We found no support for a “hierarchy of sciences” with physical sciences at the top and social sciences at the bottom. However, we found large differences in reporting practices between disciplines, with p-values between 0.041 and 0.049 over 1990–2013 being 65.7 times more prevalent in the biological sciences than in the physical sciences. The p-values near the significance threshold of 0.05 on either side have both increased but with those p-values between 0.041 and 0.049 having increased to a greater extent (2013-to-1990 ratio of the percentage of papers = 10.3) than those between 0.051 and 0.059 (ratio = 3.6). Contradictorily, p < 0.05 has increased more slowly than p > 0.05 , while the use of “significant difference” has shown only a modest increase compared to “no significant difference” . We also compared reporting of significance in the United States, Asia, and Europe and found that the results are too inconsistent to draw conclusions on cross-cultural differences in significance reporting. We argue that the observed longitudinal trends are caused by negative factors, such as an increase of questionable research practices, but also by positive factors, such as an increase of quantitative research and structured reporting.It is known that statistically significant (positive) results are more likely to be published than non-significant (negative) results. However, it has been unclear whether any increasing prevalence of positive results is stronger in the “softer” disciplines  than in the “harder” disciplines , and whether the prevalence of negative results is decreasing over time. Using Scopus, we searched the abstracts of papers published between 1990 and 2013, and measured longitudinal trends of multiple expressions of positive versus negative results, including  Fanelli’s alleged differences between Europe, Asia, and the United States are not statistically strong either. For example, p-values of 0.017 and 0.048 for paired comparisons between the three world regions, based on the results of a stepwise regression procedure that included all main effects and interactions. A second limitation is that all papers had increased over time . p-values between 0.01 and 0.10 that were reported in all articles published in 1965 and in 2005 in two psychological journals, Journal of Experimental Psychology: General (JEP) and Journal of Personality and Social Psychology (JPSP). The authors found a distinctive peak of p-values just below 0.05. Moreover, the overrepresentation of p-values just below the threshold of significance was more manifest in 2005 than in 1965. For example, for JPSP, in 2005, there were 3.27  times as many p-values in the range 0.04875 < p ≤ 0.05000 as compared to p-values in the range 0.04750 < p ≤ 0.04875, while for 1965 the corresponding ratio was 1.40 . Moreover, Leggett et al. found that in 2005, 42% of the 0.05 values were erroneously rounded down p-values, as compared to 19% in 1965. According to the authors, these longitudinal trends could be caused by software-driven statisticizing: “Modern statistical software now allows for simple and instantaneous calculations, permitting researchers to monitor their data continuously while collecting it. The ease with which data can be analysed may have rendered practices such as ‘optional stopping’  between 2000 and 2010 and reported that between 75% and 80% of the 5,322 p-values they collected were lower than the significance threshold of 0.05. These authors found that the distribution of reported p-values has not changed over those years.Nature that psychology and psychiatry are the “worst offenders”, based on All four aforementioned longitudinal analyses require updating, as they cover periods up to 2007 , 2008 P, 2005 L, and 201In summary, it is well known that positive results  are more likely to be published than negative  results e.g., . Howeverculturomics, has become an established method for investigating secular trends in cultural phenomena  was conducted as well.We investigated longitudinal trends between 1990 and 2013 for ices cf. . We alsoices cf.  method, Our searches were conducted with Elsevier’s Scopus. After trying out other search engines , we concluded that Scopus offers the most accurate and powerful search and export features.1p = 0.05” yielded 137,000 results for articles dated 2011, 113,000 results for articles dated 2012, and 96,500 results for articles dated between 2011 and 2012 (search date: 26 November 2014). These findings are logically contradictory.For example, the query “; (4) Google Scholar suffers from meta-data errors (Google Scholar indexes more papers than Web of Science and Scopus but has various disadvantages: (1) Google Scholar does not allow for nested Boolean searches; (2) Google Scholar does not provide a possibility for exclusively searching in the abstracts of papers; (3) When the total number of records is greater than 1,000, the estimate of this number is inaccuratea errors ; (5) Gooa errors ; (6) Bula errors ; and (7)a errors .Scopus has some unique strengths. By using braces ({ }), it allows for searches in which punctuation marks and mathematical operators are taken into consideration, whereas quotation marks are used for more liberal searches. Both Web of Science and Google Scholar neglect punctuation marks and mathematical operators. That is, Scopus is the only one of the three search engines that can distinguish between the search queries {p = 0.001} and {p < 0.001}. Another difference between Scopus and Web of Science Social sciences, consisting of the following Scopus subject areas: Social Sciences; Psychology; Arts and Humanities; Economics, Econometrics and Finance; Decision Sciences; Business, Management and Accounting.(2)Biological sciences, consisting of the following Scopus subject areas: Agricultural and Biological Sciences; Biochemistry, Genetics and Molecular Biology; Medicine; Neuroscience; Immunology and Microbiology; Pharmacology, Toxicology and Pharmaceutics; Veterinary; Environmental Science; Dentistry.(3)Physical sciences, consisting of the following Scopus subject areas: Physics and Astronomy; Engineering; Earth and Planetary Sciences; Chemistry; Chemical Engineering; Materials Science; Energy; Computer Science.The Mathematics and Multidisciplinary subject areas were not grouped in any of the aforementioned disciplines, to replicate To investigate whether longitudinal trends in significance reporting differ between regions of the world, we distinguished the following world regions as in (1)United States (US).(2)Fifteen European countries (EU15): Austria, Belgium, Denmark, Finland, France, Germany, Greece, Ireland, Italy, Luxembourg, The Netherlands, Portugal, Spain, Sweden, and United Kingdom.(3)Seven Asian countries (AS7): China, Hong Kong, India, Japan, Singapore, South Korea, and Taiwan.Note that a paper can belong to multiple world regions due to multiple authors with affiliations in countries from different world regions, or due to an author having multiple affiliations in countries from different world regions.The following queries were conducted using the advanced search function of Scopus for the abstracts of all papers in the database as well as for each discipline and world region defined above:(1)ABS({ .}), to extract the total number of papers with an abstract.(2)p-values between 0.041 and 0.049 versus p-values between 0.051 and 0.059, namely:a.p-values between 0.041 and 0.049, that is: ABS({p = 0.041} OR {p = .041} OR {p = 0.042} OR {p = .042} … OR {p = 0.049} OR {p = .049}), to extract the number of papers with p-values between 0.041 and 0.049, that is, just below the typical alpha value of 0.05.A query containing b.p-values between 0.051 and 0.059, that is: ABS({p = 0.051} OR {p = .051} OR {p = 0.052} OR {p = .052}…OR {p = 0.059} OR {p = .059}), to extract the number of papers with p-values between 0.051 and 0.059, that is, just above the alpha value of 0.05.A query containing (3)“Significant difference” versus “no significant difference” and variants thereof, namely:a.A query containing typical expressions for reporting significant differences, that is: ABS(({significant difference} OR {significant differences}) AND NOT ), to extract the number of papers with a textual manifestation of significant results.b.A query containing typical expressions for reporting no significant differences, that is: ABS, to extract the number of papers with a textual manifestation of non-significant results.(4)p < 0.05  versus p > 0.05, namely:a.p < 0.05, that is: ABS({p < 0.05} OR {p < .05} OR {p < = 0.05} OR {p < = .05} OR {p = < 0.05} OR {p = < .05} OR {p ≤ 0.05} OR {p ≤ .05}), to extract the number of papers reporting significance in reference to the 0.05 threshold of the alpha value.A query containing variants of the expression b.p > 0.05, that is: ABS({p > 0.05} OR {p > .05} OR {p > = 0.05} OR {p > = .05} OR {p = > 0.05} OR {p = > .05} OR {p ≥ 0.05} OR {p ≥ .05}), to extract the number of papers reporting non-significance in reference to the 0.05 threshold of the alpha value.A query containing variants of the expression The following measures were calculated for items 2–4 of the previous list, per publication year, for all papers in the Scopus database as well as per scientific discipline and per world region:(1)S/T]): the number of papers reporting significant results (S) divided by the total number of papers with an abstract (T) and multiplied by 100.Percentage of papers reporting significant results (100%*[(2)NS/T]): the number of papers reporting non-significant results (NS) divided by the total number of papers with an abstract (T) and multiplied by 100.Percentage of papers reporting non-significant results (100%*[(3)S/NS): the number of papers reporting significant results (S) divided by the number of papers reporting non-significant results (NS).Ratio of significant to non-significant results ((4)S/(S + NS)]): the number of papers reporting significant results (S) divided by the sum of papers reporting significant and non-significant results (S + NS) and multiplied by 100%.Percentage of significance-testing papers reporting significant results (100%*[S/NS) or the percentage of significance-testing papers reporting significant results (100%*[S/(S + NS)]) as dependent variable. Note that just-not-overlapping 95% confidence intervals correspond to a p-value smaller than 0.006  pure disciplines: papers classified by Scopus into subject areas that belong to the same discipline; and (2) discipline intersections: papers classified by Scopus into subject areas that belong to two or all three disciplines.Similarly, to gain insight into the number of papers with authors affiliated with countries from the same world region versus the number of papers with author affiliations spanning across world regions, we repeated the search queries described in “Longitudinal trends and their comparisons between scientific disciplines and between world regions” for: (1) pure world regions: papers all authors of which were affiliated solely with countries belonging to one and the same world region; and (2) world region intersections: papers by authors affiliated with countries belonging to two or all three world regions.p-values between 0.041 and 0.049, p-values between 0.051 and 0.059, and all abstracts, to illustrate the number of papers belonging to pure disciplines/world regions and to discipline/world region intersections. Longitudinal trends in the three pure disciplines/world regions were estimated as in “Longitudinal trends and their comparisons between scientific disciplines and between world regions”.Venn diagrams were constructed for p-values between 0.041 and 0.049 and between 0.051 and 0.059 differ from the corresponding trends of other manifestations of significance, we conducted 25 additional Scopus search queries of multiple p-values and p-value ranges /(N1990/T1990) was calculated, where N2013 and N1990 are the total numbers of abstracts with terms of one of the 83 search queries in 2013 and 1990, respectively, and T2013 and T1990 are the total numbers of papers with an abstract for these two years .For these 77 queries as well as for the 6 queries described in “Longitudinal trends and their comparisons between scientific disciplines and between world regions” , the ratio p < 0.05 but not p > 0.05, p > 0.05 but not p < 0.05, and both p < 0.05 and p > 0.05 .Yearly number of papers containing (2)p-values between 0.001 and 0.099 with a 0.001 interval, that is, p = 0.001, p = 0.002, …p = 0.099 .Yearly number of papers with abstracts containing (3)p-values between 0.041 and 0.049, p-values between 0.051 and 0.059, and the total number of papers with an abstract, in three research fields of biological sciences, proposed by one of the reviewers: psychiatry, surgery, and cardiovascular medicine. This was done by restricting our searches to the terms psychiatr*, surg*, and cardio* OR heart, respectively, in the journal name .Yearly number of papers containing p-values were conducted both for strings with and without zero before the decimal separator . For all search queries containing “<” or “>” we also included variants with “< =,” “= <,” “≤” or “> =,” “= >,” “≥.” Searches in Scopus are not case sensitive, that is, a search query in lower cases  also retrieves records with the same query in upper cases . Spaces around mathematical operators are neglected by Scopus; that is, queries with and without spaces yield the same results. All analyses were conducted for papers published in the period 1990–2013. The employed MATLAB (version R2011b) script, the CSV files with the raw data as exported from Scopus, and an Excel file with all search syntaxes and the names of the corresponding CSV files are provided as Supplemental InformationEach abstract was counted only once per search query, independent of whether it included one or more manifestations of significance. All data were extracted between 15 and 30 November 2014. All searches of numerical According to our searches, Scopus contained a total of 30,677,779 papers with an abstract published between 1990 and 2013. Of these, 3,061,170 papers belonged to the social sciences, 14,412,460 papers belonged to the biological sciences, and 15,364,142 papers belonged to the physical sciences. The sum of the number of papers in the three disciplines was greater than the total number of papers, because some papers were classified into multiple disciplines. The US were found in the affiliations of 8,073,346 papers, EU15 in 8,790,965 papers, and AS7 in 7,413,236 papers.The number of papers has increased over the years in all three scientific disciplines . Over thp-values between 0.041 and 0.049 and the p-values between 0.051 and 0.059 have increased over time  in 2013. Positive results increased 10.3-fold in the same period: from 0.030%  in 1990 to 0.314%  in 2013. In other words, the ratio of significant to non-significant results increased from 1.6  in 1990 to 4.7  in 2013  than that of p < 0.05  in 1990 to 5.6  in 2013  than the percentage of abstracts reporting only p > 0.05 (0.12%) and the percentage of abstracts reporting both p < 0.05 and p > 0.05  than that of p < 0.05 only (2013-to-1990 ratio of the percentage of papers = 1.3), in line with the results of reporting p < 0.05 versus p > 0.05.In a supplementary analysis we searched for and compared abstracts containing p-values between 0.041 and 0.049 and between 0.051 and 0.059 was rare in the social sciences and even rarer in the physical sciences . A large overlap also occurred between the social sciences and the biological sciences.The Venn diagrams in p-values between 0.041 and 0.049 versus p-values between 0.051 and 0.059, “significant difference” versus “no significant difference,” and p < 0.05 versus p > 0.05, for the three disciplines, both for cross-classified papers and pure disciplines. It can be seen that the results for pure disciplines were similar to the results for the cross-classified papers described in “p-values between 0.041 and 0.049 versus p-values between 0.051 and 0.059”–“p < 0.05 versus p > 0.05.” The 95% confidence intervals were wider and the trends were more erratic for the analysis of pure disciplines  as compared to the analysis of cross-classified papers, because the sample sizes were smaller in the former case  and “significant difference” versus “no significant difference,” with a decrease of significant (p < 0.05) results for US and EU15 over time, an increase of both significant and non-significant results for AS7 . Note that in terms of prevalence, the smaller the p-value the more frequently it was reported  intervals than the intervals in p-values between 0.041 and 0.049 versus p-values between 0.051 and 0.059, for “significant difference” versus “no significant difference,” and for p < 0.05 versus p > 0.05. The longitudinal trends were compared between disciplines and between world regions. A comparison of the reporting of a variety of p-values, p-value ranges, textual reporting of significance, and idiosyncratic expressions  in 1990 versus 2013 was conducted as well.We investigated longitudinal trends between 1990 and 2013 for p-values between 0.051 and 0.059 in the abstract has increased by a factor of 3.6 between 1990 and 2013, which indicates that negative results are not disappearing. Also striking is the 10.3-fold increase of p-values between 0.041 and 0.049 over the same time period.The percentage of papers with p-values below 0.1, the smaller the p-value the more frequently it was reported . Indeed, 84% of the physical sciences papers containing p-values between 0.041 and 0.049 were also classified into biological sciences by Scopus  research.p < 0.05 versus p > 0.05, we found that AS7 had the most rapid decline of the percentage of significant results, which is inconsistent with the hierarchy of sciences , the size and direction of the effects are currently elusive. Regional differences in significance reporting are probably obscured by important moderators such as (1) the emergence of China as the second publishing power after the US during the last decade , (2) thap-value between 0.041 and 0.049 in the abstract. Inspection of the 72 abstracts revealed one clear false positive, in the physical sciences . In the remaining 71 abstracts, p-values referred to a statistical test of some kind. However, the p-value was not always related to a clear-cut comparison between two or more groups or experimental conditions. For example, in some abstracts, the p-value belonged to a measure of association, to a variable contributing to a model, or to the assessment of model fit. Moreover, in our searches we made no distinction between unadjusted and adjusted p-values, such as those resulting from Bonferroni corrections for multiple testing. Abstracts often contain multiple p-values, some of which are overlapping or correlated, such as adjusted and unadjusted p-values of the same effect and p-values of subgroup analyses  that reported a analyses . One stranalyses  work wasp-values between 0.041 and 0.049 and the papers with p < 0.05 correspond to respectively 0.18% and 1.7% of all 1990–2013 papers with an abstract indexed in Scopus. Moreover, our searches only focused on the abstract of the papers and by no means provide an exhaustive analysis of the semantic content of papers  are broad, and differences in statistical approaches between specialties within a discipline can be expected. As a follow-up analysis, we searched for journals . We founjournals . Of courWe opted for automated searches. Automated searches have been fiercely criticized in the past. We argue that—provided that one uses a great number of diverse search terms and search strategies—an automated search should render a representative cross-sectional estimate of the trends in scientific publishing. Our manual inspection of the 72 abstracts mentioned in “Faulty inclusions” made it clear to us how difficult it really is to assess whether a paper supports a specific null hypothesis or not. We ran into three types of problems: (1) Issues of accessibility: Despite the fact that our university has 17,300 journal subscriptions, a large proportion of the full-text articles are hidden behind paywalls, which introduces serious availability bias . (2) Issues of interpretation, especially when judging papers containing specialized terminology outside one’s field. (3) The fact that many papers do not explicitly report on a hypothesis or might test multiple hypotheses at the same time. All three issues lead to problems of reproducibility. The results of an automated search, on the other hand, are highly reproducible.N = 4,656 in N = 3,701 in N = 3,627 in p-values between 0.01 and 0.10 retrieved from 2007/2008 issues of three psychological journals, Psychological Science (PS), Journal of Experimental Psychology: General (JEP) and Journal of Personality and Social Psychology (JPSP). The p-values from PS were also re-collected and re-analyzed by a blind assessor, who found a distinctive peak of p-values just below the 0.05 threshold for this journal. Recently, p-values just below the 0.05 threshold found by JPSP, do “not replicate” and that “clearly, more data is needed.” Prompted by Masicampo and Lalande’ study, p-values associated with t-tests and ANOVA from the Journal of Vision and found no peak in the p-value distribution just below the 0.05 threshold. Our automated searches allowed us to retrieve 54,330 papers with p-values between 0.041 and 0.049, 14,708 papers with p-values between 0.051 and 0.059, 319,141 papers with “significant difference,” 418,580 papers with “no significant difference,” 523,175 papers with p < 0.05, and 69,758 papers with p > 0.05. Hence, sample size is an important advantage of automated searches.For manual searches a sample size of 3,000 to 5,000 articles seems to be the maximum achieved so far  that questionable research practices have increased over the past 25 years. This interpretation should be regarded with some caution, since a change of the p-value distribution can occur for various reasons, including changes in the file drawer effect and changes in the number of studies investigating a true effect  over a narrative summary of research findings . In this line, a single falsification can be worth more than numerous confirmations of an established null hypothesis. So, perhaps some asymmetry between positives and negatives results in innovative research fields is expectable, or even desirable.In addition to questionable statistical practices, positive explanations for the observed trends are possible, particularly when one considers that not only significant but also non-significant results have increased. First, scientists may have become more knowledgeable, and therefore better able to formulate accurate predictions and design statistically powerful experiments that disprove a null hypothesis. Second, scientists may have become more likely to use, and report the results of, statistical significance testing. The observed longitudinal trends of the reporting of  instead . Third,  instead  may be c instead , and the10.7717/peerj.733/supp-1Figure S1Click here for additional data file.10.7717/peerj.733/supp-2Figure S2Click here for additional data file.10.7717/peerj.733/supp-3Figure S3Click here for additional data file.10.7717/peerj.733/supp-4Figure S4Click here for additional data file.10.7717/peerj.733/supp-5Figure S5Click here for additional data file.10.7717/peerj.733/supp-6Figure S6Click here for additional data file.10.7717/peerj.733/supp-7Supplemental InformationClick here for additional data file.


Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by synovial inflammation and joint disability. Curcumin is known to be effective in ameliorating joint inflammation in RA. To obtain new insights into the effect of curcumin on primary fibroblast-like synoviocytes , which are key effector cells in RA, we employed gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS)-based metabolomics. Metabolomic profiling of tumor necrosis factor (TNF)-α-stimulated and curcumin-treated FLS was performed using GC/TOF-MS in conjunction with univariate and multivariate statistical analyses. A total of 119 metabolites were identified. Metabolomic analysis revealed that metabolite profiles were clearly distinct between TNF-α-stimulated vs. the control group (not stimulated by TNF-α or curcumin). Treatment of FLS with curcumin showed that the metabolic perturbation by TNF-α could be reversed to that of the control group to a considerable extent. Curcumin-treated FLS had higher restoration of amino acid and fatty acid metabolism, as indicated by the prominent metabolic restoration of intermediates of amino acid and fatty acid metabolism, compared with that observed in TNF-α-stimulated FLS. In particular, the abundance of glycine, citrulline, arachidonic acid, and saturated fatty acids in TNF-α-stimulated FLS was restored to the control level after treatment with curcumin, suggesting that the effect of curcumin on preventing joint inflammation may be elucidated with the levels of these metabolites. Our results suggest that GC/TOF-MS-based metabolomic investigation using FLS has the potential for discovering the mechanism of action of curcumin and new targets for therapeutic drugs in RA. Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by synovial inflammation and hyperplasia, and concomitant destruction of the cartilage and bone. Proinflammatory transcription factors such as NF-κB and proinflammatory cytokines such as tumor necrosis factor (TNF)-α, are closely associated with the pathological process of RA .diferuloylmethane), a polyphenol derived from the rhizomes of the plant Curcuma longa (turmeric), has been used as a traditional medicine to treat many inflammatory disorders [Curcumin  and animal models in RA –12. AlthIn this study, we applied metabolomics to investigate the beneficial effect of curcumin on FLS in RA. Metabolomics is a tool for comprehensively analyzing all small-molecule metabolites generated in a given biological system, and has been widely used in several biomedical areas, such as cellular responses to drugs or nutrients and new drug development . For exaThe important pathologic feature of RA FLS is the characteristic ability to express inflammatory cytokines, chemokines, adhesion molecules, and matrix-degrading enzymes. FLS also increase in number and become a prominent component of the destructive pannus in RA ,19. AlthCurcumin was purchased from Sigma-Aldrich . Curcumin 10mg was dissolved in dimethyl sulfoxide (DMSO) 1mL, and then further diluted in phosphate-buffered saline (PBS).Because immortalized mammalian FLS cell lines in RA are not yet available for scientific research, primary FLS cultures were employed. Synovial tissues were obtained from 3 RA patients undergoing arthroscopic wrist synovectomy or total knee joint replacement. The clinical characteristics of the patients are depicted in Table A in g (relative centrifuge force) for 10 min. The pellet was washed twice with Dulbecco’s Modified Eagle’s Media  containing 10% fetal bovine serum (FBS). The resuspended cells were plated at a concentration of 2 x 106/mL in a total volume of 1 mL/200 mm2 into T-25 culture flasks. After overnight incubation, the non-adherent cells were removed by replacing the medium with fresh culture medium. The attached cells were cultured in DMEM with 10% FBS and 50 units/mL penicillin, 50 mg/mL streptomycin, and 0.025 mg/mL amphotericin B, until 90% confluent growth was achieved. Primary cultured cells were passaged three to five times over several weeks for subsequent experiments. Between the third and fourth passages, cultures were a homogeneous population of fibroblastic cells.Patients with RA were diagnosed according to the standard criteria reported by the American College of Rheumatology . SynoviaTo investigate the metabolic perturbation and anti-inflammatory effects of curcumin on RA FLS, FLS were divided into three groups as follows: 1) RA FLS pretreated with curcumin (40 μM) for 1hr before stimulation with TNF-α (100 ng/ml) for 24 hour; 2) RA FLS only stimulated with TNF-α (100 ng/ml) for 24 hours; 3) control RA FLS not treated with curcumin or TNF-α at all.g for 5 min at 4°C to remove particulate matter, and was stored at -20°C in fresh tubes. Using commercially available enzyme immunoassay kits , the culture supernatants were used to determine the quantities of IL-6, IL-8, MMP-1, and MMP-3.To study the effects of curcumin on IL-6, IL-8, and matrix metalloproteinase (MMP) production, RA FLS (N = 3) were incubated with DMSO-containing vehicle or curcumin (40 μM) for 1 h, followed by stimulation with TNF-α (100 ng/mL) for 24 h. Then, the culture supernatant was collected and centrifuged at 10,000 g for 5 min at 4°C, the supernatant was collected and completely vacuum-dried using a vacuum concentrator . The dried samples were derivatized with 5 μL of methoxyamine hydrochloride in pyridine  at 30°C for 90 min and 45 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide  at 37°C for 30 min. A mixture of fatty acid methyl esters (C08-C30) as retention index markers was added to the derivatized extract.To obtain the metabolome sample from RA FLS (N = 3), we used fast filtration methods with a slight modification of the methods used in previous studies . Brieflym/z at an acquisition rate of 17 spectra/s. The temperatures of ion source and transfer line were 250°C and 280°C, respectively, and the impact of electron ionization was at 70 eV.We used an Agilent 7890B GC  equipped with Pegasus HT TOF-MS  for sample analysis. A 1 μL sample was injected into an RTX-5Sil MS column  and an integrated guard column  on splitless mode for separation of metabolome in samples. The oven temperature was set for 5 min at 50°C, and was then increased to 330°C at the rate of 20°C/min, where it was then held for 5 min. The mass spectra were collected at the mass range of 85 to 500 http://www.metaboanalyst.ca) [P < 0.05 were considered significant.The data obtained from GC/TOF-MS was preprocessed by using Chroma TOF software . After that, we used BinBase, an in-house database for further processing of the preprocessed data . The prolyst.ca) . The meam/z values with retention time were detected. After deconvolution and alignment using ChromaTOF software and BinBase, a total of 119 metabolites were identified  of 0.73 and predictive capability (Q2) of 0.81, indicating the excellence of the PCA model. From the PCA score plots, the metabolite profiles were clearly separated between the three groups   . The priA total of 84 metabolites, including fatty acid metabolism derivatives , phosphate, adenosine-5-monophosphate, and malonate positively contributed to PC 1 . HoweverA total of 119 identified metabolites were clustered and visualized by the HCA using the Euclidean distance and average linkage method to determine possible variations in the metabolite profiling of the three groups . The clut-test at the 5% significance level was conducted. The levels of 41 metabolites, including xanthine, palmitic acid, stearic acid, pyruvate, and myristic acid were lower in the TNF-α-stimulated group than those in the control group. Twenty four metabolites, such as oxoproline, tyrosine, threonine, serine, phenylalanine, tryptophan, and valine were higher in the TNF-α-stimulated group than in the control group. Subsequently, the metabolic pattern of the curcumin-treated RA FLS before TNF-α stimulation became similar to that in the control group. Among 65 metabolites altered by TNF-α, the levels of 54 metabolites reached levels similar to those in the control group after curcumin treatment  for 24 h resulted in a 2.5-, 2-, 2.3-, and 1.8-fold induction of IL-6, IL-8, MMP-1, and MMP-3 production, respectively, compared to that in the control group, when measured in culture media using ELISA . TreatmeOur metabolomic analysis using RA FLS reaffirmed that curcumin has a protective effect against the inflammatory response of TNF-α. Exposure to TNF-α caused prominent changes in the intracellular metabolites of RA FLS that was reversed by curcumin treatment. In addition, TNF-α stimulation and curcumin treatment led to perturb metabolite profiling of amino acids and fatty acids. It is suggested that curcumin could weaken obvious metabolic effects of TNF-α exposure in RA FLS and prominent metabolic target of TNF-α and curcumin is amino acid and fatty acid metabolism.Recently, metabolomic analysis has been used to monitor the activity of tumor cells, the effects of anticancer drugs in tumor cells, and to detect specific genetic alterations in tumor cells –29. RA Fhttp://en.wikipedia.org/wiki/Anti%E2%80%93citrullinated_protein_antibody-cite_note-pmid17558653-3 The high levels of citrulline in TNF-α-stimulated FLS reduced to the level of the control group after curcumin treatment. Collectively, this suggested that the effect of curcumin on preventing joint inflammation may be associated with the restoration of amino acid metabolite levels, such as citrulline or glycine. In addition to the known mechanism of action of curcumin, our metabolomic study revealed the new mechanism of action of curcumin for reducing synovial inflammation.The pathway analysis conducted using MetaboAnalyst based on enrichment analysis and pathway topology analysis to reveal the change of metabolisms by TNF-α and curcumin. These results showed that metabolite levels, which are involved in amino acid biosynthesis, purine metabolism, glycolysis, and fatty acid metabolism, were affected by TNF-α in RA FLS , but retAccumulated data have revealed that TNF-α perturbs the normal regulation of lipid metabolism . AccordiIn conclusion, metabolomic data and multivariate statistical analysis clearly revealed strong TNF-α- and curcumin-induced effects on the central metabolism of RA FLS. A prominent metabolic target of TNF-α and curcumin is amino acid and fatty acid metabolism, as revealed by perturbation of metabolite profiles. This is the first report of GC/TOF-MS-based metabolomic application to evaluate the effect of curcumin on FLS which are key effector cells in RA. These results also suggest that metabolomic investigation of FLS has the potential for discovering new biologic targets for therapeutic drugs in RA.S1 File(DOCX)Click here for additional data file.


Diffuse large B-cell lymphoma (DLBCL) is one of the most common types of aggressive B-cell non-Hodgkin lymphoma. About one-third of patients are either refractory to the treatment or experience relapse afterwards, pointing to the necessity of developing other effective therapies for DLBCL. Human B-lymphocytes are susceptible to JC polyomavirus (JCPyV) infection, and JCPyV virus-like particles (VLPs) can effectively deliver exogenous genes to susceptible cells for expression, suggesting the feasibility of using JCPyV VLPs as gene therapy vectors for DLBCL.in vitro and in vivo.The JCPyV VLPs packaged with a GFP reporter gene were used to infect human DLBCL cells for gene delivery assay. Furthermore, we packaged JCPyV VLPs with a suicide gene encoding thymidine kinase (TK) to inhibit the growth of DLBCL in vitro. As measured by the MTT assay, treatment with tk-VLPs in combination with gancyclovir (GCV) reduced the viability of DLBCL cells by 60%. In the xenograft mouse model, injection of tk-VLPs through the tail vein in combination with GCV administration resulted in a potent 80% inhibition of DLBCL tumor nodule growth.Here, we show that JCPyV VLPs effectively entered human germinal center B-cell-like (GCB-like) DLBCL and activated B-cell-like (ABC-like) DLBCL and expressed the packaged reporter gene Our results demonstrate the effectiveness of JCPyV VLPs as gene therapy vectors for human DLBCL and provide a potential new strategy for the treatment of DLBCL. Diffuse large B-cell lymphoma (DLBCL) is one of the most common types of aggressive B-cell non-Hodgkin lymphoma (B-NHL) and accounts for about 31% of adult B-NHLs in western countries . OverallBCL-2 gene translocation is found in almost all follicular lymphomas and in some diffuse large cell lymphomas [C-MYC oncogene leads to its overexpression [BCL-2 and C-MYC are among the current gene therapy approaches for lymphomas [In its basic concept, gene therapy is transferring a piece of genetic material into a target cell for the purpose of curing or slowing the progression of disease . Much prymphomas ,11. In Bpression . Accordiymphomas ,14. Otheymphomas ,16, thatymphomas , and thaymphomas . AlthougE. coli [E. coli–based in vivo packaging system was used. Therefore, it should be possible to use JCPyV VLPs to deliver genes of interest to tissue types that are the natural hosts of this virus for gene therapy purposes.Virus-like particles (VLPs) are made of viral proteins and structurally resemble viral capsids, but do not contain viral genetic materials; yet VLPs have similar infectious pathways as virions ,20. The E. coli , yeast [E. coli , or inseE. coli  and has E. coli . It has E. coli  and achiE. coli  when an Recent research has revealed the presence of JCPyV DNA and protein in DLBCL tissues of the gastrointestinal tract , indicatHuman GCB-like DLBCL, Toledo (CRL-2631) and HT (CRL-2260), cell lines were purchased from the Bioresource Collection and Research Centre . ABC-like DLBCL, SU-DHL-2 (CRL-2956), was purchased from ATCC . Cells were maintained in RPMI 1640 medium containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1 mM sodium pyruvate and supplemented with 10% fetal bovine serum (hereinafter referred to as complete culture medium).et al. [E. coli that expressed JCPyV VP1, and the VLPs were purified from the E. coli lysates by 20% sucrose cushion and CsCl velocity gradient centrifugations. The fractions collected were dialyzed against Tris-buffered saline  and analyzed for VLP content by the hemagglutination method, and the VLPs were concentrated by using Centricon filters . VLPs packaged with pEGFP-N3 or pUMVC1-tk were named gfp-VLPs or tk-VLPs, respectively.JCPyV VLPs in which plasmids expressing green fluorescent protein  and thymidine kinase  were packaged were prepared as described by Chen et al. . In shor2 for 72 h. Green fluorescent protein expression was detected with a confocal microscope .DLBCL cells were washed twice with phosphate-buffered saline (PBS), suspended in 50 μl PBS, and incubated with 10 μg of gfp-VLPs for 1 h at 4°C. Afterward, the cells were washed twice with cool PBS and cultured in complete culture medium at 37°C and 5% CO4 cells per well in complete culture medium were pseudoinfected with tk-VLPs at 1 μg per well. After gancyclovir (GCV)  was added to a final concentration of 10 μg/ml to a subset of the wells, leaving other wells without GCV as a control, the cells were incubated at 37°C with 5% CO2. At 2, 3, 4, and 5 days after pseudoinfection, the cells were collected by centrifugation, washed twice with PBS, suspended in complete culture medium containing 100 μl of 5 mg/ml 3--2,5-diphenyltetrazolium bromide , and then cultured at 37°C with 5% CO2 for 1 h. Afterward, the cells were centrifuged to facilitate the removal of the medium and additives, 100 μl of dimethyl sulfoxide was added to the wells and allowed to incubate for 30 min, and the absorbance of each well was then measured in a spectroscope  using a 595 nm filter.The growth-inhibiting effect of tk-VLPs on Toledo cells was assessed with the MTT assay as follows. Toledo cells cultured in 96-well flat-bottom microtiter plates  at 1 × 10ad libitum and kept on a 12 h light/dark cycle. All animal procedures were performed according to approved protocols and in compliance with the recommendations for proper care and use of laboratory animals of the Institutional Animal Care and Use Committee of National Chung Cheng University.Four-week-old male SCID mice were purchased from the BioLASCO Taiwan Co., Ltd.  and housed and maintained in controlled, specific pathogen–free airflow cabinets. The mice were given water and standard chow Ten million Toledo cells were subcutaneously inoculated into each SCID mouse. After 1 month, the Toledo tumor–bearing mice were intravenously injected with gfp-VLPs at 105 μg per injection every three days, six times in total. The VLP inoculation dosage was determined by titration at the beginning of the study. After the entire course of VLP treatment was completed (18 days after the first VLP injection), the mice were anesthetized and their tumor nodules were removed and embedded in optimum cutting temperature compound . Frozen section was performed to give slices of 6 um thickness. Green fluorescent protein expression was detected with a ZEISS AXioskop2 upright fluorescence microscope .7 cells per mouse. One month later, the Toledo tumor–bearing mice were randomized into four groups of four mice each and subjected to different treatment combinations: tk-VLPs with or without GCV; control (non-transgene-carrying) VLPs with or without GCV. tk-VLPs or control VLPs were administered intravenously at 105 μg per injection every three days, three times in total. GCV was administered by intraperitoneal injection (300 mg kg−1) every three days after the first tk-VLP or control VLP injection. The mice were euthanized when the tumors reached a volume of approximately 10,000 mm3, and the tumors were removed and weighed.To induce the formation of human DLBCL tumor nodules in mice, Toledo cells were harvested from subconfluent cultures, washed once with a serum-free medium, resuspended in PBS, and injected subcutaneously into the right flank of SCID mice at 1 × 10t-test and by one- or two-way ANOVA, with a P-value <0.05 being considered to represent a significant difference.Data were expressed as mean ± standard deviation. Data analysis was performed using Student’s In order to evaluate the suitability of JCPyV VLPs as gene transfer vectors for DLBCL therapy, we first needed to determine if the VLPs can enter DLBCL cells just as native JCPyV virions can. Thus, we tested JCPyV VLPs carrying the green fluorescent protein gene, or gfp-VLPs, for gene transduction activity using Toledo and HT cells (GCB-like) and SU-DHL-2 (ABC-like) as cell models. DLBCL cells in culture were treated with gfp-VLPs, and reporter gene expression was assessed 72 h later by examination under a fluorescence microscope. As shown in Figure in vitro. As shown in Figure The DLBCL treatment strategy currently in clinical use (R-CHOP) promises greater therapeutic success by combining several chemotherapeutic agents with a monoclonal antibody, but the efficacy of this therapy has been limited by chemoresistance arising from the genetic heterogeneity of the tumor cell populations. For this reason, we chose to exploit the higher proliferative activity of all tumor cells relative to normal cells by employing the suicide gene therapy approach with the JCPyV VLP vectors. Our chosen therapeutic gene, HSV-1 thymidine kinase, is a nucleoside kinase that, by adding a phosphate group to the prodrug GCV and consequently disrupting chain elongation during DNA replication, serves as a suicide gene for actively replicating tumor cells, with relatively little effect on normal cells. We purified JCPyV VLPs packaged with the thymidine kinase gene, or tk-VLPs, and assayed them in combination with GCV for cytotoxicity in human DLBCL cells in vitro, we further assessed whether the tk-VLP treatment is similarly effective in vivo and thus potentially therapeutically useful. As before, we used SCID mice as an animal model by subcutaneously implanting Toledo human DLBCL cells into the mice, and began the testing of tk-VLPs one month later when tumor nodules had formed. tk-VLPs were injected into the mice intravenously to allow the VLPs to reach the DLBCL tumors via blood circulation and transduce the target cells with the thymidine kinase gene; GCV was injected into the mice the next day to activate the cytotoxic activity of the suicide gene, completing the first cycle of treatment. This treatment cycle was repeated every three days for a total of three cycles, after which the subcutaneous nodules were removed and weighed to measure the therapeutic effect of the tk-VLP/GCV combination. As revealed by our results, the size of the tumor nodules at the end of the treatment course averaged more than 4 g for all control treatment groups, but was on average less than 1 g for the tk-VLP/GCV combination group (Figure in vitro and in vivo, indicating the potential of our novel system to become a useful treatment strategy for DLBCL.After confirming the growth-inhibiting effect of our suicide gene approach on human DLBCL cells in vitro and in vivo that JCPyV VLPs can enter human DLBCL cells and express the exogenous genes they carry with high efficiency, as evidenced by the expression of the green fluorescent reporter, and can deliver the thymidine kinase suicide gene into DLBCL cells to induce cell death in the presence of GCV. In our animal model, tk-VLPs injected through the tail vein were able to reach xenografted DLBCL tumor nodules via blood circulation and induce cytotoxicity in the nodules in the presence of GCV, resulting in a drastic 80% reduction in tumor growth. These results strongly support the promise of tk-VLPs as a gene therapy strategy for DLBCL.In this study, we demonstrate both DLBCL is the most common, aggressive subtype of B-NHL  and is cE. coli purine nucleoside phosphorylase with its prodrug substrate 6-methylpurine-2′-deoxynucleoside [Suicide gene therapy generally consists of two steps: an exogenous enzyme-expressing gene is first transferred to tumor cells via a vector; an inactive prodrug is then administered that is converted by the exogenous enzyme into a lethal drug, which kills the tumor cells . A numbecleoside ; nitrorecleoside ; and lincleoside . Among tcleoside , which icleoside . GCV tricleoside . The antcleoside , glioma cleoside , bladdercleoside , and colcleoside . These pcleoside -72 and sE. coli expression system on a large scale at low cost [in vivo DNA packaging method developed for them [in vitro osmotic shock [in vivo. We found that tk-VLPs introduced into mice by tail vein injection were indeed able to specifically target subcutaneous human DLBCL nodules and cause them to shrink (Figure Recent research on JCPyV VLPs has shown them to be promising gene therapy vectors . JCPyV Vlow cost , and thefor them , which ric shock , not onlic shock . All thek Figure , but mouin vitro and in vivo. Although many aspects remain to be improved before practical application, tk-VLPs have the potential to become a therapeutic choice for human B-cell lymphomas in the future.In summary, we have demonstrated here that the JCPyV VLP can be used as a gene delivery vector for gene therapy for human DLBCL both


This cross-sectional study included 270 subjects  and RA patients (n = 173). RA patients were divided into four groups: the first group without IR and not using antitumor necrosis factor-α (TNF−) ; the second group without IR and using anti-TNF-α ; the third group with IR and not using anti-TNF-α ; and the fourth group with IR and using anti-TNF-α . G3 and G4 had higher (p < 0.05) advanced oxidation protein products (AOPPs) and oxidative stress index (OSI) compared to G1. G4 group presented higher (p < 0.05) AOPPs and OSI than G2. TRAP was significantly lower in G3 compared to G1. Plasma TNF-α levels were significantly higher in G4 and G2 compared to G1 (p < 0.0001) and G3 . The presence of insulin resistance was robustly associated with both oxidative stress and TNF-α levels. More studies are warranted to verify if IR can be involved in therapeutic failure with TNF-α inhibitors. This trial is registered with Brazilian Clinical Trials Registry Register number RBR-2jvj92.The aim of this study was to evaluate the involvement of TNF- In addition, RA patients have higher risk of developing cardiovascular disease (CVD) and this is related to chronic inflammation  and cortis of RA , 7 and his of RA , 8. In apatients . Abnormapatients .α) can induce higher oxidative stress by initiators of the nuclear factor kappa B activation cascade and is under its transcriptional control, constituting a positive feedback loop [α therapy can reduce oxidative stress in patients with RA [Oxidative stress has a prominent role in the etiology and pathogenesis of joint tissue injury and chronic inflammation in patients with RA, which may lead to connective tissue degradation and joint and periarticular deformities . Reactivack loop . Moreove with RA , 16.Our group has investigated the development of IR and the metabolic syndrome in chronic inflammatory diseases –20 and tα on the inflammatory process, oxidative stress, and disease activity in patients with RA.Therefore, the aim of the present study was to verify the influence of insulin resistance and TNF-n = 97) and RA patients (n = 173), aged between 18 and 70 years. The control group was selected from among blood donors of the University Hospital who did not present autoimmune disease, and RA patients were selected from among the Ambulatory of Rheumatology of the University Hospital of Londrina, Paraná, Brazil. RA patients were initially divided into two groups: the first group without IR  and the second group with IR . After that, to verify the influence of insulin resistance and also of anti-TNF-α therapy on anthropometric, biochemical, immunological, and oxidative stress parameters in patients with RA, they were divided into four groups: the first group (control group) without IR and not using anti-TNF-α therapy ; the second group without IR and using anti-TNF-α therapy ; the third group with IR and not using anti-TNF-α therapy ; and the fourth group with IR and using anti-TNF-α therapy . RA patients (G2 and G4) were using anti-TNF-α therapy at least for six months. Sex, age, and ethnicity were controlled. RA was classified according to the 2010 rheumatoid arthritis classification criteria [This cross-sectional study included 270 subjects, healthy individuals  did not smoke and did not drink alcohol regularly. None of the participants in the study presented heart, thyroid, renal, hepatic, gastrointestinal, or oncological diseases, and none were receiving estrogen replacement therapy or drugs for hyperlipidemia or hyperglycemia or antioxidant supplements. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and the Ethical Committee of the University of Londrina, Paraná, Brazil, approved all procedures involving human subjects and patients. Written informed consent was obtained from all subjects/patients.Body weight was measured in the morning to the nearest 0.1 kg by using an electronic scale with individuals wearing light clothing and without shoes; height was measured to the nearest 0.1 cm by using a stadiometer. Body mass index was calculated as weight (kg) divided by height (m) squared. Waist circumference (WC) was measured on standing subjects midway between the lowest rib and the iliac crest.μU/mL) × glucose fasting (nmol/L)/22.5. IR was considered when HOMA-IR ≥ 2.114 [α levels were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) using a commercial immunoassay ELISA . Erythrocyte sedimentation rate (ESR) was obtained by automated kinetic-photometric method .After fasting for 12 hours, serum or plasma samples were obtained and the patients underwent the following laboratory blood analysis: glucose and uric acid (UA) were evaluated by a biochemical autoanalyzer , using Dade Behring kits; plasma insulin level and anticyclic citrullinated peptide (anti-CCP) antibody were determined by chemiluminescence microparticle immunoassay . The homeostasis model assessment-IR (HOMA-IR) was used as a surrogate measurement of insulin resistance . Conside ≥ 2.114 . Serum hSamples for evaluating oxidative stress and total antioxidant capacity were performed with EDTA as anticoagulant and antioxidant. All samples were centrifuged at 3.000 rpm for 15 minutes and plasma aliquots stored at −70°C until assayed.μL), 30 mM KH2PO4/K2HPO4 buffer (pH 7.4), and 120 mM KCl with 3 mM of tert-butyl hydroperoxide in a final volume of 2 mL. Tert-butyl hydroperoxide-initiated chemiluminescence was measured in Beckman LS 6000 Liquid Scintillation Counter set to the out-of-coincidence mode, with a response range from 300 to 620 nm. The vials were kept in the dark up to the moment of assay, and determination was carried out in a dark room at 30°C. The results are expressed in counts per minute (cpm).The CL-LOOH in plasma was evaluated as described previously by Gonzalez Flecha et al. . For cheμmol/L) of chloramines-T equivalents.AOPPs were determined in the plasma using the semiautomated method described by Witko-Sarsat et al. . AOPPs rμM Trolox/mg UA. TRAP analysis in conditions associated with hyperuricemia, as in patients with MetS, may be jeopardized because uric acid concentration is responsible for 60% of plasma total antioxidant capacity. Thus, a correction of total antioxidant capacity based on uric acid concentration is needed [TRAP was determined as reported by Repetto et al. . This mes needed , 33.μmol/L) divided by TRAP (μM Trolox/mg UA), which indicates the oxidant-antioxidant ratio as a reflection of the cellular redox state.Oxidative stress imbalance was verified when OSI was calculated as AOPPs .Distribution of sex, ethnicity, and therapy was analyzed by chi-square test with Yates correction. Comparisons between groups were performed using the Kruskal-Wallis test with Dunn's posttest and data were expressed as the median (25–75%). The results were considered significant when α therapy (p = 0.043) with enhanced frequency in patients with high disease activity. In addition, IR+ group showed higher ESR (p = 0.023) and hsCRP (p = 0.040) compared to the IR− group , WC , plasma glucose , and insulin  levels and HOMA-IR  compared to the control group and IR− group, respectively  compared to the control group, whereas IR− group showed lower AOPPs (p < 0.05) levels compared to the control group. Higher AOPPs (p < 0.0001) and OSI (p < 0.001) and lower TRAP (p < 0.05) were verified in the group composed of IR+ patients in relation to IR− group (α levels were significantly higher both in IR− (p < 0.01) and in IR+ (p < 0.0001) groups compared to the control group (α levels than IR− group (p < 0.05)  .α therapy. The groups composed of patients with IR, IR+ TNF− (G3) and IR+ TNF+ (G4), had higher (p < 0.05) AOPPs and OSI compared to G1 (control group). In addition, G4 group presented higher (p < 0.05) AOPPs and OSI than IR− TNF+ (G2) group. TRAP was significantly lower in IR+ TNF− group (G3) in relation to G1. On the other hand, the groups without insulin resistance, G1 and G2, showed no differences in oxidative stress markers (p < 0.05) levels in G3 and G4 than in G1, and G4 had also increased ESR (p < 0.05) levels compared to G2. There were significantly lower (p < 0.05) hsCRP levels in G4 compared to G3  and G2 (p < 0.0001), compared to G1   .α therapy with etanercept or adalimumab are shown in p = 0.071) in patients using adalimumab and this trend was independent of BMI  (data not shown). In sum, presence of IR was related to increase in DAS 28 and ESR and hsCRP and TNF-α levels and AOPPs and OSI and decreased TRAP in patients with RA. On the other hand, IR did not have a role in changes related to RF and anti-CCP. In addition, TNF-α increase is related to IR development in patients with RA.Oxidative stress data according to anti-TNF-Several reports have shown that IR is related to chronic inflammation , 32, 33 α may have an important role in the IR pathogenesis by multiple mechanisms, such as downregulation of genes that are required for normal insulin action, direct effects on insulin signaling, induction of elevated free fatty acids via stimulation of lipolysis, and negative regulation of peroxisome proliferator-activated receptor-γ (PPARγ), an important insulin-sensitizing nuclear receptor [α therapy, high-grade inflammation was correlated negatively and independently with circulating adiponectin concentration [ In vitro studies have shown that TNF-α induced serine phosphorylation of insulin receptor substrate-1 (IRS-1) and inhibited insulin receptor tyrosine kinase, causing a change of the insulin signaling [α therapy, which is generally indicated to patients who have a severe disease not controlled by disease-modifying antirheumatic drugs (DMARDs), showed higher TNF-α levels. Even with anti-TNF-α therapy, TNF-α levels have not reached the values obtained by patients who control disease activity with conventional therapy and DAS 28 maintained higher score (≥3.3) than the recommended one for patients using or not using biological agents [α therapy, used etanercept, a soluble TNF-α receptor fusion protein. Etanercept prolongs the half-life of TNF-α with a subsequent rise in measured serum TNF-α levels; thus it renders TNF-α biologically inactive and unavailable to bind to its receptor [α increase, suggesting that chronic inflammatory process may be associated with IR development and maintenance in patients with RA. Regarding the present data, it is not possible to assure that etanercept changed TNF-α in a biologically inactive substance. However, it is conceivable to suggest that other proinflammatory cytokines, which were not evaluated in this study, may be involved in the inflammatory process verified in patients with IR.Several reports have shown the association between chronic inflammatory disease states and IR , 33, 38.receptor . In RA pntration , an impontration . In vitrignaling . In the l agents . Of notereceptor –44. In tα levels and unfavorable oxidative status. Reactive oxygen species (ROS) damage directly cellular elements in cartilage and either directly or indirectly the components of the extracellular matrix by upregulating mediators of matrix degradation. ROS impair chondrocyte response to growth factors and migration to sites of cartilage injury. In addition, ROS inhibit the synthesis of matrix components including proteoglycans by chondrocytes [α is thought to be the main contributor to increased ROS release in RA patients [α stimulation [α exerts its cytotoxic effects via generation of intracellular ROS that induce apoptosis [α can induce ROS production from neutrophils through pathway activating phagocytic NADPH oxidases in mitochondria [α combined with cytokines such as GM-CSF or G-CSF enhances O2− generation [α, interleukin 6 (IL-6), and interleukin 17 (IL-17), which can cause significant decline in insulin sensitivity [α levels because they function as a second messenger to stimulate nuclear factor kappa B dependent expression of proinflammatory cytokines [α level can be involved in IR development and maintenance and have a direct influence on oxidative stress. It seems that a cyclic and complex relationship occurs between TNF-α, oxidative stress, and IR in patients with RA.The present study demonstrated that RA patients with IR have higher TNF-drocytes . In the patients , 45, 46,patients , 48. Larpatients , and thimulation . TNF-α epoptosis , 52. Morchondria  and TNF-neration . Of notesitivity . On the ytokines . Altogetα therapy remain a matter of controversy. Kageyama et al. [α therapy was reported. Meanwhile, Biniecka et al. [α therapy. DAS 28 < 2.6 was found in seven patients who were considered as anti-TNF-α responders and DAS 28 ≥ 2.6 in 11 patients who were considered as anti-TNF-α nonresponders. There was a decrease in 4-HNE levels only in anti-TNF-α responders patients. The aforementioned study seems to suggest that anti-TNF-α therapy can decrease oxidative stress in RA patients by controlling the inflammatory process, and hence they do not act directly on the production of ROS. In the present study, most patients who used anti-TNF-α therapy were taking etanercept. Nevertheless, differently from Kageyama's et al. study [α therapy maintained DAS 28 in similar values obtained by patients who were not using anti-TNF-α therapy. Meanwhile, inflammatory process shown by increased ESR and TNF-α levels, mainly in RA patients with IR, progressed in these patients being responsible for oxidative stress increase.The administration of biological drugs seems to have a role in increasing the barrier which the body possesses against oxidative stress . Howevera et al.  showed aa et al.  did not a et al.  evaluatel. study , the patUpon looking at the results obtained in the present study, some limitations have to be considered. First, the cross-sectional design does not allow for inference causality. Second, although the minimum number of patients has been reached by the calculation of the sample size, a greater number of patients would probably confer more strength to the statistical results.This study corroborates with Binieckas et al.'s , which sα are important factors involved in redox imbalance in patients with RA and it seems to be due to the maintenance of inflammatory state and disease activity. The data from the present study suggest a complex interaction of TNF-α, oxidative stress, and IR, but the presence of insulin resistance seems to be directly associated with both oxidative stress and TNF-α levels. The differences in oxidative stress markers in RA patients with or without IR could contribute to a better design for future drugs and/or nutritional interventional studies in this population. In addition, more studies are warranted to verify if IR can be involved in therapeutic failure with TNF-α inhibitors.This study demonstrates that IR and TNF-


Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The cream containing Nano curcu was found to be effective against human bacterial pathogens and hence can be used for treatment of bacterial diseases.Curcumin is one of the polyphenols, which has been known for its medicinal use since long time. Curcumin shows poor solubility and low absorption, and therefore, its use as nanoparticles is beneficial due to their greater solubility and absorption. The main aim of the present study was the formation of curcumin nanoparticles (Nano curcu), evaluation of their antibacterial activity against human pathogenic bacteria and formulation of Nano curcu-based cream. We synthesized Nano curcu by sonication method. The synthesis of Nano curcu was assessed for their solubility in water and by UV–visible spectrophotometry. Further, the nanoparticles were characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, nanoparticle tracking and analysis, and zeta potential analysis. In vitro antibacterial activity of Nano curcu was evaluated against  Curcuma longa L. is a perennial herb, which belongs to family Zingiberaceae, and commonly known as turmeric. It occurs in tropical and sub-tropical regions throughout the world. It is commonly cultivated in Asian countries, mostly in India and China and is extensively used in ayurveda, unani, and siddha systems of medicine as one of the household therapies to alleviate different diseases -1,6-heptadiene-3,5-dione] powder was procured from Hi-Media Laboratories Ltd., Mumbai.Stock of curcumin solution (5 mg/ml) was prepared by dissolving curcumin powder in Dichloromethane (20 ml). One ml of stock solution was added to boiling water (50 ml) in drop-wise manner under ultrasonication condition with an ultrasonic power and frequency of 50 kHz. The solution was sonicated for about 30 min. After sonication, the mixture was stirred at 800 rpm for about 20 min till the orange colored precipitate was obtained. Thereafter, supernatant was discarded and the pellet obtained was used for further study.The preliminary detection of synthesized Nano curcu was carried out by UV–visible spectrophotometer , scanning the absorbance spectra in the range of 200–800 nm wavelength.k = 635 nm) was passed. Particles present within the path of the laser beam were observed via a dedicated non-microscope optical instrument LM 20 having CCD camera. The motion of the particles in the field of view (approx. 100 × 100 μm) was recorded (at 30 fps), and from the subsequent video and images, size of particles was calculated.To determine average size and size distribution of Nano curcu, NTA analysis was performed by LM 20 . Liquid sample of Nano curcu was introduced into a scattering cell through which a laser beam  mixing dried powder of nanoparticles with KBr. Spectra were taken in the range of 500–2000 cmZeta potential measurements of synthesized nanoparticles were performed with Zetasizer Nano ZS 90  by using zeta dip cells. Zeta potential of synthesized nanoparticles was analyzed to determine the charges present on the surface of nanoparticles and its stability at pH 7. The samples for analysis were prepared by mixing of Nano curcu colloid in dichloromethane in 1:10 proportion. For measuring zeta potential, 1000 µl of the sample was taken in clear disposable zeta cells.Transmission electron microscopic analysis (TEM) was useful to determine the size and topology of synthesized Nano curcu. A drop of solution containing Nano curcu was placed on the carbon-coated copper grids and kept in infrared light until sample gets dried. After drying, powder of nanoparticles was loaded on specimen holder. TEM micrographs were taken by analyzing the prepared grids on Philips CM 200 super twin’s TEM operating at 200 kV (0.23 nm resolution) instrument.X-ray diffraction is the method to determine crystalline structure or phase of crystal. Dried Nano curcu powder was used for analysis purpose. The diffraction patterns were recorded by PAN analytical X PRT PRO, D-8, Advanced Brucker instrument (The Netherlands).Escherichia coli (ATCC 14948) and S. aureus (ATCC 333591) were procured from American Type Culture Collection Center, USA, and P. aeruginosa (MTCC 4676) from Microbial Type Culture Collection, Chandigarh. The antibacterial activity of the Nano curcu and silver nanoparticles was assessed against test bacteria by using Kierby–Bauer disc diffusion method  was added and it was heated slightly to dissolve completely. It was followed by addition of methyl paraben (0.3 g) and dissolved by heating. Then, 30 g of glycerol was added followed by tween 80 (3.6 g). All the above components were mixed together (except drug) which results in the formation of water phase.−1) were added to the mixture by continuous stirring on magnetic stirrer to dissolve the drug properly.For oil phase, liquid paraffin (5 g) was taken in a separate beaker and it was heated. Then, white paraffin (10 g) was taken in another beaker and heated later, and liquid paraffin was mixed with it which results in the formation of oil phase. After the formation of both the phases, they were mixed together drop by drop with continuous stirring. After mixing of both the phases, Nano curcu  and synthesized Nano curcu showed absorbance peak at 419 nm which is the characteristic feature of Nano curcu , S. aureus (ATCC 333591), and P. aeruginosa (MTCC 4676)  against S. aureus. Silver nanoparticles were most effective against P. aeruginosa and less effective against S. aureus. Commercially available antibiotic chloramphenicol was found to be more effective against S. aureus (40 mm) and less effective against E. coli (29 mm). P. aeruginosa was susceptible to antibiotic gentamycin, whereas S. aureus was less susceptible to gentamycin  and less effective against S. aureus (20 mm) (Fig. The antimicrobial activity of Nano curcu was tested against a MTCC 466 (Fig. 7ATCC 1494, S. aure−1 can be assigned for C=C stretching, 1452 cm−1 corresponds to C=H, and the absorption at 1146 cm−1 due to C–H stretching. The absorption peak at 1037 cm−1 might be due to C–N stretch. The absorption spectra of control might be attributed to the functional group such as benzene ring, C–O–C bond, and aromatic C–H stretching. These finding are supported by many researchers (Yadav et al. To confirm the synthesis of Nano curcu, solubility testing was performed in which Nano curcu were soluble in water, while curcumin powder was not soluble in water Fig.  inset. IThe phenolic group in Nano curcu interacts with outer lipopolysaccharide layer present in the Gram-negative bacteria. Moreover, less peptidoglycan content helps in the weakening and breakage of bacterial cell wall, resulting in the death of the bacterial cell. In addition, antibacterial cream of Nano curcu possesses both the water phase and oil phase. Both the phases are stabilized by surfactant. The oil phase of the cream helps in the better interaction of Nano curcu with the outer lipopolysaccharide layer present in the Gram-negative bacteria, thereby causing the disruption of the less rigid cell wall of the Gram-negative bacteria Fig. .Fig. 10PE. coli, S. aureus, and P. aeruginosa. It can be concluded from the results that Nano curcu synthesized by sonication method inhibited activity of bacteria. The formulated cream is new generation of antiseptic cream, which could be used in the treatment of infection caused by E. coli, S. aureus, and P. aeruginosa. The Nano curcu could also be used for wound healing.Nano curcu were fabricated by physico-chemical method. It is simple, natural, and easy method used for the synthesis of Nano curcu. Synthesized nanoparticles showed its efficacy against bacteria, viz.,


Spodoptera frugiperda 9 and Trichoplusia ni cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be circumvented. Thus, it is now possible to generate tRV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain.Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P Human rotaviruses are the main cause of severe infant gastroenteritis. Rotavirus disease is associated with approximately 453 000 annual childhood deaths of which most occur in developing countries Amongst others, RV-VLPs are some of the promising candidates that are currently considered as a potential viable alternative option Rotavirus represents a genus in the Reoviridae virus family. It has a dsRNA genome consisting of 11 segments that encodes six structural  and six nonstructural (NSP1–NSP6) viral proteins. The three capsid protein layers that enclose the genome are composed of 60 dimers of the 102 kDa VP2, inner capsid in vitro environment which may cause them to have phenotypic differences to wild-type strains. Therefore, preparing RV-VLPs directly from field strains using the consensus expression system-based codon-optimised sequences derived from their dsRNA may represent a novel strategy of producing non-live RV-VLP vaccines from any human or animal field strains without the prior intermediate step of cell culture adaptation. RV-VLPs produced in this manner may not only shorten the time required to prepare RV-VLPs from wild-type strains, but also may permit production of RV-VLPs with antigenic characteristics that closely mimic the current circulating wild-type strains. In the current study, the possibility of generating chimaeric RV-VLPs using a common DS-1-like VP2/6 dRV-VLP backbone was investigated. Outer capsid proteins of selected prevalent rotavirus field strains in Africa and those that emerged in the past two decades of which their dsRNA was isolated directly from human stool samples were used.Single-layered VP2 (sRV-VLPs), double-layered VP2/6 (dRV-VLPs), and triple-layered VP2/6/7 or VP2/6/7/4 (tRV-VLPs) RV-VLPs have previously been produced successfully in baculovirus vector expression systems (BVES) by using either mono-cistronic Species A human rotavirus strains (RVA) of African origin characterised directly from stool samples  were useAutographa californica multi-capsid nucleopolyhedrosis virus (AcMNPV) promoters, into pFastBac .The Bac-to-Bac Baculovirus Expression System  was used for the production of chimaeric RV-VLPs according to the manufacturer's specifications with minor deviations. pFastBACquad pFBq,  was usedORFs coding for VP2, VP4, VP6 and VP7 derived from the consensus nucleotide sequences of the selected rotavirus strains  were codE. coli strain AcBACΔCC E.coli cells to produce the recombinant bacmids. High molecular weight bacmid DNA was isolated from five colonies per construct using the BACMAX DNA purification kit  by following the manufacturer's instructions. Selection of recombinant bacmids were carried out by PCR as described in the BEVS manual using TaKaRa Ex-Taq DNA polymerase .The Spodoptera frugiperda (Sf) 9 cells, maintained in complete TC-100 medium  containing 10% foetal bovine serum (FBS), 1% antibiotic  and 1% fungizone , were transfected with each of the five recombinant bacmid DNAs isolated for each pFBq construct. FuGENE 6 and X-tremeGENE transfection reagents  were used per manufacturer's instructions. The 6-well plates were incubated at 28°C until the cytopathic effect (CPE) was complete. The transfection supernatant was harvested from each well, centrifuged at 500×g for 2 min, and the supernatant  was stored at 4°C for infection experiments. Plaque purifications were performed and 10 separate plaques per recombinant baculovirus were selected.Expression of recombinant rotavirus proteins by the plaque purified baculoviruses was verified by harvesting cell pellets from each virus stock followed by performing SDS-PAGE and western blot analysis. The cell pellet was lysed for 1 h with 200 µl of cell lysis buffer , 0.1 mM ethylene-diamine-tetra-acetic acid , 1% sodium deoxycholate , 0.1% sodium dodecyl sulphate  and complete protease inhibitor cocktail tablets by following manufacturer's instructions . 14 µl of the cell lysate was analysed on SDS-PAGE gels which were stained with Coomassie blue RV-VLPs were produced in Sf9 or High Five (Invitrogen) cells in shaker cultures. Sf9 cells were maintained in Ex-Cell Titre High Medium  containing 10% FBS , 1% Penicillin/Streptomycin/Amphotericin B , 0.1% Pluronic F-68 solution  and 0.5–1 µg/ml Leupeptin or Aprotinin protease inhibitors . High Five cells were maintained in Express 5 serum-free medium  containing 0.6 mg/L L-glutamine , 10 µg/ml gentamycin  and 0.5–1 µg/ml Leupeptin or Aprotinin protease inhibitors . The infected cultures were incubated at 28°C and shaking at 96 rpm until CPE reached approximately 95% (between 5–6 days).6 cells/ml in 100 ml shaker cultures were infected with viral stocks confirmed to simultaneously express VP2 and VP6 proteins. Different strategies were used to produce chimaeric RV-VLPs consisting of VP2/6/4, VP2/6/7 and VP2/6/4/7 protein layers, detailed in During preliminary investigations in our laboratory, MOI of 0.1, 1, 2, 5 and 10 pfu/cell were compared for expression of recombinant rotavirus proteins and dRV-VLP formation. A MOI of 2 pfu/cell was shown to give the best yield of dRV-VLP formation . Therefore, RV-VLPs were produced by infecting either Sf9 or High Five insect cells with one or more baculoviruses containing the ORF (s) encoding one or more rotavirus structural proteins at a MOI of 2 pfu/cell. To produce dRV-VLPs (VP2/6) from the GR10924 G9Pg for 15 min at 4°C. The cell pellet was resuspended in 10 ml lysis buffer and centrifuged at 3,000×g for 15 min at 4°C to remove cell debris. The supernatant recovered from culture and cells was layered on a 40% sucrose cushion prepared in Tris-calcium buffer  followed by ultracentrifugation in a Sorvall WX Ultra Series centrifuge  using a Surespin 630 rotor (36 ml) at 135,172×g for 2 h at 4°C. The supernatant was discarded and the pellet was resuspended in 400 µl Tris-calcium buffer of which 100 µl was applied to the top of 10%–60% sucrose gradients prepared in Tris-calcium buffer g in a Sorvall TH-660 rotor  at 4°C. The bands containing RV-VLPs were visualised with the Gradient Station ip fractionator  which was also used to collect 18 fractions of 220 µl each from sucrose gradients by following the manufacturer's instructions. The protein concentration of the RV-VLPs was determined using a Bicinchoninic Acid (BCA) Protein Assay Reagent  by following the manufacturer's instructions.The cells and medium were harvested 5–6 days post-infection (dpi) followed by centrifugation at 3,000×g for 1 h at 4°C. The pellet was suspended in 100 µl Tris-calcium buffer. The structural integrity of the RV-VLPs was verified by negative staining the samples on a 0.5% Formvar-coated copper grid using 1% uranyl acetate stain  followed by examination using two transmission electron microscopes (TEM) .To verify the production of RV-VLPs, the presence of the recombinant rotavirus structural proteins was determined by screening the gradient fractions using SDS-PAGE or western blot analysis as described in section 2.3. The fractions confirmed to contain RV-VLPs were pooled and washed using Tris-calcium buffer followed by ultracentrifugation at 106,401×7 to 1×109 pfu/ml were determined using agarose-plaque assays. Recombinant VP2, VP4 and VP6 of approximately 100 kDa, 87 kDa and 45 kDa, respectively, were visualised on SDS-PAGE gels. Expression of recombinant VP7, of approximately 36 kDa, could only be confirmed with western blot  and 1.99 mg protein/L (approximately 2.42×1016 particles/L) was recovered from 1×106 of High Five and Sf9 cells, respectively. For tRV-VLPs (VP2/6/7/4), 1×106 High Five and Sf9 cells respectively yielded 14.08 mg protein/L (approximately 9.23×1016 particles/L) and 5.75 mg protein/L (approximately 3.77×1016 particles/L), Fig. S3 and 15 to 1.13×1016 particles/L and 9.23×1015 to 2.77×1016 particles/L were assumed to be complete particles in Sf9 and High Five cells, respectively.Using SDS-PAGE, proteins VP2, VP4, VP6 and VP7 could be detected in fractions 9–15 of the sucrose gradients in most cases , which cOn electron micrographs, dRV-VLPs were visible from gradient purified RV-VLPs produced through infection of insect cells with baculoviruses expressing VP2 and VP6, In the current study, RV-VLPs were derived from the consensus insect cell codon-optimised sequence of rotavirus strains characterised directly from stool samples without prior adaptation to cell culture. dsRNA of rotavirus strains  as intact 87 kDa VP4 and also an approximately 36 kDA VP7 band, similar to what Yao et al. In conclusion, the current study proves the concept of generating chimaeric tRV-VLPs from rotavirus dsRNA extracted directly from clinical specimens. Assembly of VP7 outer capsids of various genotypes  on a universal DS-1-like dRV-VLP (C2 and I2 genotypes) demonstrates the potential of using the BVES to tailor-made genotype specific RV-VLP vaccines for populations in targeted regions. The outcomes of the current study should support initiatives of developing alternative rotavirus sub-unit vaccines, especially in Africa where the burden of rotavirus diarrhoea is high. The novel approach of producing RV-VLPs using the consensus insect cell codon-optimised nucleotide sequence derived from whole-genome amplified cDNA Figure S1Map of the pFastBACquad (pFBq) baculovirus transfer plasmid used for cloning and co-expression of rotavirus proteins in insect cells. The elements of pFBq were derived from the pFastBac  and pBACgus4x-1  transfer plasmids. The two polyhedron (polh) and two p10 promoters are located in the multiple cloning site (flanked by Tn7R and Tn7L transposition elements) that allow restriction enzyme-mediated cloning of foreign genes of interest.(TIF)Click here for additional data file.Figure S2Evaluation of expression of VP7 by recombinant baculoviruses as indicated by SDS-PAGE (A) and duplicate western blot analysis (B). (I) Recombinant VP7 expressed by five plaque-purified baculoviruses that contain VP7 encoding ORFs. (II) Recombinant VP4 and VP7 expressed by five plaque-purified baculoviruses that contain VP4 and VP7 encoding ORFs. (III) Recombinant VP7 (lane 2), VP4 (lane 3), VP4 and VP7 (lane 4), VP2 and VP6 (lane 5) expressed by amplified recombinant baculoviruses containing rotavirus ORFs encoding these proteins, respectively. Lanes 1. Ladder, PageRuler Plus Prestain Protein Ladder . Wild-type, empty baculovirus used as a control.(TIF)Click here for additional data file.Table S1Protein yield of RV-VLPs obtained from SF9 and High Five cells, and the approximated theoretical number of particles.(DOC)Click here for additional data file.


We performed a meta-analysis to evaluate the effect of anti–tumor necrosis factor (TNF) therapy on the frequency of extra–articular manifestations (EAMs) in patients with ankylosing spondylitis (AS).We searched with the terms ‘ankylosing spondylitis’, ‘infliximab’, ‘etanercept’, ‘adalimumab’, ‘golimumab’, ‘certolizumab’, ‘TNF inhibitor/blocker/antagonists’ or ‘anti-TNF’ on MEDLINE, EMBASE and Cochrane Library for randomized controlled trials (RCTs) of ≥12 weeks with parallel or crossover design of TNF inhibitor versus placebo to treat uveitis, inflammatory bowel disease (IBD) and/or psoriasis of AS, published before February 2014.P = 0.01). Subgroup analysis showed receptor fusion proteins were more efficacious for uveitis than placebo , but monoclonal antibodies were not . Anti–TNF therapy and placebo group did not significantly differ in treating IBD in AS patients . In subgroup analysis, neither monoclonal antibodies  nor receptor fusion proteins  significantly differed from placebo in treating IBD. We found no suitable reports on psoriasis.We found 8 RCTs that fit our criteria. Anti–TNF therapy was associated with less uveitis than placebo in patients with AS (OR: 0.35, 95% CI: 0.15–0.81, Anti–TNF therapy was preventive for flares or new onset of uveitis in AS patients, and might be an alternative for these patients. However, monoclonal anti–TNF antibodies and TNF receptor fusion proteins were not efficacious for IBD in AS patients. Ankylosing spondylitis (AS) is a chronic, progressive, inflammatory rheumatic disease that primarily affects the sacroiliac joints . AS is pUveitis, which is characterized by pain with red eye and photophobia, increased tear production and blurring of vision , occurs IBD is characterized by a chronic inflammation of the gut mucosa and includes Crohn’s disease (CD) and ulcerative colitis (UC). A recent systematic review and meta-analysis showed the pooled prevalence of IBD in patients with AS to be 6.8% . TraditiPsoriasis is a systemic inflammatory cutaneous disease with plaque lesions and nail deformities. The pooled prevalence of psoriasis, a secondary disorder in AS, was 9.3% in patients with AS .In a 2010 update by the Assessments in Ankylosing Spondylitis International Society and the European League against Rheumatism (ASAS/EULAR) of recommendations for the management of AS , NSAIDs Infliximab (INF) is a chimeric mouse–human monoclonal immunoglobulin G (IgG) 1 antibody . AdalimuAdditionally, trials of anti–TNF therapy in AS have yielded impressive results -25 and aWe captured all relevant studies published before February 2014 on MEDLINE, EMBASE and the Cochrane Library using following search terms: ‘ankylosing spondylitis’, ‘infliximab’, ‘etanercept’, ‘adalimumab’, ‘golimumab’, ‘certolizumab’, ‘TNF inhibitor/blocker/antagonists’ or ‘anti-TNF’.Studies included in this meta-analysis met the following criteria: they were RCTs; their duration of study was ≥12 weeks; they used a parallel or crossover design of TNF inhibitor versus placebo treatment; and their data included information on uveitis, IBD and psoriasis in patients with AS.Two independent investigators determined the relevance of the cited articles by reading the abstracts. Disagreements were resolved through discussion and consensus or, as needed, with consensus with a third investigator. When there were multiple articles from the same trial, the most complete and recently reported data were included.Evaluations of methodological quality and risk of bias were performed independently by two reviewers, and disagreements between the two were resolved by consensus. Methodological quality of included articles was further assessed using modified Jadad criteria with an 8-item scale designed to assess randomization, blinding, withdrawals and dropouts, inclusion and exclusion criteria, adverse effects and statistical analysis . Score rP <0.05 was considered to be statistically significant; 95% confidence intervals (CI) were reported. Homogeneity was tested using the Q statistic (significance level at P < 0.10) and the I2 statistic (significance level at I2 > 50%) [I2 statistic was significant. Otherwise, a fixed-effects model was used. The existence of a publication bias for the meta-analysis was examined using a funnel plot. To assess the potential confounding effect of heterogeneity, subgroup analyses were performed. According to TNF-inhibitor classification, we divided those into two subgroups of TNF receptor fusion proteins (ETA) and monoclonal anti-TNF antibodies .The statistical analysis was performed using Review Manager 5.2  from the Cochrane Collaboration, 2013. Categorical dichotomous variables were assessed using the odds ratio (OR). 2 > 50%) . A randoA total of 1,395 relevant articles were retrieved from various databases of which 801 were excluded after scanning the titles; 521 after carefully reading the abstracts; and an additional 65 articles for various reasons . Six trials -36,38 reP = 0.01, Figure P = 0.04); whereas monoclonal anti-TNF antibodies did not significant differ from placebo . Analysis for IBD in these patients found that the anti-TNF therapy and placebo did not significantly differ  or receptor fusion proteins  significantly differed from placebo. Funnel plot analysis showed symmetry, which indicates that publication bias was not a significant factor in these studies (Figures Anti-TNF therapy was associated with less uveitis than placebo in patients with AS (OR: 0.35, 95% CI: 0.15–0.81, Analysis of risk of bias showed that only 3 trials reported their methods of sequence generation and allocation concealment in detail ,35,36. BThis meta-analysis compared anti-TNF therapy with placebo in patients with AS. The results indicate significant positive benefits of anti-TNF agents to treat uveitis in these patients. For IBD treatment outcomes, the anti-TNF therapy group and the placebo group did not significantly differ. However, Subgroup analysis showed the receptor fusion protein ETA was more efficacious than placebo for uveitis in this patient population, whereas monoclonal anti-TNF antibodies were not. Neither monoclonal antibodies nor receptor fusion proteins significantly differed from placebo in treating IBD.Anti-TNF therapy has been shown to be beneficial for the treatment of uveitis in patients with AS. A retrospective study  of patieet al. [Braun et al.  investiget al. ; in factet al.  or new-oet al.  in AS paAlthough anti-TNF agents are effective in treating skin and nail lesions of psoriasis ,49, treaThe present study evaluated the efficacy of anti-TNF therapy on the frequency of EAMs in patients with AS. Anti-TNF therapy including ETA could be a credible alternative for AS patients who have uveitis. However, no anti-TNF therapy was efficacious for treating IBD in patients with AS. The 8 included studies that met the inclusion criteria had high-moderate Jadad scores; therefore the conclusions of this systematic analysis are reliable. More high-quality, large prospective RCTs with long-term follow-up are needed to confirm the efficacy and outcomes of anti-TNF therapy for EAMs of AS.Compared with placebo, anti-TNF therapy including ETA was associated with significantly fewer flares and new onset of uveitis, but were not significant efficacious for treating IBD in AS patients. This meta-analysis of patient-level data from 8 RCTs significantly advances the notion that anti-TNF therapy may be a credible alternative for AS patients with uveitis. Future studies involving anti-TNF therapy for EAMs of AS are needed.


Bombyx mori in 1987, discoveries of ILPs in other insects mushroomed and propelled intense studies of the functional roles of ILPs in numerous insect species. The primary sites of ILP production are brain neuroendocrine cells, but ILPs are also produced in other tissues. Synthesis of ILPs is regulated by many factors including the insulin/insulin-like growth factor signaling pathway (IIS) itself. ILPs can function in an autocrine, paracrine, and endocrine fashion and target a diversity of cells and tissues.Since the first isolation of insulin-like proteins (ILPs) from the silkmoth, IIS is activated when ILPs bind to the insulin receptor (InR). IIS interacts with various major signaling pathways such as the Target of Rapamycin TOR (an important nutrient sensor) and FOXO . In insects, it also interacts with the signaling pathways of the developmentally significant hormones juvenile hormones (JHs) and ecdysteroids. The numerous interactions of IIS with other major signaling pathways results in a plethora of physiological actions in insects affecting a multitude of life events. The consensus is that the role of IIS in insects bears close similarities to its functional counterparts in the insulin/insulin-like growth factors of vertebrates.The present Research topic is a compendium of 14 articles on the roles of IIS in a wide spectrum of functions. Of these, seven are reviews focused on different aspects of actions of IIS, five are original research and one is an editorial highlighting the contents of the present Topic. The IIS functions presented here include nutrition, growth and development, behavior, lifespan, semi-lethality, stress, dietary restriction, reproduction, axon guidance, and circadian and seasonal timekeeping. This Research Topic illustrates the complexity of the role of IIS in the detection of environmental cues, their translation into physiological adjustments of insects and the regulation of IIS by other physiological factors involving multiple interactions with other signaling pathways. It also raises stimulating questions for future directions of research in the role of IIS in insect physiology.A historical account of the discovery of the first ILPs, the bombyxins, in the silkmoth is presented by Mizoguchi and Okamoto . BombyxiDrosophila has been extensively studied. Nässel et al. (Drosophila brain (DILPs), their axonal projections and their regulation by factors including neurotransmitters, neuromodulators, hormones, and factors from the fat body. The functional role of DILPs in behavior, lifespan, resistance to stress, and starvation is also discussed. Individual DILPs perform different physiological roles during Drosophila development. A review by Kannan and Fridell  regulates axon guidance in photoreceptor cells in the developing nervous system by binding to specific adapter protein called Dreadlocks (Dock). However, mutations in DlnR of both putative Dock binding sites did not lead to defects in axon guidance. The authors suggest that Dock may be able to bind to multiple regions of DInR in vivo to ensure proper interaction with the receptor in order to achieve appropriate axon guidance.The role of IIS in l et al.  review t Fridell  pays para et al.  report ta et al.  show thaDrosophila. An insect also used as a model animal to study the integration of nutritional information and reproduction, is the mosquito. Hansen et al.  induces expression of the clock protein PERIOD in Rhodnius circadian clock cells, demonstrating a direct effect of these hormones on the molecular oscillator and indicating interaction of the insulin and PTTH signaling pathways in the regulation of clock genes. The article also shows that the clock cells that control rhythmicity in hormones are themselves responsive to feedback from these same hormones by the IIS and PTTH signaling pathways. Sim and Denlinger (C. elegans. IIS involvement in diapause is executed in association with the JH signaling pathway.IIS is also involved in the regulation of circadian and seasonal timekeeping at multiple levels of physiological organization. The relationship between metabolic state and the circadian clock in the brain is reviewed by Erion and Sehgal . In Drosnd Steel  report tenlinger  review tManduca. Unlike Bombyx, in which this process involves intersection of the IIS pathway with the PTTH signaling pathway, both bombyxin and Manduca ILP fail to stimulate steroid synthesis in Manduca. The authors conclude that steroid synthesis in Manduca is dependent on PTTH signaling and independent of IIS and does not require interaction of these two pathways.Smith et al.  investigDrosophila, but also in other insects, the authors summarize evidence that the IIS/TOR pathways regulate plasticity of organ growth and size during development. This plasticity depends on interactions between IIS/TOR pathway with those of ecdysteroid and JH, but that there are also differences in tissue sensitivities to the pathways.The extensive variability of tissue responses to IIS raises the question of how and why insect tissues respond so differently to insulin. This is addressed in the review by Koyama et al. . Based mIn conclusion, IIS forms complex networks with several other signaling pathways. This complexity of pathway interplay and crossover introduces numerous variables that are difficult to determine or control during an experiment. Nijhout and Callier  developeThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.