionate burden of breast cancer. Age-adjusted mortality rate of this cancer is more than 40% higher in AAs than in European-ancestry populations. AA women tend to be diagnosed with breast cancer at a younger age and with more aggressive types of the disease, such as ER- (estrogen receptor negative) and ER−/PR−/HER2-  breast cancer. Within AAs, women having a higher African ancestry level, estimated by ancestry informative markers (AIMs), have been shown to have an increased likelihood of ER−/PR- versus ER+/PR+ breast cancers BRCA1, BRCA2, TP53, and PTEN) and four moderate-penetrance genes  have been discovered for breast cancer To date, four high-penetrance genes  were selected randomly from those who were cancer-free and frequency-matched to cases in a 1∶1 ratio on age at enrollment (±1 year), recruitment method, and sample type .This study uses resources from the Southern Community Cohort Study (SCCS) and the Nashville Breast Health Study (NBHS). The NBHS is a population-based case-control study Written, informed consent was obtained from all participants prior to interview, and the study protocols have been approved by Institutional Review Boards at Vanderbilt University (for NBHS and SCCS) and Meharry Medical College (for SCCS).P<10–6), 4) genotyping not concordant with previous data, 5) duplicates or “cryptic” duplicates, 6) first-degree relative, 7) ethnic outliers based on a subset of 37,000 uncorrelated markers which passed QC. SNPs were excluded for any of the following reasons: 1) call rate <95%, 2) deviated from HWE in controls at P<10–7, 3) genotyping discrepancies in more than 2% of duplicate samples. Data cleaning was conducted within the whole COGS Project. After QC, a total of 199,961 SNPs for 1,116 cases and 932 controls were included in the dataset. We then performed principal component analyses (PCA) using a set of 4,613 uncorrelated SNPs . Five additional participants (three cases and two controls) were excluded due to >6 σ away from the means of PCA1 and PCA2. In total, 1,113 cases and 930 controls from the SCCS and NBHS were successfully genotyped by the COGS SNP array.Genotyping for samples described above was conducted using the protocol for the COGS Project as described elsewhere In our previous project, nine GWAS-identified SNPs were genotyped using Taqman/Sequenom in 810 cases and 1,784 controls from the NBHS and SCCS http://med.stanford.edu/tanglab/software/frappe.html), which implements an Estimation-Maximization algorithm for simultaneously inferring each individual’s ancestry proportion and allele frequencies in the ancestral populations Individual African ancestry level was estimated from 612 AIMs included in the COGS SNP array using the program frappe  for each study participant by multiplying the number of risk alleles (0/1/2) of each SNP by the weight  for that SNP, and then summing them together. Since data for a complete set of SNPs were only available for the 1,110 cases and 929 controls genotyped using COGS SNP array, GRS analysis was conducted among these subjects.P<0.1) with direction of association consistent with previous reports. SNP rs1219648 was not included in the COGS SNP array and only 66% women of the COGS subjects had data available for this SNP, genotyped in our previous project through Sequenom (23). SNP rs13387042 was genotyped in both by the COGS SNP array and Sequenom (23). This SNP, however, did not reach P<0.1 in the samples analyzed using COGS SNP array and thus was not included in the GRS analyses. Thus, these two SNPs, rs1219648 and rs13387042, were excluded in GRS analyses. The remaining eight SNPs, rs10069690, rs999737, rs8170, rs17817449, rs13329835, rs1045485, rs4849887, and rs4808801 were included in the GRS analyses. All statistical analyses were conducted in SAS, version 9.3, with the use of two-tailed tests.We constructed a GRS using SNPs that showed a statistically significant association with breast cancer risk in this study. In the present study, ten SNPs were associated with breast cancer , and both of them were included in the present study. Similarly, in the locus 16q12/TOX3, SNPs rs4784227 and rs3803662 were discovered in Asians 2 = 1.0), was used as a substitute. SNPs rs17271951 and rs3803662 are not in LD in HapMap Africans (r2 = 0.03), and both of them were included in the final analyses. In the 10q26/FGFR2 locus, both rs1219648 and rs2981582 were discovered in Europeans but were in moderate LD in HapMap Africans (r2 = 0.25). For the 6q25/TAB2 locus, rs9485370 was used to replace the originally reported SNP rs9485372 2 = 1 in HapMap Africans). For the other 21 loci, one index SNP (reported in previous GWAS) per locus was selected. Therefore, in total, 28 SNPs in previously reported 25 loci were investigated in the present study. Five of them were deviated from Hardy-Weinberg test with P<0.05, including rs1045485, rs10995190, rs13281615, rs889312, and rs999737.Two SNPs have been discovered in the 10q21/P = 0.04, binomial sign test). Five SNPs were nominally statistically significant (P≤0.05) in the same direction as previously reported  1.19 (1.05–1.36) and P-value 0.007. Notably, the MAF is very low (0.047 in controls) for SNP rs999737 at 14q24/RAD51L1, however, a strong association was observed for this SNP, with OR (95% CI) 1.59 (1.15–2.19) and P-value 0.005. Allelic OR (95% CI) for the other 3 SNPs are 1.17 (1.04–1.33) for rs13387042 (P = 0.011), 1.17 (1.04–1.33) for rs1219648 (P = 0.011), and 1.25 (1.07–1.47) for rs8170 (P = 0.006). The CASP8 SNP (rs1045485) identified previously through a candidate gene approach has a low frequency (MAF = 0.065) in AAs. A marginally significant association was found for this SNP with OR (95% CI) of 1.25 (0.96–1.62) and P-value 0.096. Significant association was observed for SNP rs4973768 at 3p24/SLC4A7 in this study, however, in the opposite direction as previously reported. No association was observed with the other 21 SNPs. Among them, the minor allele in rs10771399 at 12p11/PTHLH is very rare in AAs with MAF = 0.034 in controls.Of the 28 SNPs, 19 SNPs had an OR of breast cancer risk in the same direction as initial reports. This is higher than expected under the null hypothesis (reported . SNP rs1P≤0.05). The OR (95% CI) was 1.21 (1.06–1.37) for rs17817449 at 16q12/FTO (P = 0.004) and 1.16 (1.01–1.32) for rs13329835 at 16q23/DYL2 (P = 0.037). A marginally significant association (P<0.10) in the same direction as in Europeans was found for two additional SNPs, with ORs (95% CI) of 1.13 (0.99–1.30) for rs4849887 at 2q14/INHBBP (P = 0.075), and 1.13 (0.98–1.29) for rs4808801 at 19p13/ELL (P = 0.091). The 22q13/MKL1 SNP rs6001930 showed an association (P = 0.035), however the direction was inconsistent with that observed in Europeans. No associations were identified for the other 37 SNPs, among which four SNPs have a MAF <0.05, including rs11552449 at 1p13/AP4B1, rs1353747 at 5q11/PDE4D, rs11780156 at 8q24/MYC, and rs11571833 at 13q13/BRCA2.Of the 42 SNPs, one for each of the 42 loci recently discovered in European-ancestry populations P≤0.05) or marginally significant (P≤0.1) association with any of these three subtypes of breast cancer are presented. Three SNPs that were not associated with overall breast cancer showed nominally statistical significance (P≤0.05) in analysis by subtypes in the same direction as previously reported. SNPs rs1011970 at 9p21/CDKN2A/2B and rs941764 at 14q32/CCDC88C were associated with ER+ breast cancer with ORs (95% CI) 1.27 (1.05–1.54) (P = 0.014) and 1.26 (1.02–1.56) (P = 0.032), respectively. SNP rs17529111 at 6q14/FAM46A was associated with ER−/PR−/HER2− tumor with OR (95% CI) of 1.97 (1.02–3.82) (P = 0.043). Differences in the strength of the association were also observed for three other SNPs across breast cancer subtypes. SNP rs17817449 at 16q12/FTO showed an association with ER+ tumor with OR (95% CI) of 1.32 (1.09–1.60) but not with ER− or ER−/PR−/HER2− tumors. Both rs10069690 at 5p15/TERT and rs999737 at 14q24/RAD51L1 showed the strongest association for ER−/PR−/HER2− breast cancer though associations were also observed for ER+ and ER− cancers.Association results between SNPs with risk of breast cancer by subtypes, including ER+, ER-, and ER−/PR−/HER2−, are presented in P = 7.8×10–10). Such associations also were observed for all three subtypes of breast cancer.Significant associations were observed between GRS and risk of breast cancer . ORs (95P<0.05) and three SNPs were marginally significantly associated (P<0.10) with overall breast cancer risk in the same association direction as previously reported. Three additional SNPs showed a significant association (P<0.05) when stratified by breast cancer subtype. GRS analyses showed significant associations with the risk of overall or subtype of breast cancer.In the present study, we investigated associations of 70 index SNPs in 67 breast cancer susceptibility loci identified to date in up to 1,231 cases and 2,069 controls of AA women. We found that seven SNPs were significantly associated  was originally discovered in a GWAS of AAs FGFR2, association was observed in our previous study BABAM1 was only investigated in a pooled study from Africans and AAs and no association was observed CASP8 SNP (rs1045485) and RAD51L SNP (rs999737) have a MAF <5% in African-ancestry populations. Therefore, it is not surprising that these two SNPs were not replicated in all previous studies of African-ancestry populations Among the ten SNPs that showed significant or marginally significant association with overall breast cancer risk in the present study, six have been investigated in previous studies of African-ancestry populations TOX3 was replicated in the WHI FGFR2) was significantly associated with breast cancer risk in two studies MRPS30KLF4We did not replicate associations for the other 60 reported SNPs with the risk of overall breast cancer. Inconsistent results were reported for some of them in previous studies of African-ancestry populations. For example, SNP rs3803662 at 16q12/In general, our results that approximately 90% of index SNPs were not replicated in AAs are consistent with results from previous studies in African-ancestry populations TNP1, 5q11/MAP3K1, 10q26/FGFR2, and 19p13/BABAM1 loci Because of the large difference in genetic architecture between African-ancestry and European/Asian ancestry populations, failure to replicate most of the reported SNPS in AAs is not surprising. Most, if not all, index SNPs identified in GWAS are associated with breast cancer risk through their strong LD with causal variants. African-ancestry populations have shorter LD and more genetic variations than European/Asian ancestry populations and may have different SNPs in LD with the causal variant. This may be the major reason why index SNPs are not replicated in African descendants. For example, in the BWHS, originally reported index SNPs rs10941679 and rs3803662 were not replicated, but other SNPs in these regions, rs16901937 and rs3104746, were associated with breast cancer To our knowledge, this is the first study in AAs that has evaluated index SNPs in all breast cancer susceptibility loci identified to date. However, the sample size in our study is relatively small, especially when stratified by breast cancer subtype. Some of the null associations observed in this study could be due to inadequate statistical power. Meta-analysis by pooling together all existing data in the AA populations will increase the statistical power to evaluate the effects of these variants in AAs. The other limitation of this study is that we only investigated index SNP in each locus. Large-scale fine mapping studies are needed to identify genetic risk variants at these loci in African-ancestry populations. Such work will be very helpful to identify causal variants for breast cancer.In summary, in this African-ancestry population study, we replicated approximately 10% of index SNPs in 67 breast-cancer susceptibility loci. Heterogeneity was observed across the breast cancer subtype. These results show the complexity in applying GWAS findings to African-ancestry populations. Large-scale studies in AAs are needed to discover genetic risk variants which impact this population.Table S1African genetic ancestry proportion of study participants estimated by AIMs.(DOCX)Click here for additional data file.Table S2Association of breast cancer risk with 60 SNPs located in reported breast-cancer susceptibility loci in African Americans with P>0.1.(DOCX)Click here for additional data file.


Flaviviridae, Flavivirus) is an arthropod-borne flavivirus transmitted by Culex species mosquitoes. We report here the complete genome of the JEV genotype I strain JEV_CNS769_Laos_2009 isolated from an infected patient in Vientiane, Lao People’s Democratic Republic (PDR) (Laos).Japanese encephalitis virus (JEV) ( Flaviviridae, Flavivirus) is an arbovirus transmitted through a zoonotic cycle involving Culex species mosquitoes, pigs, and water birds   of a patient admitted with encephalitis at Mahosot Hospital, Vientiane, Laos. This 12-year-old boy died after being admitted from Meuan District, Vientiane Province , with 3 days of fever, headache, vomiting, diarrhea, confusion, convulsions, neck stiffness, and a Glasgow Coma score of 11 of 15. The CSF white count was 335/ml . Viral RNA was extracted from cell culture supernatant at passage 3. Overlapping real-time PCR (RT-PCR) products spanning the complete coding sequence were obtained using primers designed from (i) the alignment of 8 complete open reading frames (ORF) of genotypes I, III, and V   in length. The ORF  encodes a polyprotein processed into three structural proteins, capsid (C) (127 amino acids [aa]), premembrane/membrane (prM/M) (167 aa), and envelope (E) (500 aa), and seven nonstructural proteins, NS1 (352 aa), NS2A (227 aa), NS2B (131 aa), NS3 619 aa), NS4A (149 aa), NS4B (255 aa), and NS5 (905 aa). The 5′ and 3′ noncoding regions (NCRs) are 96 and 570 nt long, respectively. Cleavage sites are identical to those reported previously, and 4 glycosylation sites  are predicted ( aa, NS4AThis constitutes the first isolation and complete characterization of a clinical strain of JEV in Laos. Phylogenetic analysis revealed that JEV_CNS769_Laos_2009 belongs to genotype I, which is consistent with previous data from neighboring countries , 7. The This full-length sequence might contribute to a better understanding of the molecular epidemiology of JEV and to the study of various aspects of JEV biology.KC196115.The GenBank accession no. of JEV_CNS769_Laos_2009 is


Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as “black” AKU chondrocytes, while those coming from the white portion were referred to as “white” AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both “white” and “black” AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in “black” AKU chondrocytes. J. Cell. Physiol. 227: 3333–3343, 2012. © 2011 Wiley Periodicals, Inc. Alkaptonuria  is an autosomal recessive inherited rare disease resulting from a deficiency of the enzyme homogentisate 1,2-dioxygenase  that normally splits the aromatic ring of homogentisic acid , an intermediary product of the catabolism of tyrosine and phenylalanine . All water used was Milli-Q .AKU chondrocytes were obtained, after informed consent in accordance with the Declaration of Helsinki, from hip cartilage fragments of three alkaptonuric patients suffering from ochronotic arthropathy and undergone surgery for hip replacement. The study received approval from the Local Ethics Committee. The characteristics of patients enrolled for the study are schematically reported below:Patient #1: female, age 62, 4/4 backbone impairment, 4/4 articular joints impairment, two orthopaedic surgical interventions, urinary HGA level (24 h): 300 mg/dl.Patient #2: male, age 60, 4/4 backbone impairment, 4/4 articular joints impairment, five orthopaedic surgical interventions, urinary HGA level (24 h): 371 mg/dl.Patient #3: female, age 69, 4/4 backbone impairment, 4/4 articular joints impairment, two orthopaedic surgical interventions, urinary HGA level (24 h): 475 mg/dl.g. Cell pellets were then re-suspended in DMEM containing 10% fetal calf serum (FCS) and 0.33 mM HGA and expanded in monolayer culture at 37°C in 95% relative humidity and in a 5% CO2 atmosphere. HGA was added to mimic in vitro the AKU pathological condition; the concentration used, 0.33 mM, is in the range of circulating HGA in AKU patients' serum  containing 2% penicillin/streptomycin solution and 0.2% amphoterycin B. Chondrocytes were isolated by sequential enzymatic digestion: 30 min with 0.1% hyaluronidase, 1 h with 0.5% pronase, and 1 h with 0.2% collagenase at 37 °C in wash solution (DMEM + penicillin/streptomycin solution + amphoterycin B). The cells suspension was then filtered twice using 70 µm nylon meshes, washed and centrifuged for 10 min at 700×The DNA content of cell pellets from each culture was used as a parameter of cell proliferation. DNA was extracted with a high pure PCR template preparation kit according to manufacturer's instructions  and quantified with Qubit™ .Apoptosis was analyzed by flow cytometry evaluating Annexin V binding . Cells were stained with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide.Nitrite levels were measured in chondrocyte culture supernatants with Griess reagent  . Cytokine concentrations were calculated using a standard curve established from serial dilutions of each cytokine standard as described in the manufacturer's protocol and expressed as picograms per milliliter of culture medium.Cells were washed twice with sterile PBS and resuspended in 50 µl of a buffer containing 65 mM DTE, 65 mM CHAPS, 9 M urea, and 35 mM Tris-base. Cell disruption was achieved by sonicating briefly in an ice bath. Protein content in cell lysates was assessed according to Bradford  membranes] or 50 µg (2D gels to be silver stained) of proteins were adsorbed onto Immobiline Dry Strips  for 10 h. Isoelectric focusing (IEF) was carried out with a Protean IEF cell (Bio-Rad). The voltage was linearly increased from 300 to 3,500 V during the first 3 h and then stabilized at 5,000 V for 22 h .Prior to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), IPG were equilibrated in 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.05 M Tris–HCl pH 6.8 containing first 2% (w/v) DTE, and later 2.5% (w/v) iodoacetamide (IAA). SDS–PAGE was carried out applying 40 V per gel until the dye front reached the bottom of gels.2 for 75 min. Protein transfer was checked by staining with 0.2% Ponceau S in 3% (v/v) TFA for 3 min and de-staining with water.Silver ammoniacal staining was carried out according to Switzer  in 5% (v/v) TFA to allow derivatization of protein carbonyls  or by gel matching with proteomic reference maps already produced, calibrated, and characterized in our laboratories  and analyzed with Image Master™ Platinum (Amersham Biosciences). For comparative proteomic analysis, spot% relative abundance was adopted; for multiple spots identified as different molecular species of a same protein, the average% relative abundance was calculated. For the quantitative analysis of immunorevealed protein carbonyls, the intensity of bands, which is automatically normalized by Image Master™ Platinum against the surrounding background, was chosen as the reference parameter.t-test and multiple-measurement ANOVA analysis followed by the Bonferroni-type multiple comparison were used when necessary. Differences with at least a P-value ≤0.05 were considered significant. For comparative proteomic and redox proteomic analyses, only representative gels and films are shown.All of the experiments were carried out in triplicate; data are presented as average values with standard deviation. Student's In this work, we characterized two distinct types of primary chondrocytes (namely “white” and “black” AKU chondrocytes) obtained from AKU patients suffering from ochronotic arthropathy who underwent surgery. TEM analysis revealed that both “white” and “black” AKU chondrocytes contained intracellular ochronotic pigments  and showBoth types of AKU cells showed a proliferation rate higher than the control  and “white” AKU chondrocytes had a proliferation rate higher than “black” ones (with a +6.1-fold change) A. Since To evaluate the inflammatory status of AKU ochronotic chondrocytes, we measured the levels of IL-1β, IL-6, IL-8, IL-10, and TNFα in cell culture supernatants. With the only exception of IL-1β in “black” AKU chondrocytes, which did not show any difference with respect to the control, for each assayed cytokine we found significantly higher levels in AKU cells, as shown in IL-6 levels were significantly increased in AKU “white” and “black” chondrocytes with respect to the control , similarly to what we observed for IL-8 levels (+7.7-fold-change in “white” and +4.7-fold-change in “black” AKU chondrocytes). IL-6 is a co-factor of the catabolic effects of IL-1β , that is thought to contribute to the structural integrity of cartilage tissue, allowing a suitable response of load bearing joints to mechanical stress , glutathione S-transferase P (GSTP1), heat shock 70 kDa protein 4 (HSP74), and mitochondrial superoxide dismutase (SODM). The simultaneous deficiency of these proteins reinforce the hypothesis that AKU cells cannot adequately control reactive oxygen species (ROS)- and quinone-mediated toxicity.Differently from what observed with the comparative proteomic analysis, when we investigated protein oxidation through immunorevelation of carbonylated proteins we found that “white” and “black” AKU chondrocytes showed quite distinct patterns . Gel-matenergy) and VIME (cell organization). In AKU cells, whereas “white” chondrocytes had no resolved immunoreactive spots but only high molecular weight protein aggregates stacked on the top of the SDS–PAGE gel, “black” AKU chondrocytes showed, together with carbonylated high molecular weight protein aggregates, the following carbonylated proteins: G3P (energy), EF1A1 , VIME (cell organization), CATA and CH60 .In control chondrocytes, we could identify as carbonylated only the proteins G3P . This, together with a lower expression of proteins promoting protein folding, points towards an increased protein aggregation which might lay the basis in vivo for the production and deposition of ochronotic pigments.


Sargassum muticum (SME) against ultraviolet B (UVB)-induced cell damage in human keratinocytes (HaCaT cells). SME exhibited scavenging activity toward the 1,1-diphenyl-2-picrylhydrazyl radicals and hydrogen peroxide (H2O2) and UVB-induced intracellular reactive oxygen species (ROS). SME also scavenged the hydroxyl radicals generated by the Fenton reaction (FeSO4 + H2O2), which was detected using electron spin resonance spectrometry. In addition, SME decreased the level of lipid peroxidation that was increased by UVB radiation, and restored the level of protein expression and the activities of antioxidant enzymes that were decreased by UVB radiation. Furthermore, SME reduced UVB-induced apoptosis as shown by decreased DNA fragmentation and numbers of apoptotic bodies. These results suggest that SME protects human keratinocytes against UVB-induced oxidative stress by enhancing antioxidant activity in cells, thereby inhibiting apoptosis.The aim of this study was to investigate the cytoprotective properties of the ethyl acetate fraction of  Ultraviolet (UV) radiation is the primary environmental cause of skin damage, leading to conditions such as skin carcinogenesis, inflammation, solar erythema and premature senescence –7. Many Sargassum muticum, is widely distributed on the seashores of the southern and eastern parts of Korea. The extracts of S. muticum demonstrated various biological activities, including antioxidant, antimicrobial and anti-inflammatory properties  bromide (MTT) and Hoechst 33342 dye were purchased from Sigma Chemical Company . Cu/Zn SOD and CAT antibodies were purchased from Biodesign International . Diphenyl-1-pyrenylphosphine (DPPP) was purchased from Molecular Probes . All other chemicals and reagents were of analytical grade.2. Cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal calf serum, streptomycin (100 μg/mL) and penicillin (100 units/mL).Human keratinocytes (HaCaT cells) were obtained from the Amore Pacific Company . Cells were maintained at 37 °C in an incubator with a humidified atmosphere of 5% COS. muticum (SME) at concentrations of 12.5, 25, 50 and 100 μg/mL were added to a 1 × 10−4 M solution of DPPH in methanol. The resulting reaction mixture was shaken vigorously. After 3 h, the amount of DPPH remaining was measured at 520 nm using a spectrophotometer.The ethyl acetate fraction of 2O2-treated cells, cells were seeded at a density of 1.5 × 105 cells/well. Sixteen hours after plating, cells were treated with SME at concentrations of 12.5, 25, 50 and 100 μg/mL. After 30 min, H2O2 (1 mM) was added to the plate. Cells were incubated for an additional 30 min at 37 °C. For detection of ROS in UVB-exposed cells, cells were treated with SME as described above. After one hour, cells were exposed to UVB radiation at a dose of 150 mJ/cm2. The UVB source was a CL-1000M UV Crosslinker , which was used to deliver an energy spectrum of UVB radiation . Cells were incubated for an additional 24 h at 37 °C. After the addition of DCF-DA solution (25 μM) for 10 min, the fluorescence of 2′,7′-dichlorofluorescein was detected using a PerkinElmer LS-5B spectrofluorometer . Image analysis for the generation of intracellular ROS was achieved by seeding the cells on a cover-slip loaded six-well plate at 2 × 105 cells/well. Sixteen hours after plating, the cells were treated with SME at a concentration of 100 μg/mL. One hour following SME treatment, the plate was irradiated with UVB. Twenty four hours later, DCF-DA (100 μM) was added to each well, and cells were incubated for an additional 30 min at 37 °C. After washing with phosphate-buffered saline (PBS), the stained cells were mounted onto a microscope slide in mounting medium . Images of the cells were collected using the laser scanning microscope 5 PASCAL program  on a confocal microscope.The DCF-DA method was used to detect intracellular ROS levels in HaCaT keratinocytes . For det2O2 + FeSO4) and then reacted with a nitrone spin trap, DMPO. The resultant DMPO/·OH adducts was detected using a JES-FA electron spin resonance (ESR) spectrometer  [4, 10 mM H2O2, and SME (100 μg/mL). The ESR spectrometer parameters were set at a magnetic field of 336 mT, power of 1.00 mW, frequency of 9.4380 GHz, modulation amplitude of 0.2 mT, gain of 200, scan time of 0.5 min, scan width of 10 mT, time constant of 0.03 sec and a temperature of 25 °C.Hydroxyl radicals were generated by the Fenton reaction  ,30. The Lipid peroxidation was estimated using DPPP, a fluorescent probe . After tg for 15 min. The supernatants were then collected from the lysates, and the protein concentrations were determined. Aliquots of the lysates (40 μg of protein) were boiled for 5 min and electrophoresed on a 10% SDS-polyacrylamide gel. The electrophoresed proteins were transferred onto nitrocellulose membranes, and the membranes were subsequently incubated with primary antibodies against Cu/Zn SOD and CAT. The membranes were further incubated with secondary immunoglobulin-G-horseradish peroxidase conjugates . The protein bands were detected using an enhanced chemiluminescence Western blotting detection kit .Cells were treated with SME (100 μg/mL) for 1, 6, 12 and 24 h; alternatively, cells were treated with SME (100 μg/mL) for 1 h and then exposed to UVB and incubated at 37 °C for 24 h. The harvested cells were then lysed on ice for 30 min in lysis buffer (100 μL) containing 120 mM NaCl, 40 mM Tris (pH 8.0) and 0.1% NP 40. Cells were centrifuged at 13,000 × Cells were treated with SME at a concentration of 100 μg/mL for 24 h. One hour later, they were exposed to UVB and incubated at 37 °C for 24 h. SOD activity was measured using a colorimetric assay kit  according to the manufacturer’s protocol. The kit utilized the tetrazolium salt WST-1, which produces a water-soluble formazan dye upon reduction with the superoxide anions. The formazan dye product was detected at 450 nm. SOD activity was calculated on the basis of the percent inhibition of the superoxide anions.2O2 to produce formaldehyde. Formaldehyde is then measured colorimetrically at 540 nm using 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole as the chromogen. CAT activity was expressed in nmol/min/mL.CAT activity was measured using a colorimetric assay kit  according to the manufacturer’s protocol. Briefly, CAT reacts with methanol, an electron donor, in the presence of H5 cells/mL. Sixteen hours after plating, SME was added at a concentration of 100 μg/mL and cells were exposed to UVB radiation at a dose of 150 mJ/cm2 one hour later and incubated at 37 °C for 48 h. Fifty microliter of MTT stock solution (2 mg/mL) was added to each well to yield a total reaction volume of 200 μL. After incubating the cells for 4 h, the plate was centrifuged at 800 × g for 5 min, and the supernatants were aspirated. The formazan crystals in each well were dissolved in dimethylsulfoxide (150 μL), and the absorbance at 540 nm was read on a scanning multi-well spectrophotometer [Cells were seeded in a 96-well plate at a concentration of 1 × 10otometer .Cells were treated with SME at a concentration of 100 μg/mL and exposed to UVB one hour later. Cells were incubated for an additional 48 h at 37 °C. The DNA-specific fluorescent dye Hoechst 33342 (1.5 μL of a 10 mg/mL stock solution) was added to each well, and the cells were incubated for 10 min at 37 °C. The stained cells were then visualized under a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera to examine the degree of nuclear condensation, and the apoptotic cells were quantified.in situ cell death detection kit  according to the manufacturer’s instructions [5 cells/well. Sixteen hours after plating, cells were treated with SME. Forty eight hours later, chamber slides were fixed with 4% paraformaldehyde for 1 h at 15–25 °C, and cells were permeabilized for 2 min in 0.1% sodium citrate solution containing 0.1% Triton X-100. After washing in PBS, sections were incubated with the TUNEL reaction mixture for 1 h at 37 °C. After washing with PBS, the stained cells were mounted onto a microscope slide in mounting medium . Cells were then observed under a fluorescent microscope  and quantified.The TUNEL assay was performed using an ructions . BrieflyCellular DNA fragmentation in HaCaT cells was assessed by analyzing cytoplasmic histone-associated DNA fragmentation using a kit from Roche Diagnostics  according to the manufacturer’s instructions.p-value of <0.05 was considered statistically significant.All values were expressed as means ± standard error. The results were subjected to an analysis of variance (ANOVA) using the Tukey’s test to analyze difference. A SME protected cells against UVB radiation via free radical scavenging capacity, induction of antioxidant enzymes and inhibition of apoptosis. Therefore, these results suggest that SME may be developed as a protective agent against UVB damage.