Tumor necrosis factor (TNF) is a pleiotropic cytokine involved in many aspects of immune regulation. Anti-TNF biological therapy has been considered a breakthrough in the treatment of chronic autoimmune diseases, such as rheumatoid arthritis (RA). In this review, because of the major involvement of T cells in RA pathogenesis, we discuss the effects of anti-TNF biotherapy on T-cell responses in RA patients. We also outline the potential fields for future research in the area of anti-TNF therapy in RA.This could be useful to better understand the therapeutic efficiency and the side effects that are encountered in RA patients. Better targeting of T cells in RA could help set more specific anti-TNF strategies and develop prediction tools for response. The discovery of the role of tumor necrosis factor (TNF) in the pathogenesis of rheumatoid arthritis (RA) has led to anti-TNF biological therapy as a breakthrough in the treatment of chronic autoimmune diseases, such as RA, Crohn’s disease, psoriatic arthritis, and spondyloarthritis . VariousTNF is a pleiotropic cytokine involved in many aspects of immune regulation . TNF is In this review, because of the major involvement of T cells in RA pathogenesis, we discuss the effects of anti-TNF biotherapy on T-cell responses in RA patients. This could be of help for the interpretation of the clinical effects (or lack thereof) of anti-TNF treatments, as well as being useful to better understand the side effects which are encountered in RA patients.Much has been learned from mouse models in the understanding of RA, especially regarding the role of T cells. Collagen-induced, K/BxN, IL-1 RA-KO, and SKG models were shown to depend on T lymphocytes . More spThere has been an ongoing debate over the respective importance of macrophages and T cells. The presence of T cells in joints and the expansion of clonotypic T cells, as a result or a cause of inflammation, in the synovium of RA patients has fueled that debate. The role of the HLA-DR shared epitope in the development of RA is a strong indication for the role of T cells . There iTNF has been shown to be essential in many stages of T-cell development. In the thymus, TNF promotes the apoptosis of triple-negative CD3/CD4/CD8  and doubSecondary lymphoid organs (SLO) are crucial for the development of efficient adaptive immune responses. Organized in well-demarcated T-cell zones and B-cell follicles, SLO bring the antigen that is trapped by various subsets of dendritic cells (DCs) in close contact with the immune cells, provide costimulatory signals from DCs, and thereby initiate an appropriate immune response.TNF-mediated signaling is crucial for the development of some and for structural maintenance of most of the SLO. Distinct cellular sources and molecular forms of TNF contribute to the organization of SLO microarchitecture. TNF from B and T cells cooperates to maintain the structural integrity in lymph nodes, which are indispensable for the generation of efficient local immune responses.The requirement of TNF signaling for organized lymphoid structures in mice was confirmed by studies in humans. Rheumatoid arthritis patients receiving ETA lack germinal center development in their tonsils . SimilarAltogether, TNF controls the development and organization of SLO structures and, thereby, influences the development of adaptive immune responses. This could be of importance during the follow-up of RA patients, especially children, treated with anti-TNF.Activation of naive T cells is initiated during their encounter with antigen peptide presented by mature DCs. This activation is dependent on coactivation mediated by the membrane interaction between members of the TNF/TNF-R family other than TNF cytokine on T cells and DCs. As a cytokine, TNF contributes to efficient antigen presentation by inducing DC maturation.Interaction of T cells with antigen presenting cells leads to differentiation into effector and memory T cells (reviewed in ). To und+ and CD8+ T cells during viral infection through apoptosis, thus subsequently limiting the T cell memory compartment [RM) with CD69 and CD103 expression [RM in turn contributes to the maturation of DCs and efficient Ag presentation for recall T-cell activation. Anti-TNF biologics are thus expected to modulate the effector and the memory T-cell response during infections and vaccination (vide infra).TNF is reported to negatively regulate the expansion of effector CD4partment . TNF, acpression . These TTo invade inflamed tissue, T lymphocytes must have the capacity to traffic through endothelial cell junctions. This phenomenon, called diapedesis, has been shown to depend on TNF and interferon (IFN)-γ . Thus, aHowever, TNF has a contrasting role in T-cell activation , 19. TheToxicity of IFX for T cells is minimal and the metabolism of T cells is not significantly altered by anti-TNF . HoweverAnother possible mechanism of action of anti-TNF on T cells is the regulation of cell death. The action of anti-TNF drugs on cell death was tested using the Jurkat T-cell line transfected with tmTNF . Due to +CD25+ cells was evaluated at the start of treatment with IFX and after 3 months of treatment [+CD25+ cells, which was increased in RA patients compared with healthy donors, was reduced after treatment with IFX [In patients with active RA, spontaneous apoptosis of CD4There is now a growing literature in RA patients on increased T helper (Th)1 –29 and THull et al. reported that patients responding to ADA or ETA had an increase in circulating Th17 . Convers+CD4+ T cells expressing IL-10 in RA patients [Furthermore, all the anti-TNF drugs IFX, ADA, CZP, and ETA induce IL-17patients . The ind+ T-cell phenotyping techniques, on the biologic administered, and the methodology used.In conclusion, Th17 and Th1 compartments are increased in response to TNF inhibitors but a definitive answer as to whether they are linked to good or poor responses is needed. This is likely to depend on Th CD4STAT6, which is associated with the Th2 response, was also induced in T cells from patients treated with ADA . This wo+ T cells, called T peripheral helper (Tph) cells, has been identified in the synovial membrane of RA patients using mass cytometry technology [+ T cells that express high levels of the checkpoint protein PD-1 and, contrary to T-follicular helper cells (Tfh), do not express CXCR5. Tph cells induce the differentiation of plasma cells through IL-21. The inhibition of Tph by anti-TNFs [The development of paradoxical psoriasis as a side effect of anti-TNF  treatment in RA patients has been observed. The mechanism has been shown to involve IFN-α produced by plasmacytoid dendritic cells whose maturation is inhibited by anti-TNF  and not chnology . Tph celnti-TNFs  may prevnti-TNFs .+ T cells [There are 2 types of CD4CD25 FoxP3-positive Tregs, inducible (iTregs) and natural (nTregs). Inducible Tregs depend on TNF-R2 as exemplified by the observation that TNF-R2 is critical for stabilization and homeostasis of Tregs . TNF has T cells . However T cells , T-effec T cells , and thi T cells . This ca T cells . Such a On the T lymphocyte side, the role of soluble versus tmTNF has been explored in several models. T lymphocyte-monocyte contact is important in inflammation. This involves, in part, tmTNF interaction with TNF-R2 on adjacent cells . BlockinIn T cells, IFX but not ETA induces IL-10 production through reverse signaling, showing disparity in the efficacy of biologics regarding molecular mechanisms  but suggReactivation of tuberculosis during anti-TNF therapy by monoclonal antibodies and, to a lesser extent, by ETA has been a major drawback of biotherapies of rheumatic diseases . Product+ terminally differentiated effector memory CD45RA+ T cells (TEMRA cells) with antimicrobial activity against mycobacterium tuberculosis and is responsible for impairing the T-cell defense against microbes [IFX triggers a reduction in CD8microbes .+ T-cell proliferation and IFN-γ production against tuberculosis PPD and CFP-10 antigens were shown to be impaired by a 14-week treatment with anti-TNF in patients with a positive test for prior tuberculosis infection [CD4nfection . The inh+-derived TNF is essential for antilisteria activity in mice. Patients treated with IFX are at higher risk for infections with listeria, another intracellular bacteria, than those treated with ETA [CD8with ETA .+ and CD8+ T lymphocytes through their cytotoxic activity and their release of cytokines such as TNF and IFN-γ [+ response in RA patients treated with IFX, ADA, or ETA is conserved [Viral infections are controlled at least in part by CD4nd IFN-γ . Anti-TNnd IFN-γ  and we honserved . HoweverHepatitis B infections are controlled by T lymphocytes. Depletion of the T-cell response by anti-TNF treatments may explain the resurgence of hepatitis B chronic infections which may occur more frequently with antibodies than with ETA . There iRecommendations with regard to hepatitis B and C infections in patients treated with anti-TNF have been proposed . Caution+ T cells activated by cytokines, among them TNF, can provide help to B cells for the production of IgM, IgG, and IgA [+ T cells expressing tmTNF provide a costimulatory signal for B cells [TNF is involved in T cell-dependent B-cell responses. Resting memory CD45RO and IgA . CD4+ T  B cells .Regarding response to vaccines, clinical studies performed with influenza and pneumococcal  vaccinat+ T cells. Such immunogenicity of anti-TNF antibodies in RA patients suggests that there is no profound decay of T cell-mediated B-cell immunity. Thus, from a functional point of view, only a partial decrease of the B-cell response is observed in RA patients treated with anti-TNF. Although cumbersome, a way of reducing the immunogenicity of anti-TNF antibodies would be to identify T cell epitopes and to modify them accordingly. From a clinical point of view, prescribing anti-TNF with methotrexate, an immunosuppressive drug that reduces the production of Th1 cytokines [A proportion of RA patients treated with anti-TNF biologics develop antidrug antibodies that can hamper the efficiency of treatments . Due to ytokines , decreasFigure Differences between antibodies (IFX and ADA), and monovalent CZP, and ETA were outlined in the present review. They are summarized in Table The mode of action, especially on T cells, of TNF-inhibitors is still not completely understood. For example, reverse signaling induced by TNF inhibitors must be explored in more detail. Future therapeutic strategy for RA should still take TNF inhibitors into account despite the availability of other biologics targeting other cytokines such as IL-6 and the more recent advent of JAKi. The choice of molecule should depend on better knowledge of the mode of action of the various TNF inhibitors. Nonspecific effects of anti-TNF antibodies on the immune system plead for a more targeted action such as bispecific antibodies targeting cells on the one hand and proinflammatory cytokine on the other .


Ankylosing spondylitis (AS) results from the combined effects of susceptibility genes and environmental factors. Polymorphisms in genes regulating inflammation may explain part of the heritability of AS.Using a candidate gene approach in this case-control study, 51 mainly functional single nucleotide polymorphisms (SNPs) in genes regulating inflammation were assessed in 709 patients with AS and 795 controls. Data on the patients with AS were obtained from the DANBIO registry where patients from all of Denmark are monitored in routine care during treatment with conventional and biologic disease modifying anti-rheumatic drugs (bDMARDs).The results were analyzed using logistic regression (adjusted for age and sex).p < 0.05). The polymorphisms were in genes regulating a: the TNF-α pathway (TNF -308 G > A (rs1800629), and − 238 G > A (rs361525); TNFRSF1A -609 G > T (rs4149570), and PTPN22 1858 G > A (rs2476601)), b: the IL23/IL17 pathway (IL23R G > A (rs11209026), and IL18–137 G > C (rs187238)), or c: the NFkB pathway (TLR1 743 T > C (rs4833095), TLR4 T > C (rs1554973), and LY96–1625 C > G (rs11465996)).Nine polymorphisms were associated with risk of AS (TLR1 743 T > C (rs4833095) (odds ratios (OR): 2.59, 95% confidence interval (CI): 1.48–4.51, p = 0.04), and TNFRSF1A -609 G > T (rs4149570)  were associated with increased risk of AS and the combined homozygous and heterozygous variant genotypes of TNF -308 G > A (rs1800629)  were associated with reduced risk of AS.After Bonferroni correction the homozygous variant genotype of TNF (rs1800629), TNFRSF1A (rs4149570), and IL23R (rs11209026). Furthermore, we identified novel risk loci in TNF (rs361525), IL18 (rs187238), TLR1 (rs4833095), TLR4 (rs1554973), and LY96 (rs11465996) that need validation in independent cohorts. The results suggest that genetically determined high activity of the TNF-α, IL23/IL17, and NFkB pathways increase risk of AS.We replicated associations between AS and the polymorphisms in  Ankylosing spondylitis (AS) is a type of spondyloarthritis in which hallmark clinical features are inflammation at entheses and subchondral bone of the pelvic and spinal joints with subsequent abnormal new bone formation at these sites. Ultimately, this leads to ossification of entheses and joints resulting in loss of joint mobility. The incidence varies between 0.1 and 1.8% with the highest incidence in Scandinavia. Onset is typically in young adults with a male predominance. Medications used include non-steroid anti-inflammatory drugs (NSAIDs), and biological disease-modifying anti-rheumatic drugs (bDMARDs), i.e. tumor necrosis factor-α inhibitors (anti-TNF) and more recently also an interleukin(IL)-17A inhibitor (secukinumab) [The cause of AS is unknown but is believed to involve a combination of genetic and environmental factors . The herhttp://www.bu.edu/nf-kb/gene-resources/target-genes/). The TNF-α and NFkB pathway are intertwined and TNF-α can feedback stimulate NFkB by binding to TNF receptors (TNFR1 or TNFR2), resulting in a kinase cascade similar to, but distinct from, the pathway induced by TLRs [TNF-α can be activated by Pathogen-Associated Molecular Patterns (PAMPs) such as bacterial or viral DNA, flagellin, or lipopolysaccharide (LPS), through the NFkB pathway. PAMPs can be recognized by Toll-like receptors (TLRs) thereby initiating a kinase cascade which phosphorylates and degrades the NFkB inhibitor IkBα . This re by TLRs .The IL23/IL17 pathway can also stimulate TNF-α activity. The pro-inflammatory cytokine IL-17 enhances the production of other pro-inflammatory cytokines including TNF-α, and the secretion IL-17 itself can be enhanced by IL-23 .PAMPs can also be recognized by intracellular Nod-like receptors (NLRs). In turn, NLRs can activate pro-inflammatory cytokines including IL-18 . IL-18 iThe aim of this study was to assess whether functional single nucleotide polymorphisms.(SNPs) in genes involved in the TNF-α, IL23/IL17, NFkB, and other pro- and anti-inflammatory pathways were associated with risk of AS.The DANBIO registry includes prospectively collected clinical data on patients with inflammatory joint diseases including smoking status, disease characteristics e.g. HLA-B27 status, disease activity, treatment, and treatment outcomes. Patients from all of Denmark are monitored in routine care during treatment with conventional and biologic disease modifying anti-rheumatic drugs (bDMARDs) .Mycobacterium tuberculosis) were collected from all patients screened for tuberculosis at Statens Serum Institut  from 01.09.2009 to 31.01.2013; the Department of Respiratory Diseases B and the Department of Clinical Microbiology, Aarhus University Hospital  from 01.01.2011 to 31.01.2014; the Department of Clinical Biochemistry, Herlev and Gentofte Hospital  from 01.03.2012 to 31.01.2014; the Department of Biochemistry, Hospital of Lillebaelt ; and the Department of Biochemistry, Hospital of Slagelse  from 01.01.2014 to 31.01.2014. Furthermore, from 01.01.2013 to 31.12.2013 blood samples were collected from all patients with AS treated with or without anti-TNF drugs at the Department of Rheumatology, Frederiksberg Hospital .Screening for tuberculosis before initiation of treatment with biological drugs is routinely performed in Denmark. Left over blood clots  from each blood sample with the clinical data from DANBIO, 709 patients with AS (ICD-10: M45.9) were identified. The control group consisted of 795 healthy blood donors recruited from Viborg, Denmark.Fifty-one SNPs in genes involved in the TNF-α, IL23/IL17, NFκB, and other pro- and anti-inflammatory pathways were assessed. A list of all SNPs studied and genotype distribution is presented in Table Bank et al. [http://www.lgcgenomics.com/).DNA extraction  was performed as described by k et al. . For thek et al. –17. Comphttp://zzz.bwh.harvard.edu/gpc/cc2.html). The lowest minor allele frequency (MAF) of the studied SNPs was 0.10. The ‘high-risk allele frequency’ was set to 0.10, the ‘prevalence’ was set to 0.0018 [The Genetic Power Calculator was utilized for power analysis of discrete traits .Among the patients with AS the median age was 32 years (SD: 11.5) and 68% (483/709) were males. The healthy controls had a median age of 43 years (SD: 11.5) and 52% (411/384) were males. Among the patients 37% (118/323), 23% (73/323), and 41% (132/323) and among the controls 26% (207/788), 24% (189/788), and 50% (392/788) were current smokers, former smokers and never smokers, respectively. HLA-B27 staus was available for 498 patients of which 83% (411/498) were positive. Sixty percent (427/709) of the patients were treated with anti-TNF.TLR1 (743 T > C (rs4833095)) (p = 0.03), TLR2 ) (p = 0.02), TLR4 (rs1554973 T > C) (p = 0.03), TLR9 (1174 G > A (rs352139)) (p = 0.02) and TGFB1 (− 509 C > T (rs1800469)) (p = 0.02). After correction for multiple testing, all SNPs studied were in Hardy-Weinberg equilibrium.The genotype distributions among the healthy controls deviated from Hardy-Weinberg equilibrium for TLR1 743 T > C (rs4833095)  and the combined homozygous and the heterozygous variant genotypes of TNFRSF1A -609 G > T (rs4149570)  were associated with increased risk of AS. The homozygous variant genotype of TLR4 T > C (rs1554973)  and LY96–1625 C > G (rs11465996) , and the combined homozygous and the heterozygous variant genotypes of TNF -308 G > A (rs1800629) , TNF -238 G > A (rs361525) , PTPN22 1858 G > A (rs2476601) , IL18–137 G > C (rs187238) , and IL23R G > A (rs11209026)  were associated with reduced risk of AS   and TNFRSF1A -609 G > T (rs4149570)  were associated with increased risk of AS and the combined homozygous and the heterozygous variant genotypes of TNF -308 G > A (rs1800629)  were associated with reduced risk of AS  was associated with reduced risk of AS  compared to the haplotype combination 11. In TNF all haplotype combinations were associated with reduced risk of AS compared to the haplotype combination 11 (rs361525GG and rs1800629GG).The TLR2 or IL1B.No associations were found for haplotype combinations of TNF (rs1800629 and rs361525), TNFRSF1A (rs4149570), and PTPN22 (rs2476601)), b: the IL23/IL17 (IL23R (rs11209026), and IL18 (rs187238)), or c: the NFkB (TLR1 (rs4833095), TLR4 (rs1554973), and LY96 (rs11465996)) pathways were associated with risk of AS.In this case-control study, polymorphisms in a: the TNF-α (TNF (rs1800629) [TNFRSF1A (rs4149570) [IL23R (rs11209026) [NLRP3 (rs4612666) was associated with increased risk of AS in Chinese patients [NLRP3 (rs4612666) with increased risk of AS (p = 0.06). However, our results are in contrast to a meta-analysis of the PTPN22 (rs2476601) polymorphism that did not find an association with AS [TNF (rs361525), IL18 (rs187238), TLR1 (rs4833095), TLR4 (rs1554973), and LY96 (rs11465996) that need validation in independent cohorts.The found assocaitions for 1800629) –22, TNFR4149570) , and IL21209026) –33 are i with AS . FinallyTNF (rs1800629 and rs361525) polymorphisms suggest that reduced TNF-α mRNA level and expression of TNF-α was associated with reduced risk of AS [TNF rs1800629 and rs361525 were associated with reduced risk of AS. Likewise, the associations observed for the TNFRSF1A (rs4149570) polymorphism indicates that increased expression of the TNF-α receptor 1 was associated with increased risk of AS [PTPN22 (rs2476601) polymorphism suggests that reduced TNF-α serum level was associated with reduced risk of AS [Most of the SNPs assessed in our study have known biological effects thus allowing a biological interpretation of the observed associations based on increased or reduced gene activity as summarized in Table sk of AS , 49. Thisk of AS . Furthersk of AS . Taken tIL23R (rs11209026) polymorphism suggests that reduced IL-17 serum level, and thus reduced TNF-α activity, was associated with reduced risk of AS [IL18 (rs187238) polymorphism indicates that reduced IL-18 expression, and thus reduced IL-17 and TNF-α activity, was associated with reduced risk of AS [IL23R (rs11209026) and the IL18 (rs187238) polymorphisms thus suggest that a genetically determined high activity of the IL23/IL17 pathway was associated with increased risk of AS. The two SNPs furthermore support that genetically determined high activity of the TNF-α pathway was associated with increased risk of AS. The observed associations between the polymorphisms in IL23R and IL18 and risk of AS are in line with previous studies pointing out the IL23/IL17 pathway as central to the pathophysiology of AS [IL-17 is known to induce the production of many cytokines including TNF-α . IL-18 isk of AS . Furthersk of AS , 54. Thegy of AS .TLR1 (rs4833095) polymorphism suggests that increased TLR1 level was associated with increased risk of AS [, the associations observed for the LY96 (rs11465996) polymorphism suggests that increased MD-2 (LY96) and TNF-α level was associated with a reduced risk of AS [TLR4 (rs1554973) polymorphism was associated with reduced risk of AS which was supported by the haplotype results (Table TLR4 (rs1554973) polymorphism is unknown, however, the result supports the notion that the NFkB pathway may be involved in the etiology of AS.This study also suggests that the NFkB pathway may be involved in the etiology of AS. The associations observed for the sk of AS . High lesk of AS . FinallyTLR1 rs833095 poTLR1, TLR4, LY96, TNF, TNFRSF1A, IL18, and IL23R found in this study, could potentially – in part – explain this altered cytokine milieu present in AS patients.Both TNF-α  and inteTNF (rs1800629) polymorphism [TNF polymorphisms, as well as the HLA-B27 locus, are located on chromosome 6, and there is a risk that even a minor linkage disequilibrium could have confounded our results [TLR1 (rs4833095), TLR2 (rs4696480), TLR4 (rs1554973), TLR9 (rs352139), and TGFB1 (rs1800469) were not in Hardy-Weinberg equilibrium among the healthy controls. Due to the number of polymorphisms analyzed this is probably a type II error. The polymorphisms do not deviate from Hardy-Weinberg equilibrium when corrected for multiple testing. We cannot exclude that some of our positive findings may be due to chance due to the obtained p-values and the number of statistical tests performed. When the results were corrected for multiple testing only the variant allele of TLR1 (rs4833095) and TNFRSF1A (rs4149570) were associated with increased risk of AS and the variant allele of TNF (rs1800629) was associated with reduced risk of AS.There are aspects of this study which should be interpreted with care. Conflicting results have been reported for the morphism , 49, 65.F rs180069 polymorA major strength of this study was that the cohort was rather large including 709 patients with AS and 795 healthy controls and the associations that we report were biologically plausible. Also, the validity of the diagnosis is expected to be high, since the patients were identified via a clinical database that the rheumatologist use for prospective monitoring of patients as part of routine care .TNF (rs1800629), TNFRSF1A (rs4149570), and IL23R (rs11209026). Furthermore, we identified novel risk loci in TNF (rs361525), IL18 (rs187238), TLR1 (rs4833095), TLR4 (rs1554973), and LY96 (rs11465996) that need validation in independent cohorts. The results suggest that genetically determined high activity of the TNF-α, IL23/IL17, and NFkB pathways increase the risk of AS.In conclusion, we replicated associations between AS and the polymorphism


Saprolegnia parasitica is a fish pathogen that causes severe economic losses worldwide. Virkon-S is a well-known disinfectant known to exhibit antimicrobial activities against bacteria, viruses, and fungi. In this study, we tested the anti-fungal activity of Virkon-S against S. parasitica, the major causal agent of saprolegniasis. (EPC) cells grown in L-15 medium and acute toxicity tests were carried out with cultured fingerlings of common carp for 96 h. Artificial infection with S. parasitica was performed by placing the fish in tanks containing zoospores of S. parasitica after descaling and wounding at three positions. The diseased fish were kept in tanks containing 2, 4, and 10 ppm of Virkon-S for 10 days to observe the treatment effect.The lowest concentration of Virkon-S that prevented germination or the visible growth of spores and the percent spore germination were determined using potato dextrose agar plates containing different concentrations of Virkon-S. The cytotoxic effect was evaluated using the Ez-Cytox Cell Viability Assay with epithelioma papulosum cypriniin vitro assay results showed that Virkon-S could inhibit spore germination and the resulting mycelial growth at a concentration as low as 4 ppm. No cytotoxic effect on EPC cells was observed even at a concentration as high as 100 ppm. Additionally, no acute toxicity in the common carp was observed at 10 ppm following 96 h exposure. Ten days of treatment with 4 and 10 ppm Virkon-S resulted in complete reversal of artificially-induced saprolegniasis in the common carp.The This data indicates that Virkon-S can be used for the control of saprolegniasis without harmful effects in fish. However, further research on the effect in humans and food supplies is necessary. Saprolegnia infections are visible to the naked eye as white patches on the skin of the infected fish or as ‘cotton wool’ on fish eggs. From these eggs the fungus can spread to live eggs via positive chemotaxis, meaning some chemical signals from the live eggs cause the fungus to move towards them . In Korea, Virkon-S has been approved as a quasi-drug for animals and was used as a disinfectant for aquaculture facilities in 2016.Virkon-S was originally developed by Antec International  and launched in 1986 for use in farming and livestock production. It is regarded as one of the most advanced farm disinfectants. It was one of the first oxidative disinfectants to be used on the farm and continues to lead the way in livestock production and farm biosecurity, having been successfully deployed against 500 disease-causing pathogens including viruses, bacteria, and fungi, which cause foot and mouth disease, avian influenza, lobacter . Due to Saprolegnia. In this study, we tested the antifungal activity of Virkon-S against S. parasitica in vitro and in vivo to determine the possibility of using this material for the control of saprolegniasis in the future.Despite the wide spectrum of antimicrobial activity of Virkon-S, there have been no reports of its antifungal activity against The animal protocol used in this study has been reviewed and approved by the Pukyong National-Institutional Animal Care and Use Committee (PKNU-IACUC), which outlines the ethical procedures and scientific care of animals used in studies .S. parasitica was purchased from the Korean Collection for Type Cultures (KCTC 46452) and cultured on potato dextrose agar (PDA) at 25 °C. This strain was isolated from a farmed rainbow trout from an aquaculture farm in Wonju, Gangwondo, Korea in 2016 before deposition.Cyprinus carpio, with an average size and weight of 11.5 ± 1 cm and 17.6 ± 3 g, respectively, were obtained from Namsangju Aquaculture Farm, located in Sangju-si, Gyeongsangbuk-do, Korea. Each fish was examined for infection and acclimated in 450-L rearing tanks at 22 ± 2 °C for 10 days. During the acclimatization period, fish were fed twice daily with a proper diet according to In total, 100 fingerlings of common carp, Virkon-S was purchased from Bayer Korea  and 1 kg contained triple salt 500 g, hexametaphosphate 181 g, sodium dodecyl benzene sulphonic acid 150 g, malic acid 100 g, sulphamic acid 50 g, and sodium chloride 15 g. A 10% stock was prepared with distilled water and further dilutions were made when necessary.S. parasitica was cultured on PDA at 25 °C for 12 days to induce spores. Approximately 10 ml of distilled water was added to each 87-mm diameter Petri dish and the mycelium and spores were scraped and filtered through eight layers of sterile cheesecloth. The final concentration of spores was adjusted to approximately 1 × 106 spores/ml using a hemocytometer. A 10 µl sample of spore suspension was spotted on the center of a 87-mm diameter Petri dish containing 20 ml of PDA with 0, 2, 4, 10, 20, or 100 ppm Virkon-S, and incubated at 25 °C. After 72 h of incubation, the diameter of mycelial growth was measured. Inhibition of spore germination was also determined by percent spore germination as described by The inhibitory effects of Virkon-S on spore germination were tested using two methods. First, the minimum fungicidal concentration (MFC), defined as the lowest concentration of a chemical that prevents germination or the visible growth of spores, was determined as described previously . Spore sS. parasitica mycelial growth was tested on PDA plates containing different concentrations of Virkon-S as described by aprolegnia-colonized PDA block of approximately 5 × 5 mm was placed on the center of the prepared plates. The plates were incubated at 25 °C for 72 h and the diameter of the mycelial growth was measured. The percentage of fungal inhibition was calculated based on the percent inhibition of radial growth (PIRG) as described by Inhibition of 5) were grown in L-15 medium supplemented with 10% fetal bovine serum, penicillin (62.5 µg/ml), and streptomycin (100 µg/ml) in 96-well plates overnight. For treatment, the cell medium was replaced with medium (100 µl) containing 10, 100, 500, 1,000, 5,000, and 10,000 ppm of Virkon-S. Non-treated cells were used as a negative control. After 24 h of incubation, 110 µl of medium containing 10 µl of water-soluble tetrazolium solution was added to each well, and the plates were incubated for a further 4 h. The absorbance at 460 nm was measured using an enzyme-linked immunosorbent assay reader , and relative cell viability was calculated using cells treated with medium only as a control.The cytotoxic effect of Virkon-S was evaluated using the Ez-Cytox Cell Viability Assay Kit  with EPC cells by following the procedures described by Cyprinus carpio, using five concentrations of Virkon-S  and three fish per concentration. Healthy and disease-free fish that were not previously exposed to any pollution agents or toxicants were selected based on their activity and external appearance. The fish were acclimated for 7 days at 18 °C under constant light and feeding with commercial feed. Three fish were placed in a 3-L glass flask containing different concentrations of Virkon-S and kept for 96 h in aerated water without any water changes or feeding. Dead fish were removed from the flask to prevent possible deterioration of the water quality. The percentage of fish mortality was calculated for each concentration after 24, 48, 72, and 96 h of exposure. After 96 h of exposure, all of the survivors were transferred to a 50-L tank equipped with aeration and observed for 40 days for any post-exposure effects.Water-only toxicity tests were carried out with cultured fingerlings of the common carp, S. parasitica and treatment with Virkon-S. Three groups were artificially infected and treated with 2, 4, and 10 ppm Virkon-S. Fish in the positive control group were infected with S. parasitica but not treated with Virkon-S. Fish in the negative control group were neither infected with S. parasitica nor treated with Virkon-S. Fish were kept in a 150-L glass fiber tank separated into three sections with a metal screen with six or seven fish in each section. The water temperature was fixed at 17 ± 1 °C using a room air conditioner. All fish were observed for behavioral and clinical signs of infection.Five groups of 20 fish were used for artificial infection with S. parasitica, fish were descaled and wounded in three places on one side of the body  using a sharp scalpel. The wounded area was approximately 1 cm2. Spores of S. parasitica were prepared as above and added to tanks containing the three treatment groups and the positive control group at a final concentration of 1 × 105 spores/ml  and enumerating spore germination on PDA plates of half thickness. There was no sign of mycelial growth on plates containing 4, 10, 20, and 100 ppm Virkon-S 72 h after incubation at 25 °C . In contrast, most of the spores on control plates germinated at a rate of 96.2% (51 out of 53 spores). Therefore, the inhibition rate of spore germination was calculated as 59% at a concentration of 2 ppm [(96.2–39.2)/96. 2 × 100].S. parasitica on PDA containing 2, 4, 10, 20, and 100 ppm Virkon-S was observed after inoculation at 25 ° C for 72 h. No growth of mycelia was observed in the plates containing 10, 20, and 100 ppm Virkon-S  inhibition was observed on a PDA plate containing 2 ppm Virkon-S. Therefore, the MFC was determined to be 4 ppm on PDA plates. When the inhibition of spore germination experiment was repeated on thin PDA plates for enumeration, complete spore germination inhibition still occurred at 4 ppm and 59% inhibition at 2 ppm was obtained. Although a concentration of Virkon-S as low as 4 ppm was proven to be effective to inhibit germination in our experiment, the concentration and contact time required for fungi inactivation seems to be specific to each fungus. It has been reported that 1 min of contact with 1% Virkon-S is sufficient to inactivate  species . On the A. fumigatus, and 18.67 mm (±1.15) for Penicillium chrysogenum. Furthermore, 5, 3, and 1% Virkon-S had no effect on the growth of either fungi. Therefore, it seems that the anti-fungal effect of Virkon-S against each target fungus needs to be tested for practical application of Virkon-S.Similarly, Pimephales promelas. No cytotoxic effect was observed at 10 and 100 ppm, although there was a slight decrease in cell viability (97%) at 500 ppm (To use Virkon-S for the control of saprolegniasis, it should be safe for fish or treated embryos. The cytotoxicity of Virkon-S was tested with EPC cells, which originated from the fathead minnow,  500 ppm . There hS. parasitica without any cytotoxic effects at a concentration that can inhibit the fungus. This was further confirmed by acute toxicity tests. No toxic effect was observed in fish kept at 2, 4, and 10 ppm for 96 h (Our results indicated that 4 ppm was sufficient to inhibit spore germination and mycelial growth. Therefore, it seems that Virkon-S could be used to control the growth of for 96 h . MoreoveS. parasitica. Fish that were wounded and inoculated with S. parasitica spores showed clear symptoms of saprolegniasis 10 days after inoculation, which was confirmed by microscopic observation and colony morphology on PDA plates. Fish treated with 2, 4, and 10 ppm Virkon-S showed 50, 100 and 100% disease recovery, respectively after 10 days of treatment. The concentrations required for the control of the disease coincided with the concentrations obtained from the in vitro assay. In addition to 100% survival of the treated fish, all recovered fish showed regeneration of scales on the wounded area, which is an indication of successful treatment.The efficacy of Virkon-S was tested with fish that were artificially infected with Virkon-S is approved as a quasi-drug that can be used as a disinfectant for the treatment of aquaculture facilities and equipment in Korea. The results of this study indicate that 4 ppm of Virkon-S can also be used for the control of saprolegniasis with no toxic effects on cultured fish cells or fish in tanks. Although further research on the effect in humans is necessary, Virkon-S is a good candidate for the control of saprolegniasis, which causes huge economic losses in the aquaculture industry.10.7717/peerj.5706/supp-1Supplemental Information 1Raw data for cell viability test of EPC cells at different concentration of Virkon-S.Click here for additional data file.


Osteoblastoma (OBL) and osteoid osteoma (OO) are usually solitary tumours, only rarely being multicentric. Herein, we report an unusual case in which a typical OBL of the spine was followed by an OO of the femur after a disease-free interval of 5 years. We believe that our unique case represents the first report of a metachronous OBL and an OO, and this presentation may confirm the close correlation between these two rare entities. OBLs and OOs are usually solitary lesions, and are only rarely multicentric.T1 with a mildly increased T2 signal .1 In a review of the English literature, there are only eight patients with multifocal OO: three had synchronous and five had metachronous lesions.Although both OBL and OO are most often monostotic, they may rarely occur in more than one bone, either synchronously or metachronously.In the case of our patient, the tumours were anatomically distant and were separated in time by a 5-year interval. It is possible that this occurrence represents nothing more than a statistical coincidence. However, although the aetiology of OOs and OBLs remains unknown, it is possible that a metachronous presentation of these rare tumours may be a manifestation representing a genetic correlation between them. We believe that our case report is important not only to confirm the possibility of a metachronous presentation of mesenchymal tumours, but also to underline a possible pathogenetic correlation between an OO and an OBL as part of the same family of tumours producing osteoid and woven bone.9 The loss or rearrangement of the long arm of chromosome 22 was found in both OBLs and OOs.7 showed the same alteration involving the long arm of chromosome 22 in two cases of OOs. They suggested that this chromosomal alteration may affect critical genes involved in the regulation of cell proliferation.7 The available data also indicate that a candidate target gene for OBL development may reside in the long arm of chromosome 22.9 The pathogenetic importance of these aberrations is not known but the possible deleted genes seem to be involved in osteogenesis and tumorigenesis.9The genetic profile of an OO and an OBL has not yet been established but there are a few reports showing genetic aberrations of both tumours.11We can speculate that the data provides two different possibilities: either the patient has two different tumours that are histologically identical and similar in their genetic profile or the OBL and OO have the same pathology, suggesting that the differential diagnosis based on size is weak. Furthermore, similar to other benign mesenchymal tumours, their metachronous presentation may represent a multifocal involvement of the same disease. In fact, the possible existence of benign metastasis is supported by the behaviour of giant cell tumours and epithelioid haemangioma.Our case report shows that a metachronous presentation of OBLs and OOs is an exceptional event, but must be considered in cases of patients with a history of previous OBL or OO who present with a deep and continuous pain in a different site, increasing in intensity during the night and relieved by oral consumption of non-steroidal anti-inflammatory drugs. The genetic mechanisms underlying OBL and OO development are largely unknown and no obvious genetic correlation between these two rare entities has been identified. However, we believe that the case of our patient represents the first report of metachronous OBL and OO, and this presentation may confirm the close correlation between these two rare entities.A metachronous presentation of OBLs and OOs is an exceptional event, but must be considered in cases of patients with a history of previous OBL or OO who present with a deep and continuous pain in a different site, increasing in intensity during the night and relieved by oral consumption of non-steroidal anti-inflammatory drugs.A metachronous presentation of OBLs and OOs may confirm the close correlation between these two rare entities and underlines a possible pathogenetic correlation between an OO and an OBL as part of the same family of tumours producing osteoid and woven bone.It is also possible that a metachronous presentation of OBL and OO may be a manifestation representing a genetic correlation between them.


However, how anti-TNF-α mAb regulates the functions of neutrophils is still unknown. Herein, we found that anti-TNF-α therapy significantly downregulated infiltration of neutrophils in inflamed mucosa of IBD patients. Importantly, anti-TNF-α mAb could inhibit neutrophils to produce proinflammatory mediators, such as ROS, calprotectin, IL-8, IL-6, and TNF-α. These data indicate that TNF-α plays a critical role in the induction of mucosal inflammatory response, and that blockade of TNF-α modulates intestinal homeostasis through balancing immune responses of neutrophils.Neutrophils have been found to play an important role in the pathogenesis of inflammatory bowel disease (IBD), and anti-TNF- Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), are chronic relapsing inflammatory disorders affecting the gastrointestinal tract. The incidence of IBD in China keeps increasing in recent decades . Althougα mAbs, infliximab  is the earliest commercialized biological molecular agent for the treatment of IBD. It is a chimeric mouse-human monoclonal antibody, which is effective for blocking both soluble and transmembrane TNF-α (tmTNF-α). Previous studies have demonstrated that IFX can induce and maintain remission in 45%–70% of IBD patients who previously failed to steroids or immunosuppressants. Patients with recurrent IBD can maintain long-term mucosal healing with IFX and avoid or postpone surgery. By the neutralization of TNF-α, IFX has been found to be attributed to several biological effects in the treatment of IBD patients, such as induction of proinflammatory T helper (Th) cell apoptosis, inhibition of growth factor, and antibody production and regulation of adhesion molecular expression. Moreover, IFX can also promote regulatory IL-10-producing macrophages, which can further induce regulatory Th cell differentiation [As one of the anti-TNF-ntiation . Howeverβ, IL-6, TNF-α, and IFN-γ) and chemokines , which further recruit neutrophils and monocytes/macrophages to the inflamed sites [+ neutrophils are one of the main sources of IL-22 in the intestine, which is of importance in guaranteeing epithelial barrier integrity [Neutrophils play a beneficial and detrimental role in intestinal inflammation . On one ed sites . Our recntegrity . The regα mAb, and explored the regulatory role of anti-TNF-α in the production of proinflammatory mediators. Our results demonstrated that anti-TNF-α therapy significantly downregulated the infiltration of neutrophils in inflamed mucosa in CD patients who responded to IFX. Importantly, anti-TNF-α mAb markedly suppressed production of proinflammatory mediators, such as MPO, calprotectin, IL-8, IL-6, and TNF-α. Moreover, blocking TNF-α could remarkably induce neutrophil apoptosis in CD patients. These data indicate that anti-TNF-α therapy ameliorates mucosal inflammation of IBD patients by downregulating activation and migration of neutrophils in gut mucosa.In this study, we investigated the migration of neutrophils in IBD patients before and after treatment with anti-TNF-n = 26), patients with CD in remission , patients with active UC , patients with UC in remission , and healthy volunteers  after overnight fasting. The diagnosis of CD or UC was based on clinical characteristics, radiological examination, endoscopy, and histological findings. The clinical characteristics of these patients with IBD are shown in All peripheral blood and intestinal tissue samples were collected from IBD patients and healthy donors in the Department of Gastroenterology, the Shanghai Tenth People's Hospital of Tongji University  from May 2016 to March 2018. EDTA anticoagulated blood samples (10–15 ml) were harvested from patients with active CD  at weeks 0, 2, and 6. Peripheral blood samples and paired intestinal mucosal biopsies were collected before and 12 weeks after the first IFX administration. According to the criteria of CDAI, these patients were classified into 2 groups, including the response group (15 patients (68.2%) achieved clinical response to IFX) and the failure group (7 patients (31.8%) failed to respond to IFX). Active CD was defined as CDAI > 150. Clinical remission was defined as CDAI < 150, and clinical response was defined as CDAI decreased by at least 70 points at the evaluation time point compared to the baseline index.22 patients with active CD were recruited and treated with anti-TNF-μm-thick sections. After deparaffiniztation and rehydration, antigen retrieval was performed with pH = 6 sodium citrate buffer or pH = 9 Tris-EDTA buffer. Endogenous peroxidase was inhibited by 3% H2O2. After blocking with 10% normal goat serum for 30 min at room temperature, these sections were incubated with rabbit anti-human CD66b mAb , rabbit anti-human MPO , or control IgG at 4°C overnight. After rinsing in 0.03% Triton-PBS buffer, the sections were incubated with HRP-conjugated anti-rabbit/mouse IgG (Dako) at room temperature for 40 min. After staining with diaminobenzidine (DAB), the sections were counterstained with haematoxylin.Fresh intestinal biopsies from IBD patients and healthy donors were fixated with 10% paraformaldehyde for 24 h and embedded with paraffins followed by slicing to 5 Peripheral neutrophils from patients with IBD were isolated with Ficoll-Hypaque  density gradient centrifugation as described previously . Briefly−ΔΔCt method.Total RNA of neutrophils was extracted with TRIzol. The concentration and purity of RNA were determined by a NanoVue spectrophotometer . The cDNA was synthesized with a 5x All-In-One RT MasterMix Kit (abm) based on the manufacturer's instructions. PCR reaction conditions were manipulated as follows: 25°C for 10 min, 42°C for 15 min, and 85°C for 5 min. qRT-PCR was performed according to the SYBR green (TaKaRa) instructions and the reaction was performed by BD 7900. RT-PCR reaction conditions were as follows: 95°C for 1 min, 95°C for 15 s, and 60°C for 30 s, repeated for 40 cycles. All primers were synthesized by Huada BioTech  and GAPDH was used as housekeeping gene. qRT-PCR analysis was calculated with the 2Protein levels of cytokines and chemokines were measured by ELISA according to the manufacturer's instruction (BioLegend). Briefly, captured antibodies were incubated in 96-well plates at 4°C overnight. Nonspecific antigens were blocked with assay diluents. The standard and samples were added and incubated at 37°C for 2 h. After thoroughly washing with 0.05% Tween-PBS, the plates were incubated with detection antibodies for 1 h and HRP for 30 min. Finally, the color was developed with TMB and the value of OD was detected with a microplate spectrophotometer (BioTek).6 peripheral neutrophils were cultured in vitro in 1 ml RPMI 1640-10% FBS and LPS (200 ng/ml) was added to stimulate the cells in the presence of IFX (50 μg/ml) or control human IgG (HIg 50 μg/ml) at 37°C in 5% CO2 humidified air for 3 h. Cells were then collected for qRT-PCR. For the detection of protein levels of cytokines produced by neutrophils, 2 × 106 neutrophils were stimulated with LPS (200 ng/ml) in the presence of IFX (50 μg/ml) or control human IgG (HIg 50 μg/ml) for 3 h. The culture medium was then replenished with 1 ml fresh RPMI 1640-10% FBS and cultured for another 24 h. Supernatants were collected for ELISA. For the detection of ROS and MPO produced by neutrophils, 2 × 106 neutrophils were incubated in the presence of human IgG (HIg 50 μg/ml) or IFX (50 μg/ml) for 3 h. 1 × 104 cells then were collected and incubated in HBSS in the presence of peroxidase and Amplex@ Red reagent in 96-well plates according to the manufacturer's instruction illustrated in the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Life Invitrogen).5 × 10n = 13) and A-UC (n = 13) during endoscopy and cultured ex vivo in 1 ml RPMI 1640-10% FBS medium in the presence of IFX or HIg (both at 50 μg/ml) at 37°C in 5% CO2 humidified air for 24 h. Tissues were then collected for qRT-PCR.Fresh colon biopsies were obtained from patients with A-CD  in patients with CD. Interestingly, we found that the percentage of CD66b+ neutrophils and expression of CD66b were significantly decreased in peripheral blood and inflamed mucosa of patients with active CD at the 12th week after anti-TNF-α therapy in the response group  and MPO in inflamed mucosa in the response group  were significantly suppressed by IFX treatment, while TGF-β and IL-17A were not altered. Moreover, we also observed that MPO and ROS production were remarkably downregulated under treatment with IFX  are associated with this phenomenon , 35. It α therapy, which contributes to the induction of remission in active IBD. We found that the impact of IFX on neutrophils is mainly dependent on the restriction of MPO, ROS, calprotectin, and proinflammatory cytokine/chemokine production, inhibition of migration to inflamed intestinal mucosa, and induction of neutrophil apoptosis. These findings indicate that blockade of TNF-α modulates immune response of neutrophils in intestinal mucosa of patients with IBD and maintains intestinal homeostasis.This study establishes that production of proinflammatory mediators and migration of neutrophils are remarkably suppressed by anti-TNF-


Clear writing is the key to success. In my early years I always thought of myself as an intellectual, and like my mother, I was a snob. As a teenager I read the classics and listened to Bach, Beethoven, and Mozart . I visited art museums and sought out mainstream and avant garde films and plays. At age 22 I would have been appalled to know that, many years later, I would follow professional sports and take life lessons from the commentators on sports radio . Maybe this isn’t entirely surprising because I played tennis, volleyball, basketball, and soccer for many years.One of the most common discussion points on sports radio is the difference in attitude and behavior shown by rookies (young first-year players) and the seasoned veterans (who were once themselves rookies). It is commonly accepted that a healthy and productive team needs to take advantage of the strength, enthusiasm, and agility of its youngest members. Nonetheless, the commentators often discuss the necessity that the new players learn from the veterans, and they are convinced that the successful teams are those that profit from the seamless combination of exuberance and experience. Moreover, the long-term success of a team derives in large part from its ability to motivate the veterans to mentor the younger players, and to signal to the rookies that they have much to learn from their older team mates. Sustained excellence requires generational transfer of knowledge and best practices, both in sports and science.The future of our community rests with the success of our graduate students, postdocs, and beginning faculty members. It is disconcerting to discover, therefore, that many of our brightest and most innovative young scientists struggle with their first papers and first grant applications, and will often, almost proudly, say that they hate writing. I don’t mind knowing that they find writing difficult (it certainly can be!), but no active and creative scientist can afford to hate writing, because our published work is the currency of our profession.Data show that early-career scientists are at a disadvantage in getting grants and in publishing their first papers. Some posit that early-career scientists are disadvantaged because they are less well known than more senior scientists. Although, that is certainly part of the answer, many of the difficulties that early-career scientists face come from what more seasoned scientists recognize as 'rookie errors' because we have seen them over and over in first papers and first grant proposals.An example: many years ago I was on an NIH Study Section and was assigned a grant from a new investigator. It seemed at first glance to suggest way too much work (being too ambitious is the most common failing of first grant applications). In this case, I estimated that it would take approximately 250 person years to do the experiments proposed for four people over a five year grant period. Why was this such a blunder? It was lethal for the proposal because it signaled to the reviewer that the principal investigator had not carefully thought through what it would take to do the work. Or even worse, he or she did not intend to do the work carefully and completely. Obviously, most successful grant applications propose more than can be done in the time allotted: however, being ambitious by a factor of two is within expected norms, whereas being ambitious by a factor of ten or more only erodes the credibility of the proposer. And while a grant panel might be willing to give a senior investigator a break by saying their past track record indicates that they will be able to overcome unforeseen obstacles, if a new investigator loses credibility by proposing vastly too many experiments, the panel will be unable to judge what he or she actually intends to do, and will consequently score the application poorly.Similar issues arise during manuscript review. Many manuscripts are plagued by errors of 'overselling', in which authors enthusiastically argue that their papers are paradigm shifting and will change the entire trajectory of science in a given field. Needless to say, this is rarely the case. Sadly, early-career scientists are often taught by their experienced mentors that overselling is necessary to get a paper accepted by a prestige journal. In reality, at eLife the most common complaint of reviewers of manuscripts from authors at all career stages is that the manuscript is 'oversold'. Reviewers are often content with authors just describing what they did and why. That said, there is a fine line between overselling and articulating clearly the new messages of the work. We all struggle with this as we try to frame our work for the reader. It is very hard to position a paper honestly so that its new contribution is evident, without crossing the line into 'overselling'.While some postdocs do write their own papers, many are part of large groups and the paper emerges from ensembles of authors within a framework devised by one or more senior authors. This experience may not provide much training for the postdoc when approaching his or her own first papers from their independent laboratory.eLife we often see papers from early-career scientists that suffer because the authors assume too much from the reader, or because the paper does not do justice to the intellectual framework that motivated the work. And we see papers that are missing appropriate controls, or papers in which the authors seem to be confused about what they are trying to say or what they can conclude from their experiments: I should stress that the latter problem is not restricted to early-career authors. I have found that my most interesting and important papers were some of the most difficult to write, precisely because we were struggling to understand the implications and logic of the findings. In many cases, I have understood the work through the act of writing about it, even sometimes rewriting after an initial version was rejected.At A large fraction of submitted papers are criticized for being poorly written or unclear. But, as in so many things, practice and experience do help. I have my own simple suggestions that can help an author to write a clear paper. 1) Start writing the paper before the experiments are 100% complete, as often a missing piece only becomes apparent when you are 'telling the story'. 2) Write the first draft quickly so that you remember what the beginning says as you work on the middle and end of the manuscript. It is very hard to get the rhythm of a piece right if you put it down for too long. If you write the Introduction in a few hours and realize it isn’t working, it is much easier to throw it away and start over than if you have spent two weeks on it. 3) Be prepared for lots of drafts. Some papers can be completed in a few drafts, but others require many more. As part of this, you have to be prepared to delete the perfect sentence or paragraph when it isn’t serving its function. 4) When you think you have a semi-final draft, go through it and get rid of excess words, sentences, and paragraphs. Read the text aloud to yourself, as your ears are more likely to pick up awkward syntax than your eyes. 5) Find a ruler and measure the number of inches or centimeters that you are dedicating to the different points in the paper, and make sure that the paper’s real message is not buried in pages of less interesting details or caveats.But most importantly, writing is the medium that allows you to explain, for all time, your new discoveries. It should not be a chore, but an opportunity to share your excitement, and maybe your befuddlement. It allows each of us to add to and modify the conceptual frameworks that guide the way we understand our science and the world. Without papers, the data are unanchored. And surely, one of the great pleasures of doing science is seeing your work change the way others think about an interesting and previously mysterious question. It is not an accident that some of our best and most influential scientists write elegant and well-crafted papers. So, work to make writing one of the great pleasures of your life as a scientist, and your science will benefit.


With a tremendous growth in the number of scientific papers, researchers have to spend too much time and struggle to find the appropriate papers they are looking for. Local citation recommendation that provides a list of references based on a text segment could alleviate the problem. Most existing local citation recommendation approaches concentrate on how to narrow the semantic difference between the scientific papers' and citation context's text content, completely neglecting other information. Inspired by the successful use of the encoder-decoder framework in machine translation, we develop an attention-based encoder-decoder (AED) model for local citation recommendation. The proposed AED model integrates venue information and author information in attention mechanism and learns relations between variable-length texts of the two text objects, i.e., citation contexts and scientific papers. Specifically, we first construct an encoder to represent a citation context as a vector in a low-dimensional space; after that, we construct an attention mechanism integrating venue information and author information and use RNN to construct a decoder, then we map the decoder's output into a softmax layer, and score the scientific papers. Finally, we select papers which have high scores and generate a recommended reference paper list. We conduct experiments on the DBLP and ACL Anthology Network (AAN) datasets, and the results illustrate that the performance of the proposed approach is better than the other three state-of-the-art approaches. The rapid development of science and technology leads to a substantial increase in the number of scientific papers. Researchers have to spend a lot of time and effort to find relevant papers. Many researchers use keyword search via scholar search engines, such as Google Scholar and Microsoft Academic Search, but there exist many instances where such a keyword query is either over broad, returning many papers that are loosely relevant to what the researcher really need, or too narrow, filtering many potentially relevant papers out or returning nothing at all –5. To alMost local citation recommendation approaches utilize scientific papers' and citation context's text content only , 16–18 aAn attention mechanism is introduced in the encoder-decoder framework; the attention mechanism combines author and venue information.An attention-based encoder-decoder (AED) model is proposed for local citation recommendation.Experimental results illustrate the efficiency of the proposed AED model.Recent studies show the encoder-decoder framework performs well in the field of machine translation –21 as itWe organize the rest of this paper as follows. Local citation recommendation is to find an ordered list of scientific papers from a dataset, and these scientific papers can be acted as candidate citations for a given citation context , 22. TheHe et al.  evaluateAs there exists vocabulary gap between the paper's content and the citation context's content, Lu et al.  proposedInspired by the encoder-decoder framework that has been successfully applied in neural machine translation , 34, we l, we denote the word embedding of the t-th word in the citation context as xtc and denote the embedding of the citation context as xl1:c=x1c ⊕ ⋯⊕xlc. A convolutional filter Wc ∈ Rk·g (g is the dimension of word vector) slides over k words at a time over all possible window lengths, i.e., {xk1:c, xk+12:c,…, xl−k+1:lc}, and the convolutional layer is defined asoi is the i-th feature map, and o ∈ ℜl−k+1. We apply max-pooling operation to obtain the largest feature value extracted by each filter and repeat p times yielding Xc as a low-dimensional vector representation sj, i.e.,For each given citation context, we use TDNN  to obtaif(Xc) gets different granularity of phrases  by using a variety of filters L={l1,…, lL||}. The phrase level representation captured by the TDNN can improve the performance of semantic representation and meanwhile reduce computational time.Finally, Ac, Ad, and Vd denote the vector representation of the authors of citation context, the vector representation of the scientific paper's author, and the vector representation of scientific paper's venue. The interaction of citation context, authors, and venues is occurred in decoding process.The authors and the venue of a scientific paper have essential impact on the scientific paper. Usually, researchers will track other researchers or research groups which have similar research interests with them. Similarly, the venue of the scientific paper also plays a vital role in whether the scientific paper should be cited, and the authoritative venues are far better than general venues. Therefore, we construct an attention mechanism based on the citation context's authors, the papers' authors, and the papers' venue. We concatenate the vector representations of citation context, citation context's author, the paper's authors, and the paper's venue, yieldingxid as the word embedding of the i-th word in the scientific papers and apply gated recurrent unit (GRU)  and denote the non-personalized citation context as c2=[ct]. When an academic newcomer, who does not have any publication, submits a citation context, the personalized AED based approach automatically reduces to non-personalized AED-based approach, since the citation context contains only content information ct. Besides, we investigate whether personal information based on citation context can result in more relevant and individualized references. We denote the personalized citation context as To validate the efficiency of the proposed AED-based approach, we compare it with the following approaches: (1) Context-Aware Relevance (CAR) model , which iFrom the table, we found that CAR-based approach performs poorest; this can be due to the CAR approach using vector representation based on bag-of-words that loses the valuable semantic information between scientific papers and the given citation contexts. TR approach is better than CAR approach as the TR approach evaluates the semantic relevance between scientific papers and the given citation contexts. However, both CAR approach and TR approach perform worse than NCN approach. This is because CAR approach and TR approach concentrate on scientific papers' content information only, but NCN approach utilizes author information besides content information. We found that the AED approach performs best in these four approaches. We attribute it to that the AED approach simultaneously utilizes multiple information related of scientific papers.We develop an attention-based encoder-decoder (AED) model for local citation recommendation in this paper; it first constructs an encoder to represent a citation context as a vector in a low-dimensional space, then it constructs an attention mechanism integrating venue information and author information, and uses RNN to construct a decoder; finally, it maps the decoder's output into a softmax layer and scores the scientific papers via the given citation context, author information, and venue information. A recommended reference paper list is generated based on scientific papers which have high score values. We conduct experiments on the DBLP and ACL Anthology Network (AAN) datasets, and the results illustrate that the performance of AED based approach is better than the other three baseline approaches.


Maceration with pectinase resulted in liqueurs with higher quercetin-3-O-glucopyranoside content. In relation to mango varieties, Haden liqueurs presented higher bioactive content than Tommy Atkins liqueurs. The liqueurs presented high antioxidant activity. The main bioactive compounds found were flavanols , flavonols (quercetin-3-O-glucopyranoside and rutin), and phenolic acids . The present study showed that the production of liqueur enabled the recovering of an important part of the bioactive content of mango peels, suggesting an alternative for the recovery of antioxidant substances from this by-product. The present work had the objective of producing liqueurs from mango peels (varieties “Haden” and “Tommy Atkins”) by processes of alcoholic maceration and maceration with pectinase, as well as to evaluate bioactive compounds by reversed-phase high-performance liquid chromatography coupled to diode array detection and fluorescence-detection (RP-HPLC/DAD/FD) and in vitro antioxidant activity (AOX), for by-product potential reuse. Alcoholic maceration in wine ethanol (65%  Mangifera indica L.) is one of the most popular edible fruit and its production, at present, ranks seventh in world fruit production, just after watermelons, bananas, apples, grapes, oranges, and coconuts [Mango  by processes of alcoholic maceration and maceration with pectinase, as well as to evaluate bioactive compounds by HPLC and in vitro antioxidant activity, in order to reuse this by-product.v/v was obtained from winery Ouro Verde-Miolo Wine Group . Folin-Ciocalteu reagent, ethanol, potassium persulfate, phosphoric acid, and potassium phosphate monobasic were purchased from Merck . Trolox  and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and 2,2-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) (ABTS) were supplied by Sigma-Aldrich . Methanol was from J.T. Baker . Ultrapure water was obtained with a Marte Científica purification system . External standards of gallic acid, syringic acid, benzoic acid, p-coumaric acid, o-coumaric acid, cinnamic acid, procyanidin B1, catechin, procyanidin B2, and caffeic acid came from Sigma-Aldrich . Epigallocatechin gallate, epicatechin, epicatechin gallate, procyanidin A2, quercetin 3-glucoside, rutin, kaempferol 3-glucoside, myricetin, and isorhamnetin 3-glucoside, quercetin-3-O-glucopyranoside came from Extrasynthese . The trans-Resveratrol was obtained from Cayman Chemical Company .Wine alcohol 65% To elaborate the liqueurs, “Haden” and “Tommy Atkins” mangoes were used, originating from the São Francisco Valley in the Northeast of Brazil. The fruits were harvested in March 2016, at full ripeness stage. The meteorological conditions at harvest were: rainfall 0.0–4.2 mm; average temperature 29.9 °C; relative humidity 51%; solar radiation 462.2 ly/day; and insolation 8.5 h/day. Meteorological measurements were taken at the agro-meteorological station of Bebedouro .The mangoes were sanitized in chlorinated solution (50 ppm), then peeled manually. The peels were cut into 1.0 × 1.0 cm and used to obtain liqueurs by alcoholic maceration and maceration with pectinase.Alcoholic maceration: Mango peels (1000 g) were placed to macerate in one liter of wine alcohol (65% v/v) where they remained for 30 days at room temperature. After maceration the liquid was drained (extract) and mixed with sucrose syrup until a standardized liqueur containing 15% total sugars and 18% ethanol was obtained. Maceration with pectinase: The peels (1000 g) were mixed in one liter of water with 100 μL of pectinase ). The maceration was carried out at 60 °C for 90 minutes. After maceration, the extract was drained and cooled to room temperature. To obtain the liqueur, a mixture of sucrose syrup and wine alcohol (65% v/v) was added to the extract to provide a beverage containing 15% total sugars and 18% alcohol.® , and placed to mature for 180 days in an environment protected from light and at a temperature of 25 ± 3 °C. The treatments consisted of producing four liqueurs from mango peels, denominated as: Haden by alcoholic maceration (HDA), Haden by maceration with pectinase (HDP), Tommy Atkins by alcoholic maceration (TAA), and Tommy Atkins by maceration with pectinase (TAP). Each treatment was performed in three replicates, totaling 12 produced liqueurs.For the standardization of the sugar content and alcoholic degree of the liquors, mass balance calculations were performed to measure the sugar and alcohol contents in the obtained extracts, and at the final concentrations desired. The liqueurs were packed in 300 mL colorless glass bottles, manufactured by Saint-Gobainv/v), titratable acidity, and total sugars %, following the methodologies described in Association of Official Analytical Chemists International (AOAC) [To obtain the basic analytical characteristics of the liqueurs, classical pH analyzes were carried out; alcohol (%l (AOAC) . Color a−1 of potassium dihydrogen phosphate with the pH adjusted to 2.05 with phosphoric acid, solvent B was methanol, and solvent C was acetonitrile. The oven temperature was maintained at 40 °C and the solvent flow at 0.6 mL min−1, with a total run time of 65 min. The detection and quantification of the compounds was carried out using external standards. The individual phenolic compounds in the liqueurs were determined by reversed-phase high performance liquid chromatography (RP-HPLC) on a Waters Systems instrument (model Alliance e2695) coupled to diode array detection (DAD) and fluorescence detection (FD). For the separation of compounds, a Gemini NX C-18 column (150 mm × 4.6 mm × 3 μm) and a Gemini NX C-18 guard column (4.0 mm × 3 mm × 3 μm) were used, both manufactured by Phenomenex . The gradient used was 0 min: 100% A; 18 min: 87.5% A, 2.5% B, 10.0% C; 30 min: 83.5% A, 3.2% B, 13.3% C; 36 min: 75.0% A, 5.0% B, 20.0% C; 48.5 minutes: 65.0% A, 8.3% B, 26.7% C; 50 min: 65.0% A, 8.3% B, 26.7% C; and 65 min: 100% A. Solvent A consisted of a solution of 25 mmol LThe analysis was performed according to the methodology described by Natividade et al. , using t−1 GAE). The total phenolic content of the liqueurs was determined by the spectrophotometric method with Folin-Ciocalteu . The abs−1). For the analysis, solutions of DPPH 1.0 mmol and ABTS radicals were prepared in ethanol and diluted to an absorbance of 0.90 ± 0.05 and 0.70 ± 0.03, respectively. The antioxidant activity of samples was assessed through the rate of decay in absorbance at 517 nm for DPPH and at 754 nm for ABTS. In the DPPH method, absorbance was measured at time t = 30 min after the addition of liquors. In the ABTS method, absorbance was determined at time t = 6 min after the addition of samples. All the analyses were performed in triplicate. Absorbance readings were performed in a spectrophotometer UV-Vis 2000A Instrutherm® .To measure the antioxidant activity in vitro, we used the methods of free radical scavenging with DPPH  and ABTS 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) ,19. The The results obtained from the analysis of the samples were submitted to analysis of variance (one-way ANOVA) and compared using the Tukey test at 5% of error probability using a SPSS program, Version 17.0, statistical package for Windows . A principal component analysis between the phytochemical profile and antioxidant activity was realized. −1), while maceration in ethanol resulted in liquors with pH and acidity of 4.84–5.01 g L−1 and 1.0–1.1 g L−1, respectively. The lower values of pH and higher values of titratable acidity in the liqueurs obtained by maceration with pectinase can be explained by the higher solubility of organic acids of the peels in water (solvent of maceration with pectinase), as citric acid and malic acid [The results of the classical analyses of the liqueurs are presented in lic acid . A highelic acid . L* values varied between 55–58.7 and 62.3–70; a* varied between 8.2–8.6 and 6.1–1.2; and b* varied between 80.7–63.3 and 71.5–37.6, for liqueurs obtained by maceration in ethanol and pectinase, respectively. The liqueurs obtained by maceration in pectinase had lower values of L* and a*, and higher values of b*, which represents a darker yellow color than the liqueurs obtained with ethanol.There were no significant differences in the values of total sugars and alcoholic strength of the liqueurs produced, with this result being expected due to the standardization performed in the elaboration of beverages. Regarding color, −1 for the Tommy Atkins and Haden varieties, respectively. A comparison of the preparation methods showed that there was no significant difference between pectinase and ethanol for the Haden variety; for the Tommy Atkins variety, however, the maceration method in ethanol obtained a higher total phenolic content. In relation to the total phenolics quantified by HPLC, the liqueurs obtained in wine alcohol also presented higher values, especially the Haden variety, with a total of 10,523 μg 100 mL−1.The total phenolic content and bioactive profile pertaining to the families of flavanols, flavonols, stilbene, and phenolic acids in the liqueurs are shown in −1 in mango varieties “Peach”, “Phiva”, “Rosa”, “Saber”, “Tommy Atkins”, and “Zill”. In the work of Sultana et al. [−1 dry mass. The values obtained for total phenolics by the Folin-Ciocalteu method and phenolic total quantified by HPLC showed that the production of this beverage from mango peels considerably recovered the bioactive content of this fruit. The total phenolic content obtained in the Tommy Atkins and Haden mango peel liqueurs was considered high in relation to the study by Sellamuthu et al. , who repa et al. , the tot−1 for the Haden and Tommy Atkins varieties, respectively , the following were also quantified: catechin (8–141), epicatechin (13.3–22), procyanidin A2 (14–78), procyanidin B1 (29.3–88), and procyanidin B2 (0–10).The mango peel liqueurs obtained by alcoholic maceration, in general, presented higher values for sums of quantified flavanols by HPLC, where the values varied from 950 to 1254 μg 100 mLectively . In liqu−1 in dry extract of “Keitt”, “Osteen”, and “Sensación” mango varieties, respectively. In the study of Dorta et al. [These values obtained for flavanols in the liqueurs were in line with those reported in the work of López-Cobo et al. , who foua et al. , differeO-glucopyranoside was the predominant flavanol in the liqueurs obtained with pectinase, in values ranging from 343.3 to 348.7 μg 100 mL−1, and was also the substance responsible for the highest total of flavonols on the liquors obtained with pectinase. For trans-resveratrol, the values obtained were low, varying from 2.0 to 4.0 μg 100 mL−1, and practically did not differ between the liqueurs produced. For liqueurs obtained with wine alcohol, the quantified flavonols were rutin (39.3–58 μg 100 mL−1), kaempferol 3-glucoside (30.7–46 μg 100 mL−1), isorhamnetin 3-glucoside (24–30.7 μg 100 mL−1), quercetin-3-O-glucopyranoside (14–42.7 100 mL−1), myricetin (8–13 μg 100 mL−1), and quercetin 3-glucoside (6.0 μg 100 mL−1). Comparing the mango varieties studied, in general, the differences in the profiles of the flavonols were not relevant.In relation to flavonols, liqueurs obtained by maceration with pectinase presented a higher value for sum quantified by HPLC . Quercet−1), quercetin 3-xyloside (7000 µg kg−1), quercetin 3-arabinoside (1800 µg kg−1), quercetin 3-glucoside (500 µg kg−1), and kaempferol 3-glucoside (100 µg kg−1). Based on the values of quercetin 3-glucoside and kaempferol mentioned by Meneses et al. [O-glucopyranoside content of the liqueurs studied.Several flavonols were quantified by Meneses et al.  in ethans et al.  and on t−1 for Tommy Atkins and Haden, respectively (−1) in the liqueur obtained by alcoholic maceration. Gallic acid has also been reported as one of the main phenolic compounds present in “Ataulfo” mango extracts, in values ranging from 43 to 62 μg mL−1 at different maturation stages [−1 (dry extract) for mango peels “Keitt”, “Osteen”, and “Sensación” varieties, respectively.Among the phytochemicals quantified by HPLC, phenolic acids were the main compounds present in the liqueurs studied, mainly in the liqueurs those obtained by alcoholic maceration, with values of 2530 and 9269 μg 100 mLectively . The main stages . In the −1 of liqueur (µmol TEAC 100 mL−1) (−1 (DPPH), and 631 and 648 μmol TEAC 100 mL−1 (ABTS). The liqueurs obtained with Tommy Atkins presented AOX values of 256 and 447 μmol TEAC 100 mL−1 (DPPH), and 275 and 466 μmol TEAC 100 mL−1 (ABTS). Regarding the elaboration methods, the liqueurs obtained by maceration in ethanol presented higher AOX than those obtained by maceration with pectinase, for the two mango varieties studied.The antioxidant activity in vitro (AOX) of the liqueurs were measured by free radical sequestration methods with DPPH and ABTS, and the results expressed as equivalent to micromoles of Trolox per 100 mL00 mL−1) . Compari−1 dry mass. Comparing the values mentioned by these authors with the values obtained in the liqueurs in the present study, it is evident that the use of the peels to produce liqueurs is an alternative for the recovery of antioxidant substances from this by-product. According to Meneses et al. , the AOX−1) were considered high, compared with a study by Leeuw et al. [−1. The antioxidant activity (DPPH) of liqueurs of red fruits, such as raspberry, strawberry, cornelian cherry, blackcurrants, blackberry, sour cherry, mahonia, sloe, chokeberry, and black rose, ranged from approximately 250 to 3250 μmol TEAC 100 mL−1 [The AOX values obtained in mango liqueurs  . In the p-Coumaric acid (pCa), kaempferol 3-glucoside (Kae), isorhamnetin 3-glucoside (Iso), myricetin (Myr), total phenolic by HPLC (TFQ), total phenolic (TP) and procyanidin B1 (PB1) (positive loadings), and trans-resveratrol (Res) and epicatechin (Epc) (negative loadings). For PC2, the variables that contributed most to the separation (loading > 0.70) were: quercetin 3-glucoside (Que), o-coumaric acid (oCa), p-coumaric acid (Cac) and quercetin-3-O-glucopyranoside (QuP) (positive loadings), and rutin (Rut), epicatechin gallate (EpG), gallic acid (Gac), and procyanidin B2 (PB2) (negative loadings). PC1 and PC2 separated on the positive part the Haden mango peel liqueur obtained by alcohol maceration (HDA), correlated with the highest content of EGC, pCa, Cac, Kae, Iso, Myr, TFQ, TP, PB1, Que, oCa, QuP, DPPH, and ABTS, which was the liqueur that presented the highest bioactive content related to phytochemicals and antioxidant activity. PC2 separated on the negative part of the liqueurs of Tommy Atkins mango peel liqueurs (TAM) and Haden by maceration with pectinase (HDP) correlated with the higher content of Rut, EpG, Gac, and PB2. PC1 separated in the negative part of the liqueur of Tommy Atkins mango peel obtained by maceration with pectinase (TAP) associated with the higher content of Epc and Res, and lower values for the content of bioactive compounds and antioxidant activity. In general, liqueurs obtained by alcohol maceration obtained higher values of phenolic compounds and antioxidant activity in vitro, and Haden mango variety presented higher bioactive content compared to Tommy Atkins. The method of maceration with pectinases was inferior to maceration with wine alcohol 65% v/v, but this is a promising technique, and in the Haden variety it presented a satisfactory bioactive content and AOX. In a study by Cosmulescu et al. [v/v) were evaluated in obtaining extracts to produce walnut liqueurs, reporting that ethanol 70% promoted a greater extraction of phenolic compounds, such as catechin, epicatechin, quercetin, myricetin, caffeic acid, and syringic acid, which corroborates with the results obtained in the present study.The factor analysis showed that the variables with higher contribution for the separation of the maceration treatments on PC1 with loading > 0.70 were: antioxidant activity (DPPH), antioxidant activity (ABTS), epigallocatechin (EGC), u et al. , differev/v) produced liqueurs with higher content of phytochemicals and antioxidant activity than maceration with pectinase. Maceration with pectinase resulted in liqueurs with high values of quercetin-3-O-glucopyranoside. In relation to the mango varieties studied, Haden presented higher phytochemical content than Tommy Atkins. The liqueurs presented high antioxidant activity in vitro, where the main bioactive compounds found in terms of quantity were flavanols , flavonols (quercetin-3-O-glucopyranoside and rutin), and phenolic acids . The values obtained for phenolic compounds quantified by HPLC and antioxidant activity showed that the production of liqueur enabled the recovering of an important part of the bioactive content of mango peels, suggesting an alternative for the recovery of antioxidant substances from this by-product. The present study showed that with the use of mango peels to produce liqueurs it is possible to recover much of the bioactive content of the mango peels, which are one of the main by-products of the fruit industry. Alcoholic maceration (wine alcohol 65%