The COVID-19 pandemic has resulted in school closures and distancing requirements that have disrupted both work and family life for many. Concerns exist that these disruptions caused by the pandemic may not have influenced men and women researchers equally. Many medical journals have published papers on the pandemic, which were generated by researchers facing the challenges of these disruptions. Here we report the results of an analysis that compared the gender distribution of authors on 1893 medical papers related to the pandemic with that on papers published in the same journals in 2019, for papers with first authors and last authors from the United States. Using mixed-effects regression models, we estimated that the proportion of COVID-19 papers with a woman first author was 19% lower than that for papers published in the same journals in 2019, while our comparisons for last authors and overall proportion of women authors per paper were inconclusive. A closer examination suggested that women’s representation as first authors of COVID-19 research was particularly low for papers published in March and April 2020. Our findings are consistent with the idea that the research productivity of women, especially early-career women, has been affected more than the research productivity of men. During the COVID-19 pandemic, many governments have shuttered schools and implemented social distancing requirements that limit options for childcare, while simultaneously requiring researchers to work from home . Robust Here, we focus on the published medical research literature, where it may be possible to provide an early evaluation of whether the gender gap in academic productivity is widening. The medical literature now includes a substantial number of articles directly relating to COVID-19, mostly generated rapidly after the broader social restrictions came into being, in most US states, in March 2020. We identified 15,839 articles on COVID-19 published between 1 January 2020 and 5 June 2020, including 1893 articles that had a first author and/or last author with an affiliation in the US. Here we report the results of an analysis that compared the proportion of women scientists in various author positions in this sample and a sample of 85,373 papers published in the same journals in 2019 .In The crosses and error-bars in https://arxiv.org/abs/2005.06303v2) based on COVID-19 papers published between 1 January 2020 and 5 May 2020 suggested larger differences than those reported here. Specifically, we found that women's share of first authorships, last authorships and general representation per author group was 23%, 16% and 16% lower for COVID-19 papers compared to 2019 papers published in the same journals. The present analysis covers a larger publication window of COVID-19 research (between 1 January 2020 and 5 June 2020), which has increased the sample from 1,179 US-based COVID-19 papers to 1893 (61% increase). Moreover, the present analysis is restricted to COVID-19 papers authored by US-based first and/or last authors, while the prior analysis included all papers with at least one US-based author. While this difference in sampling criteria might explain part of the observed variation in outcomes, we wanted to examine whether there has been a change over time. In An earlier iteration of this study . Only journals with at least five papers on COVID-19 were included in the analysis , 12,855 of 15,843 papers (81.1%)). We extracted author names for both treatment and control, and used these to determine author gender as in prior work  of the 300 publications. Of these, 129 (84.3%) were submitted after 15 March 2020, and 276 of the 300 (92.0%) were published after this date.We used mixed logit models with random intercepts and random slopes to estimate the relationship between the dichotomous intervention variable  and (i) women’s share of first authorships , (ii) women’s share of last authorships , and (iii) women’s overall representation per article  from PubMed to create dichotomous variables for COVID-19 studies published in March/April 2020 and May 2020. Following the procedure specified above, we used mixed logit models to estimate the relationship between these time-specific dichotomous variables and the three outcome measures.The statistical analyses were conducted in R version 4.0.0. For the mixed logit models, we used the ‘lme4’ v. 1.1–23 package in R. We used the ‘emmeans’ v. 1.4.7 package to produce adjusted means and ‘ggplot2’ v. 3.3.0 to produce figures.To produce  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by the eLife Features Editor (Peter Rodgers). The reviewers have opted to remain anonymous. Both reviewers were positive about the article, but they raised a small number of concerns that we would like you to address in a revised manuscript.Thank you for submitting your article "Meta-Research: Is Covid-19 Amplifying the Authorship Gender Gap in the Medical Literature?" for consideration by Summary:Reviewer #1: This is an analysis of whether COVID-19 is affecting the gender gap in medical publishing. The authors identify papers published on COVID-19 in 2020 and analyze whether women are underrepresented as first author, last authors or general authorship by comparing their representation in the same journals for all of 2019. They find that women are underrepresented, most strongly in the first author category. Further, this disparity is unlikely to be explained by the underrepresentation of female authors in specific fields that characterize papers published on COVID-19. It's a very clever and convincing analysis but some details need to be clarified.Essential revisions:1) The full dataset used in the study , as well as the code used to analyze the data, need to be made available to allow others to reproduce or expand the analysis.2) The robustness of author gender determination is not clear, and the methods should be described in more depth. For example, the text states "Gender was reliably estimated for 81.9% of the entire sample": what does "reliably estimated" mean here? How is the accuracy of the gender estimation tool established? What is the likelihood that a given gender was determined correctly? Doing spot checks of a random sample of authors can be helpful. Additionally, adding more information on the specific methods used for inferring gender from author names would be useful.3) The same issue might lead to bias in the analysis: only 81.9% of authors are included in the study, and these 81.9% represent a biased sample of all authors - those with names that are more common or more recognizable by the gender estimation tool. Thus, the study likely under-represents authors from certain countries, nationalities, and social backgrounds. Is there a way to assess the impact of this bias on the results and analysis? At a minimum, this should be discussed as a caveat.4) I am not convinced by the authors' argument that the observed differences are not due to a specialty bias. By looking at COVID-19-specific papers only, they are looking at the gender distribution of authors in a specific field, and comparing them to the gender distribution of authors in a variety of other fields who happen to publish in similar journals. It is entirely possible that there is a gender bias in coronavirus research, for example- this would not be captured by journal specialty, but would be seen in gender proportions for COVID-19 papers. Please add additional columns to table 1 looking at all research published in these journals in 2020 (not just COVID-19-related research), and/or discuss at greater length the possibility of specialty bias in your results.5) The authors limited their samples to papers with at least one US author. It could be useful to know, of the 1179 papers in the treatment group, how many papers had more than one US author? I'm concerned that this limitation may introduce confounding factors. Knowing how many of these papers were published by teams of scientists primarily working in the US, vs a single scientist working in the US with a larger international team, would address this concern and more strongly support the authors' point that the disparity in female authorship can be potentially linked to the social restrictions in the US. We want to thank the editor and the reviewers for their excellent comments and suggestions. We have gone over them all and made changes accordingly. Most importantly, some of the comments have caused an update of the data set, which changes some of the findings and allows us to fine-tune the analysis. Since we initially harvested data in April 2020, PubMed has changed its interface and now provides better (and more consistent) information on author affiliations. This allows a much more precise analysis of author country, as requested by one of the reviewers. This was not possible previously.We address this data update in the paper, in an effort to be as transparent about the process as possible, but for good measure, we would like to mention here the original sample, which was 9,050 COVID-19 papers, of which 1,179 had gender for first and last author, and at least one US-based author. These numbers are now 15,839 and 1,893 respectively. When analyzing first-authors, we restrict to US-based first authors and vice versa for last authors.Below, we list the comments from the reviewers and editor in italics and our responses in regular font face.Essential revisions:1) The full dataset used in the study , as well as the code used to analyze the data, need to be made available to allow others to reproduce or expand the analysis.We have uploaded the data and the analytical scripts to OSF. The project is private right now, but we will make it public when/if our paper is published. An R script is provided, which will allow full analysis from the most raw data set we are able to provide (gender is inferred and information from PubMed is extracted into a table).2) The robustness of author gender determination is not clear, and the methods should be described in more depth. For example, the text states "Gender was reliably estimated for 81.9% of the entire sample": what does "reliably estimated" mean here? How is the accuracy of the gender estimation tool established? What is the likelihood that a given gender was determined correctly? Doing spot checks of a random sample of authors can be helpful. Additionally, adding more information on the specific methods used for inferring gender from author names would be useful.Checking the correctness of gender assignment is of course important. Our data relies on an algorithm, which we have previously used and checked for robustness. We realize that this was not apparent from the very short comment in the paper, and have added a little more detail about which algorithm we used and that robustness checks have previously been performed.3) The same issue might lead to bias in the analysis: only 81.9% of authors are included in the study, and these 81.9% represent a biased sample of all authors - those with names that are more common or more recognizable by the gender estimation tool. Thus, the study likely under-represents authors from certain countries, nationalities, and social backgrounds. Is there a way to assess the impact of this bias on the results and analysis? At a minimum, this should be discussed as a caveat.We have adjusted the method so that we now only compare US first and last authors. We believe this is a strength of the study because it allows us to isolate the effect to a single country and the timing of both disease and social constraints. For the analysis of the full author group, there could potentially be a bias, which we address in the limitations.4) I am not convinced by the authors' argument that the observed differences are not due to a specialty bias. By looking at COVID-19-specific papers only, they are looking at the gender distribution of authors in a specific field, and comparing them to the gender distribution of authors in a variety of other fields who happen to publish in similar journals. It is entirely possible that there is a gender bias in coronavirus research, for example- this would not be captured by journal specialty, but would be seen in gender proportions for COVID-19 papers. Please add additional columns to table 1 looking at all research published in these journals in 2020 (not just COVID-19-related research), and/or discuss at greater length the possibility of specialty bias in your results.While this is certainly an important point, we also have to disagree, for two reasons. COVID-19 specific papers are not from a specific field. On the contrary, the vast majority of medical specialties have picked up research on COVID-19 in one way or another. The search for cures or vaccines are likely limited, but discussions about implications for treatment of other diseases span all of clinical medicine. Secondly, our reason for using COVID-19 research as the “treatment” case was not to look specifically into this, but rather to have a sample of papers where we knew the research would have to be done in 2020. If we introduced a new column of “other” 2020 papers from the same journals, it would be impossible to know which of these had been in the pipeline since 2019 or earlier, and which were actually based on research from 2020.5) The authors limited their samples to papers with at least one US author. It could be useful to know, of the 1179 papers in the treatment group, how many papers had more than one US author? I'm concerned that this limitation may introduce confounding factors. Knowing how many of these papers were published by teams of scientists primarily working in the US, vs a single scientist working in the US with a larger international team, would address this concern and more strongly support the authors' point that the disparity in female authorship can be potentially linked to the social restrictions in the US.We hope that the changes to how we analyze first and last authors  also addresses this point. For the share of women in the full group, this remains a valid concern, but not one we are currently able to address. While PubMed has now introduced per-author affiliations, it is not realistic to use these to make statistical analyses. When we claim to look only at papers with US-authors, this is really a conservative claim; there are certainly more papers with US addresses, but where the country or state is not included in the affiliation, or included in non-standardized forms. Our database contains more than sixty thousand “countries” from the affiliations, as there is no standard formatting of PubMed affiliations. We have trawled all of these for combinations of “USA”, “United States”, “America”, state names, names of major cities etc., to find the most complete set of affiliations related to the USA. However, we are not able to provide a reliable estimate of the national distribution of affiliations of all authors.


P5S form of the RNA-binding Cold Shock Domain-containing E1 protein, which promotes escape as an inhibitor of VSV replication by disrupting viral transcription. Given time, VSV-IFNβ evolves a compensatory mutation in the P/M Inter-Genic Region which rescues replication in CSDE1P5S cells. These data show that CSDE1 is a major cellular co-factor for VSV replication. However, CSDE1P5S also generates a neo-epitope recognized by non-tolerized T cells. We exploit this predictable neo-antigenesis to drive, and trap, tumors into an escape phenotype, which can be ambushed by vaccination against CSDE1P5S, preventing tumor escape. Combining frontline therapy with escape-targeting immunotherapy will be applicable across multiple therapies which drive tumor mutation/evolution and simultaneously generate novel, targetable immunopeptidomes associated with acquired treatment resistance.In our clinical trials of oncolytic vesicular stomatitis virus expressing interferon beta (VSV-IFNβ), several patients achieved initial responses followed by aggressive relapse. We show here that VSV-IFNβ-escape tumors predictably express a point-mutated CSDE1 Oncolytic viruses, such as vesicular stomatitis virus (VSV), are a promising class of cancer therapeutics. Here the authors report that a mutation in the CSDE1 gene renders cancer cells resistant to VSV replication and oncolysis, but a mutation-derived escape-associated neoantigen could be exploited for immunotherapy against treatment-resistant tumors. Hence, strategies that reduce treatment failure through tumor escape would be highly significant.Escape from frontline therapy is a major cause of treatment failure in cancer patients12, due to mutational plasticity and selection of treatment-resistant clones11. These treatment-escape phenotypes arise, at least in part, through the evolution of a pool of mutated tumor cells from which highly aggressive, treatment-resistant clones are rapidly selected11. In this respect, APOBEC  cytosine deaminases provide an endogenous source of DNA mutation-driving cancer evolution in response to a variety of different frontline treatments20. APOBEC3 cytosine deaminases act as innate antiviral restriction factors and catalyze cytosine to uracil deamination of ssDNA (C–T transitions and C–G transversions)17. Although the human genome encodes seven APOBEC3 enzymes (3A–H), the mouse encodes a single gene, mAPOBEC321, which has similar activities to those of human APOBEC3B (hAPOBEC3B)10. Consistent with APOBECs as drivers of tumor escape, the APOBEC3B signature is associated with therapeutic resistance in multiple cancers20.We have shown that treatment-escaped tumors differ significantly from primary tumors immunogenically, genetically, and phenotypically11. With respect to oncolytic virotherapy, we, and others, have developed vesicular stomatitis virus (VSV), a single-strand negative sense RNA virus , as an oncolytic platform for clinical testing31. VSV, which is highly sensitive to inhibition by interferon (IFN), shows selective replication in Type I IFN-defective tumor cells, while being rapidly shut down in (IFN-responsive) normal cells24. For the clinical development of VSV32, we overexpressed the IFNβ gene in the virus25 to enhance safety  and to increase the immunogenicity of infected/dying tumor cells, as IFNβ acts as a key signal 3 cytokine to facilitate priming of tumor-reactive T cells33.We have shown that APOBEC3 induction following frontline treatment with adoptive T-cell therapy, chemotherapy, or oncolytic virotherapy has profound consequences for the generation of escape variants from all three types of therapy31, several patients achieved initial responses followed by aggressive escape. To understand the mechanistic basis of these effects, we established in vitro and in vivo models in which suboptimal levels of VSV infection leads to the escape of virus-resistant cells10. We showed that suboptimal infection with VSV induced Type I IFN-dependent hAPOBEC3B- or mAPOBEC3-induced mutation of the cell genome, degradation of the viral genome, and escape of virus/oncolysis-resistant (VSV-ESC) cells10. VSV-ESC cells10 carried stable APOBEC3B mutational signatures in multiple genes11, some of which might be critical for escape. Simultaneously, we reasoned that some of these mutations may also induce neo-antigenesis—the generation of neo-epitopes with increased major histocompatibility complex (MHC) binding and immunogenicity36, rendering VSV-ESC cells susceptible to T-cell attack. Consistent with this hypothesis, we identified a C–T mutation in the CSDE1 gene (CSDE1C-T) 11 in VSV-ESC cells11, which generated a heteroclitic39 neo-epitope, which primed T-cell responses against both itself (CSDE1P5S) and, to a lesser extent, wild-type CSDE1 (2 CSDE1-specific siRNA)All analysis was performed within GraphPad Prism software (Graphpad). Multiple comparisons were analyzed using one-way or two-way analysis of variances with a Tukey’s post hoc multi-comparisons test. Survival data were assessed using the log-rank test using a Bonferroni correction for multiple comparisons. Data are expressed as group mean ± SD.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Ctenopharyngodon idella) and causes economic losses in grass carp aquaculture. Recent epidemiological investigations showed that GCRV genotype II is the dominant subtype in China. Therefore, it is very important to develop a novel vaccine for preventing diseases caused by GCRV genotype II. In this study, we employed a bac-to-bac expression system to generate GCRV-II-based virus-like particles (VLPs). Previous studies have shown that the structural proteins VP3, VP4, and VP38 encoded by the segments S3, S6, and S10 of type II GCRV are immunogenic. Hence, the GCRV-VLPs were produced by co-infection of sf9 cells with recombinant baculoviruses PFBH-VP3, PFBH-VP4, and PFBH-VP38. The expressions of VP3, VP4, and VP38 proteins in GCRV-VLPs were tested by IFA and Western blot analysis. By electron microscopic observations of ultrathin sections, purified VLPs showed that the expressed proteins are similar in shape to GCRV genotype II with a size range from 40 nm to 60 nm. The immunogenicity of GCRV-VLPs was evaluated by the injection immunization of grass carp. The analysis of serum-specific IgM antibody showed that grass carp immunized with GCRV-VLPs produced GCRV-specific antibodies. Furthermore, injection with GCRV-VLPs increased the expressions of immune-related genes  in the spleen and kidney. In addition, grass carp immunized with a GCRV-VLPs-based vaccine showed a relative percent survival rate (RPS) of 83.33% after challenge. The data in this study showed that GCRV-VLPs demonstrated an excellent immunogenicity and represent a promising approach for vaccine development against GCRV genotype II infection.Grass carp reovirus (GCRV) leads to severe hemorrhagic disease in grass carp ( Ctenopharyngodon idella) is one of the most important aquaculture species in China. It has been widely cultivated over the last 60 years and has a great commercial value [Aquareovirus of the family Reoviridae and was the first viral pathogen identified from aquatic animals in China in 1983 [Grass carp  serve as an ideal platform overcoming the limitations of traditional vaccines. The generation of VLPs was first described with the reconstitution of the tobacco mosaic virus (TMV) in 1955 . Since tPrevious studies have shown that the structural proteins VP3, VP4, and VP38 encoded by the segments S3, S6, and S10 of GCRV genotype II have a good immunogenicity. The helicase VP3 is encoded by the segment S3, which is involved in the transcription and capping of viral RNAs . The maiIn this study, we developed VLPs containing VP3, VP4, and VP38 structural proteins of the HuNan1307 strain of GCRV genotype II, which was employed as the VLP -based vaccine to immunize grass carp. Then, the immunogenicity and protection ability of the GCRV VLP-based vaccine were evaluated against a virus challenge. The data showed that the GCRV VLP-based vaccine could provide protection to grass carp and represents a novel approach for vaccine development against GCRV genotype II infection.GCRV-HuNan1307 used in this study was isolated from infected grass carp in a fish farm located in Zhuzhou . The complete genome sequences of HuNan1307 are publicly available . Insect cells (sf9) were cultured at 28 °C in Sf9-900 II complete medium .TM HTA  , the PCR products were ligated to the plasmid pMD18-T , namely pMD18T-VP3, PMD18T-VP4, and pMD18T-VP38. Subsequently, the plasmids pMD18T-VP3, pMD18T-VP4, and pMD18T-VP38 and the pFastBacTM HTA were respectively digested by restriction enzyme Not I, Kpn I, Bam I, Xho I, Not I, and Xho I . Then, the enzyme-digested fragments were purified and ligated together using T4 DNA ligase  at 16 °C for 12 h to generate recombinant plasmid pFastBacHTATM -VP3, pFastBacHTATM -VP4, and pFastBacHTATM -VP38. The target genes were inserted into the pFastBacHTATM vector under the control of polyhedron promoter (PpH). Next, the ligation mixtures were transformed into E. coli DH5α , the DNA was extracted from positive clones and confirmed by sequence analysis, and enzymatic digestion was used for identification. Then, the pFastBacHTATM -VP3, pFastBacHTATM -VP4, and pFastBacHTATM -VP38 plasmids were employed to transform DH10BacTM competent cells  to produce bacmids. Using the blue-white screening to detect colonies containing recombinant bacmid DNA, they were identified by PCR amplification using the M13 primers. The recombinant bacmids were then isolated and purified using EndoFree Plasmid Maxi kit  according to the protocol of manufacture.GCRV genomic dsRNA was extracted using a Trizol Reagent kit  according to the protocol of manufacture. Afterward, reverse transcription was performed to produce cDNA, which was used to clone the segments S3, S6, and S10 of the isolate GCRV-HuNan1307 . The target genes were amplified by PCR using primers with appropriate restriction enzymes according to the multiple cloning sites of the donor plasmid, pFastBac6 Sf9 cells were incubated into 6-well plates at 27 °C for 24 h before replacing with a ratio of 1:4 Sf9-900 II complete medium and Grace’s Insect Medium . The mixed medium was supplemented to 1.8 mL, and the cells were incubated for 30 min at room temperature. Then, 1 μg bacmid plasmids and 8 μL Cellfectin II reagent were added to 100 μL of the Grace’s Insect Medium, respectively, and incubated for 30 min at room temperature. Next, the two reagents were gently mixed and incubated for 30 min at room temperature before dispensing onto the Sf9 cells. The whole mixture of approximately 210 μL was added to the cell culture plate and incubated at 28 °C for 4 h before replacing with 2 mL Sf9-900 II complete medium. The viral stock of first passage (shortly named-P1) was harvested from the cell culture until a significant characteristic of viral infection was exhibited and named PFBH-VP3, PFBH-VP4, and PFBH-VP38. Then, amplified baculovirus was stocked until P3 viral stock.To produce the recombinant baculoviruses, Sf9 cells were seeded into six-well plates and transfected with Bacmid-VP3, Bacmid-VP4, and Bacmid-VP38 using Cellfectin II reagent . In detail, 1.0 × 105 Sf9 cells were cultured in 24-well plates and infected with the third-generation recombinant baculoviruses. Uninfected Sf9 cells were used as a blank control group. At 4 days of infection, the culture medium was discarded and PBST was used to wash the Sf9 cells three times. Then, the cells were fixed with methanol previously cooled for 10 min at −20 °C. Methanol was discarded and air-dried. The cells were washed three times with PBST for 5 min. Next the cells were incubated with 0.5%Triton for 10 min to permeabilize the cells in a shaking bed at 120 r/min. The solution was discarded and washed three times with PBST for 5 min each. The samples were blocked with 5% skim-milk powder for 30 min at 37 °C, the blocking solution was discarded, and each well was washed three times with PBST as described above. After that, the Sf9 cells were incubated with rabbit anti-VP3, anti-VP4, and anti-VP38 serum (developed in our laboratory) in a dilution of 1:200 for 1.5 h at 37 °C. The cells were washed three times with PBST and incubated with the secondary antibody, a FITC-conjugated goat anti-rabbit antibody , diluted 1:3000 in PBS, for 50 min at 37 °C. Then, PI  diluted at 1:10,000 was added to stain cell nuclei for 10 min in the dark at room temperature. All the dilutions were made using PBS. Finally, the cells were cleaned three times with PBST and observed under a fluorescence microscope.A density of 4 × 106 cells/mL and co-infected with PFBH-VP3, PFBH-VP4, and PFBH-VP38 at multiplicity of infection (MOI) of 2:2:1. All infected cultures were incubated for 1 h at 28 °C. After that, Sf9-900 II complete medium was added to the cell cultures and incubated at 28 °C. The Sf9 cells lysates were harvested when an obvious cytopathic effect occurred, which was regarded as the first GCRV-VLPs stock, and amplified GCRV-VLPs were stocked until the third GCRV-VLPs stock. To purify the VLPs, the cell supernatants were subjected to sucrose gradient ultracentrifugation after two repeated freeze-thaw cycles at −20 °C. Cells debris was removed by centrifugation at 5000 r/min for 30 min. Then, the VLPs in the supernatant were pelleted by ultracentrifugation at 37,000 r/min for 2 h at 4 °C. The pellets were resuspended in 500 μL PBS and loaded onto a 20–30–45–60% discontinuous sucrose gradient. The VLPs were collected in different fractions from top to bottom after ultracentrifugation at 37,000 r/min for 1 h at 10 °C and mixed with PBS, respectively. Then, the sample was pelleted by ultracentrifugation at 37,000 r/min for 15 min at 4 °C and resuspended in 100 μL PBS.To generate the GCRV-VLPs, Sf9 cells were adjusted to 1.0 × 10The purified VLPs were identified by Western blot analysis. The samples mixed with 5× Loading Buffer were heated at 100 °C for 10 min and centrifuged for 1 min, then separated by 12% SDS-PAGE, and transferred onto NC membranes by electrophoresis. The membranes were then blocked with 5% skim-milk powder in PBS for overnight at 4 °C and then treated with rabbit anti-VP3, anti-VP4, and anti-VP38 sera (developed in our laboratory) diluted at 1:200 for 1.5 h at room temperature. Membranes were rinsed three times with PBST (5 min per wash), followed by incubation for 45 min at room temperature with the goat anti-rabbit secondary antibody in a dilution of 1:5000. Afterward, the membranes were washed three times with PBST (5 min per wash), and the Horseradish Peroxidasechromogenic reagent  was added for development until the bands had been seen clearly. Then, the NC membranes were placed in PBS to terminate the reaction. To investigate whether VLPs were self-assembled correctly, ultrathin sections were analyzed by electron microscopy (EM). Furthermore, negative staining transmission electron microscopy (TEM) was performed to analyze the shape and size of purified GCRV-VLPs. One microliter of purified VLP selected fraction was loaded onto a copper gird and fixed for 5 min, removing the VLPs and loading phosphotungstate on the gird for 10 s. Then grids were air-dried and observed by an electron microscope for TEM analysis.7.2 TICD50/mL of 200 μL commercial attenuated vaccine GD1108 strain .The grass carp with body weight of 20 ± 0.5 g and a body length of 10 ± 0.5 cm was purchased from a grass carp farm in Guangzhou, Guangdong, China. They were acclimatized at 28 °C under laboratory conditions for 2 weeks before experimental manipulation, then were maintained in aerated water and fed daily with commercial dry feed pellets . During this observation period the grass carp did not display any clinical signs. Additionally, samples taken from these fish were negative by qRT-PCR for detection of GCRV. The grass carp were divided randomly into five groups (50 fish/group) and injected intraperitoneally for the experiments. For the GCRV-VLPs group, and 30 μg of GCRV-VLPs was used only for the GCRV-VLPs + adjuvant group, 30 μg of GCRV-VLPs was mixed with 100 μL of Montanide IMS 1312 VG adjuvant  after vortexing with a final volume of 200 μL. The attenuated vaccine group was immunized with 10 The PBS group was injected with 200 μL of PBS. The PBS + adjuvant group was injected with PBS mixed with an equal volume of Montanide IMS 1312 VG adjuvant. A boost immunization proceeded 21d later with the same antigens used as the primary immunization. Serum, spleen, and kidney were sampled from five fish in each group three-weeks after immunization and boost immunization and stored at −80 °C.All animal procedures were conducted according to animal welfare standards and approved by the Ethical Committee for Animal Experiments of Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, China. All animal experiments complied with the guidelines of the Animal Welfare Council of China.The grass carp serum samples were collected to detect specific IgM antibody by indirect ELISA. The virion protein was purified in our laboratory previously. Briefly, 100 μL of purified virion protein was coated onto an ELISA plate as antigen with a concentration of 100 ng/mL and incubated overnight at 4 °C. The next day, the plates were blocked with 5% skim-milk powder in PBS for 2 h at 37 °C. The sera were diluted to 1:200 and were added (100 μL/well) in three replicates and incubated for 1.5 h at 37 °C. Then, 100 μL of HRP-labeled mouse anti-grass carp IgM diluted to 1:10,000 in PBS was added to each well. After an hour of incubation at 37 °C, 90 μL of TMB chromogenic reagent  was added to each well for 10 min in the dark and then stopped with the TMB stop solution. Optical density (OD) values were measured at a wavelength of 450 nm (OD450) 5 min after the stop solution was added. After each incubated step, the plates were washed with PBST five times (5 min per wash) in a shaking bed at 200 r/min.TM RT Master Mix  and stored at −20 °C. The primers for detecting immune genes are listed in −ΔΔCt method.Total RNA samples were extracted from spleen and kidney of the grass carp from different groups by Trizol Reagen kit , according to the manufacturer’s instructions. The cDNAs were synthesized using the PrimeScript50 of 200 μL HuNan1307. The fish were maintained at 28 °C. The mortality and clinical signs such as dark body coloration and hemorrhage of the fines and gill covers were recorded daily for two weeks post-challenge. The relative percent survival (RPS) was calculated as follows: RPS =  × 100%.On 21 days post-immunization (dpi) after the booster immunization, all groups were challenged intraperitoneally with 10 LDp-values of <0.05 were considered significant.The experimental results are shown as means ± SD of the different groups. All statistical analyses were implemented in GraphPad Prism (7.01) and analyzed based on one-way analysis of variance and Tukey tests. TM were digested to construct the recombinant plasmids pFastBacHTATM -VP3, pFastBacHTATM -VP4, and pFastBacHTATM -VP38. Correct sizes of the recombinant plasmids were identified by dual enzyme digestion, and the results confirmed that the recombinant plasmids were successfully constructed . On 21 dpi after booster immunization, the antibody titers of the GCRV-VLPs group and GCRV-VLPs + adjuvant group increased significantly compared to the PBS control group, while in the attenuated vaccine group the IgM level was also up-regulated (p < 0.001)  .p < 0.01). After booster immunization, the expression levels of IFN in every immunized groups were increased higher than that of the PBS group (p < 0.05); in addition, the expression level of IFN in the GCRV-VLPs + adjuvant group was above 20-fold higher than that of the PBS group. In the kidney, the expression level of IFN in the GCRV-VLPs + adjuvant group increased significantly compared with PBS group after booster immunization (p < 0.01) (p < 0.001). In the kidney, the relative expression level of IgM in the GCRV-VLPs + adjuvant group increased significantly compared with PBS group on 21 dpi of both immunization (p < 0.01), being approximately 20-fold higher than that of the PBS group (p < 0.01) compared to the PBS group. The expression levels of TLR3 and TLR7 in the GCRV-VLPs group were not up-regulated compared to the PBS group. After booster immunization, the expression levels of TLR3 and TLR7 in spleen and kidney in the GCRV-VLPs + adjuvant group and attenuated vaccine group increased significantly compared to the PBS group (p < 0.001)  A. The reBS group B. The ex< 0.001) C,D. The To evaluate the protective effectiveness of the GCRV-VLPs, each group was challenged with a lethal dose of GCRV on 21 dpi after booster immunization. The typical clinical signs of GCHD, including dark body coloration, hemorrhage of the fines and gill covers, began to appear 4 days post-challenge in PBS control group fish. Dead fish samples were confirmed positive of GCRV infection by qRT-PCR. The results showed that the RPS of GCRV-VLPs + adjuvant group was 83.33%, and the RPS of GCRV-VLPs group was 75%; the attenuated vaccine group showed a higher level of protection with 91.67% . All theInfections and diseases induced by GCRV result in extremely high mortality among grass carp, which causes severe economic losses for the grass carp farming industry. Vaccination is a proven efficient way to control virus infection, which makes vaccines a weapon against viral disease. VLP-based vaccines can provide advantages over traditional technologies . The safIn our study, a GCRV-VLPs was developed using a bac-to-bac insect expression system. Recombinant baculoviruses PFBH-VP3, PFBH-VP4, and PFBH-VP38 were generated. Then, sf9 cells were co-infected with recombinant baculoviruses to produce GCRV-VLPs. Purified GCRV-VLPs were similar in morphology to the native virus, as assessed by TEM . WesternThe immunogenicity of the GCRV-VLPs based vaccine was explored through the immunization of grass carp. IgM is the major antibody in teleosts and is regarded as an indicator for the specific immune response ,38. In tThe IFN system plays an important role in innate immunity as the first line of defense against viral infections . In prevAdditionally, the protective effectiveness of the GCRV VLPs-based vaccine was evaluated the against GCRV challenge. The results showed that the GCRV-VLPs group exhibited RPS of 75% of individual fish, and the GCRV VLPs-based vaccine combined with adjuvant increased the RPS of fish challenged with GCRV to 83.33%. The RPS of the PBS + adjuvant group was 12.5%. The results showed that the adjuvant can improve the protective effectiveness, and the GCRV VLPs-based vaccine can confer effective protection against GCRV infection. Meanwhile, the attenuated vaccine group exhibited RPS of 91.67%. The results showed that the VLPs-based vaccine is not as effective as the attenuated vaccine, while the VLP-based vaccine can overcome the potential risk of reversion to virulence; therefore, safety performance is fine. Although GCRV-HuNan1307 is a representative strain of genotype II that is isolated from grass carp in Hunan province, HuNan1307 could not cause higher mortality rate in our challenge studies, which may partially inflate the survival benefits of the VLP vaccine.In summary, the present study demonstrated that the GCRV-VLPs based vaccine induced an immune response and conferred effective protection against GCRV infection in grass carp. Therefore, GCRV-VLPs can serve as a potential vaccine with developmental prospects.


Leishmania amazonensis (IC50 = 0.25 μmol L–1). In comparison with amphotericin B, a second-line drug approved for leishmaniasis treatment, (IC50 = 0.63 μmol L-1), Fc-RP1 was more active and showed a 2.5-fold higher selectivity index. The RP1 peptide presented a MIC of 4.3 μmol L-1 against S. agalactiae, whilst Fc-RP1 was four times more active (MIC = 0.96 μmol L-1), indicating that ferrocene improved the antimicrobial activity against Gram-positive bacteria. The Fc-RP1 peptide also decreased the minimum inhibitory concentration (MIC) in the assays against E. faecalis (MIC = 7.9 μmol L-1), E. coli (MIC = 3.9 μmol L-1) and S. aureus (MIC = 3.9 μmol L-1). The cytotoxicity of the compounds was tested against HaCaT cells, and no significant activity at the highest concentration tested (500 μg. mL-1) was observed, showing the high potential of this new compound as a possible new drug. The coupling of ferrocene also increased the vesicle permeabilization of the peptide, showing a direct relation between high peptide concentration and high carboxyfluorescein release, which indicates the action mechanism by pore formation on the vesicles. Several studies have shown that ferrocene destabilizes cell membranes through lipid peroxidation, leading to cell lysis. It is noteworthy that the Fc-RP1 peptide synthesized here is a prototype of a bioconjugation strategy, but it still is a compound with great biological activity against neglected and fish diseases.Parasitic diseases are a neglected and serious problem, especially in underdeveloped countries. Among the major parasitic diseases, Leishmaniasis figures as an urgent challenge due to its high incidence and severity. At the same time, the indiscriminate use of antibiotics by the population is increasing together with resistance to medicines. To address this problem, new antibiotic-like molecules that directly kill or inhibit the growth of microorganisms are necessary, where antimicrobial peptides (AMPs) can be of great help. In this work, the ferrocene molecule, one active compound with low levels of in vivo toxicity, was coupled to the N-terminus of the RP1 peptide (derived from the human chemokine CXCL4), aiming to evaluate how this change modifies the structure, biological activity, and toxicity of the peptide. The peptide and the conjugate were synthesized using the solid phase peptide synthesis (SPPS). Circular dichroism assays in PBS showed that the RP1 peptide and its conjugate had a typical spectrum for disordered structures. The Fc-RP1 presented anti-amastigote activity against  Leishmania spp., is endemic in 98 countries and its cutaneous form presents an incidence of 0.7 to 1.2 million new cases per year  (in deg cm2 dmol-1) .,47.L. amL. amazonensis at stationary growth phase at a proportion of 10 promastigotes: 1 macrophage and incubated for 8 h at 37°C in an atmosphere of 5% CO2. Non-internalized promastigotes were removed by PBS washing, when the infected culture was treated with different concentrations of peptides or the positive control amphotericin B (0.075 to 10 μmol L-1), dissolved in fresh complete RPMI medium and incubated, as above described, for 24 h. After incubation, the cells were methanol fixed and followed by Giemsa staining. The number of intracellular amastigotes was counted under a microscope. At least 200 macrophages were counted in each of the three slides and the determination of the ratio between infected and uninfected macrophages, enabling the determination of the IC50-AMA in relation to drug-free control [50-AMA) in BioEstat software was determined by nonlinear regression, the data was expressed as mean ± SD of two independent replicates, and the obtained results converted to the infection index values, which were obtained as the ratio between the percentage of infected macrophages and the average number of amastigotes per macrophage.Adherent macrophages were infected with 2-air mixture. The viability of the cells was then analyzed by the colorimetric MTT -2,5-diphenyl-2H-tetrazolium bromide) assay as previously described [Murine macrophages were incubated for 6 h in 96-well flat-bottom plates (TPP) for cell adhesion in complete RPMI. Peptides were diluted in complete RPMI and incubated for 24 h, 37 ºC, in 5% COescribed . Because50 in μM and determined by non-linear regression using Bioestat® software. Additionally, the Selective Index (SI = CC50macrophages/IC50leishmania) was determined, indicating the selectivity of the sample to the parasite.The peptide concentration corresponding to 50% cells growth inhibition was expressed as CC®PCS200-011TM) obtained from the cell bank of Rio de Janeiro (BCRJ), the cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% FBS and antibiotics . Cultures were maintained in an atmosphere of 5% CO2 at 37° C, until the cells reached the cellular density of (5x105 cells mL-1) [4 cells per well), followed by 24 h incubation for complete cell adhesion. Afterwards, the culture medium was changed to a fresh one and the cells were treated with different concentrations of compounds: for RP1 varying from 231 μmol L-1 to 0.72 μmol L-1; Fc-RP1 210 μmol L-1 to 0.65 μmol L-1; and Fc 2174 μmol L-1 to 6.78 μmol L-1. The plates were further incubated for 24 h and the cytotoxicity performed by alarmable® (Thermo Fisher Scientific) cell viability assay [For assays with HaCaT  . To adjuty assay . After tty assay . Cell vity assay , 50. TheOreochromis niloticus were collected, the plasma separated by centrifugation at 350 x g for 10 min at 28°C, and washed three times with PBS. Different concentration of the peptides was incubated with 4% (v/v) of RBC for two hours at 28°C under agitation (150 rpm) and the absorbance of supernatants was measured at 560 nm. As a positive control, a 1% Triton X-100 solution was used to obtain 100% hemolysis. The negative control was performed using 0.15 mol L-1 PBS pH 7.2. The selectivity index was calculated based on the ratio between HA/MIC [Fresh red blood cells (RBC) from the Nile tilapia n HA/MIC .Large unilamellar vesicles (LUVs) composed by POPC  or by POPC and POPS  (9:1) were prepared according to the method described in Lorenzón, EN et al 2014.-1 and 50 μmol L-1, Fc-RP1 at 0.1 μmol L-1, 1 μmol L-1 and 10 μmol L-1 and ferrocene carboxylic acid at 0.7 mmol L-1. The data were acquired using a Fluorolog-3 FL3-122 spectrofluorometer equipment (Horiba Jobin Yvon) and all experiments were performed in duplicate at 25°C.The release rate of CF from vesicles was measured by the fluorescence intensity with wavelength set at 495 nm excitation and 517 nm emission after the addition of the peptides RP1 at 10 μmol LThe peptide RP1  was syntIn order to obtain the RP1 coupled to ferrocene, the bioinorganic compound was coupled to the N-terminus of the peptide using three-fold excess of ferrocene carboxylic acid and HCTU, and six- fold excess of DIPEA for 4 h. The Fc-RP1 conjugated derivative was successfully obtained as demonstrated in . The pre-1, which is below the critical micellar concentration (CMC) [-1 of LPC, above the CMC, the peptide increased the content of α-helixes structures [222) and 208 nm (θ208) lead to a θ222/θ208 ratio less than one, suggesting absence of aggregation among the peptide chains [For PBS solution, RP1 and Fc-RP1 peptides presented disordered structures . In the on (CMC) , RP1 preon (CMC) , 55. In ructures , showingructures , 57. Thee chains , 59.Micelles are not real mimetics of membranes. Thus, in order to discard any influence of the high curvature of these structures, the peptides were evaluated in unilamellar vesicles, mimicking both microorganisms/human eukaryotic (POPC–zwuitterionic lipid) or fungal/parasitic (POPC:POPS–negative charge) cell membranes , 61. TheL. amazonensis. The results indicate that Fc-RP1 (IC50 = 0.25 μmol L –1) is more potent than both RP1 (IC50 = 1.25 μmol L–1) and ferrocene carboxylic acid (IC50 = 4.4 μmol L–1). The activity of the compounds on non-infected macrophages was also evaluated. The CC50 showed high values for all compounds as compared with IC50. In this experiment the Fc-RP1 conjugate was the more active one. Although the conjugate presents a higher activity against amastigotes, its selective index was 70, a small difference in relation to the peptide RP1 (SI = 80). In this case, the peptide was more selective to the amastigotes rather than Fc-RP1 , indicating that ferrocene improved the antimicrobial activity against the Gram-positive bacteria (A. hydrophila (Gram-negative bacteria)  .S. aureus and E. faecalis (Gram-positive) and E. coli (Gram-negative), RP1 peptide was able to completely inhibit the growth of both E. fecalis and E. coli (MIC = 8.7 μmol L-1). The peptide also showed activity against S. aureus (MIC = 69.4 μmol L-1) , E. coli (MIC = 3.9 μmol L-1) and S. aureus  , but in S. galactiae, i.e., 4.30 μmol L-1 for RP1 and 0.96 μmol L-1 for Fc-RP1. The peptide and the conjugated also did not present any cytotoxicity at the highest concentration tested (220 μmol. L-1)  of the RP1 and Fc-RP1 on fish RBCs, both compounds caused less than 10% of hemolysis when exposed to a concentration corresponding to the MIC obtained against -1) Figs  against -1 cause the release of 10% of CF from POPC, while 1 μmol L-1 of Fc-RP1was sufficient to release 70% POPC content  release from LUVs were investigated using POPC and POPC: POPS (9:1) vesicles containing this fluorescent marker. RP1 at 50 μmol L content . Similartent . Previous studies showed that compounds conjugated with organometallics, like ferrocene, has great biological potential , 70–73.Leishmania amazonensis . However, the Fc-RP1 conjugated showed higher toxicity (17.3 μmol L –1) when in contact with macrophage as compared with the same tests for RP1 peptide and ferrocene carboxylic acid molecule (>100 μmol L-1). It is well-known that bioconjugates containing organometallic compounds such as the ferrocene have great biological potential [The RP1 peptide and Fc-RP1 presented anti-amastigote activity in otential , 71, 73.otential . The phyotential , may expotential , 70, 75.Compounds containing Fc have shown anti-proliferative activity on hormone-dependent breast cancer cell line MCF-7 , corrobo50 = 0.63 ± 0.72 μmol L-1), the Fc-RP1 was more active, also showing a 2.5-fold higher selectivity index. However, the treatment with amphotericin B presents serious side effects. It is done intravenously and can cause toxicity issues. The cost of treatment using amphotericin B is another obstacle. These problems make that the mortality caused by leishmaniasis continues to rise, requiring more selective and more affordable treatments [We have also compared the results of the conjugated peptide Fc-RP1 against amphotericin B, which is the second-line drug approved for leishmaniasis treatment. In comparison with amphotericin B . The bacteria species S. agalacitiae and A. hydrophila are pathogenic for the fish Nile tilapia (Oreochromis niloticus), causing a foodborne disease in humans [S. aureus is a Gram-positive bacterium, considered an opportunistic microorganism and is one of the major causative agents of community and hospital infections worldwide [E. coli is a Gram-negative bacterium found in the intestinal tract, which is also known to cause infectious endocarditis, bacteremia, sepsis and urinary tract infections [E. faecalis. Additionaly to these diseases, this microorganism is also associated with endodontic infections [In order to evaluate the application of the compounds against bacteria, the antibacterial potential was tested in human pathogenic bacteria: n humans , 82. Oftorldwide  Several orldwide . Meanwhifections , 85, 86.fections –89.-1 against S. agalactiae, whilst Fc-RP1 was four times more active (MIC = 0.96 μmol L-1), indicating that ferrocene improved the activity against Gram-positive bacteria. The satisfactory activity of the peptide and conjugate on Gram-positive bacteria can be explained by the presence of teichoic acids in their cell membrane, which confers a negative net charge on the bacterial surface, enabling the interaction [The antibacterial activity of the peptide in Gram-positive strains can be seen in eraction . Furthereraction . It is weraction . AnotherE. faecalis (MIC = 7.9 μmol L-1), E. coli (MIC = 3.9 μmol L-1), and S. aureus (MIC = 3.9 μmol L-1), as can be seen in Figs -1) and Fc-RP1 (MIC = 7.9 μmol L-1) remained similar when tested against E. faecalis strain, showing that the optimized antimicrobial effect of the Fc-RP1 peptides on the bacteria may be dependent on the evaluated strain. The data also shows that the conjugation of ferrocene to RP1 peptide is an interesting strategy for increasing the bioactivity of the AMP peptides [The coupling of ferrocene in RP1 also decreases the minimum inhibitory concentration (MIC) in the assays against peptides –95.E. coli and not against A. hydrophila could be explained by the interaction of the peptide with other cell wall components. Lorenzon et al. (2013) showed that the dimerized AMP aurein promoted the aggregation of Candida albicans via interactions with mannans found in the cell wall. Moreover, the changes in the activity against these strains can be attributed to the capacity of Gram-positive bacteria to modify the composition of their cell membrane [Additionally, the activity of peptides against membrane . This moThe cytotoxicity of the compounds was tested against HaCaT cells , helpingIn order to clarify a possible mechanism of action of the Fc-RP1 peptide, the permeabilization assays were performed. The data shows that the coupling of ferrocene to the RP1 peptide significantly increases the permeabilization of the LUVs. In this study, the ferrocene coupled to the peptide improved the lysis of the bacterial mimetic membrane, in accordance with the antibacterial activity obtained. Besides, several studies have shown that ferrocene destabilizes cell membranes through lipid peroxidation, leading to cell lysis. In order to confirm this hypothesis, vesicle permeabilization assays were carried out in the presence of 0.7 mmol L-1 ferrocene carboxylic acid, which corresponds to a concentration that is seventy times higher than the one used when testing the permeabilization potential of Fc-RP1 . The ferIt is noteworthy that even with the Fc-RP1 peptide being a prototype of a bioconjugation strategy; it still is a compound with great biological activity against neglected tropical diseases and fish diseases.E. coli, S. aureus and, E. faecalis, as well as, the activity against a bacterial fish pathogen, S. agalactiae. At the same time, the conjugate peptide preserved its low cytotoxicity against HaCaT and fish blood cells. In overall, the obtained data reveals the potential of the Fc-RP1 peptide to be used in antimicrobial therapy, including a possible usage in pisciculture.The solid phase peptide synthesis protocol enabled the development of the conjugate, Fc-RP1, composed of an antimicrobial peptide and the organic molecule ferrocene. The comparison between the AMP and the conjugated Fc-RP1 showed that the presence of the organic molecule increased the antimicrobial activity against Purity analyzes of the RP1 and Fc-RP1 peptides and their mass spectra are found in the supplemental material  Figs.S1 Fig-1. (c) Peptide mass spectrum profile. The peaks of 721.80; 541.36 and 433.46 represent the mass-to-charge ratio of the peptide RP1 with charge of +3, +4 and +5, respectively.(a) HPLC profile of the crude (a) and purified (b) peptide, with retention time at 10.5 min Analytical HPLC was performed on Shimadzu spectrometer, with C18 reverse phase Ultraspehere Phenomenex column , detection at 220 nm and gradient method of 5 to 95% solvent B in 30 min with flow rate of 1 mL min(TIF)Click here for additional data file.S2 Fig-1 flow. A peak with retention time at 10.5 min indicates the presence of the uncoupled RP1 peptide. (b) HPLC profile of the pure peptide Fc-RP1. (c) Peptides mass spectrum profile. The peaks of 792.46; 594.61 and 475.86 represent the mass-to-charge ratio of the conjugated with charge of +3, +4 and +5, respectively.(a) HPLC profile of the crude Fc-RP1 peptide (Retention time: 14 min) in analytical mode using Shimadzu spectrometer with C18 reverse phase Ultraspehere Phenomenex column , detection at 220 nm, using a gradient method of 5 to 95% solvent B in 30 min with 1 mL minThe (TIF)Click here for additional data file.S3 FigIn (a) the compounds were incubated in neutral pH solution  and in (b) were incubated in acid medium (Water + 0.045% Trifluoroacetic Acid), both images represent the percentage of remaining compounds in different degradation intervals.A. hydrophila (Gram-negative bacteria). It is worth to mention that the ferrocene carboxylic acid did not cause any effect on bacteria growth (a growth .(TIF)Click here for additional data file.S4 FigPeptides diluted in ultrapure water and positive control (bacterium and Mueller Hinton broth). Mean (n = 3) with respective standard deviation. Different letters indicate that the values differ by the Tukey method (p <0.05).(TIF)Click here for additional data file.S5 FigAntibacterial assays with molecule Fc (a) E. faecalis, (b) S. aureus, and (c) E. coli. The methodology followed the guidelines of the Clinical and Laboratory Standers Institute . Three independent experiments were performed, and the data were analyzed by applying the one-way ANOVA with Tukey's post hoc test using GraphPad Prism Version 5.01 software . The maximum coefficient of variation accepted was 25%, the confidence level was 95% (p <0.05) and ***p<0.001, *p<0.05; **p<0.01; ns: not significant.(TIF)Click here for additional data file.


We are pleased to present a special issue of Data Science and Engineering (DSE), which contains a collection of papers from the DASFAA 2020 conference. Due to publication schedule, we publish four of these papers here, and postpone the publication of three additional ones to the coming DSE issues. In this issue, we also include three other papers selected from regularly submitted papers.The International Conference on Database Systems for Advanced Applications (DASFAA) provides a leading international forum for discussing the latest research on database systems and advanced applications. The conference’s long history has established the event as the premier research conference in the database area. DASFAA 2020 focuses on research, development, and applications in relation to database, including a wide range of topics, such as neural network, knowledge graph, time series, social networks, attention mechanism, graph mining and crowdsourcing. DASFAA 2020 was held during September 24–27, 2020, in Jeju, Korea. The conference was originally scheduled for May 21–24, 2020, but postponed due to the outbreak of COVID-19 and its continual spreading all over the world. DASFAA 2020 attracted a total of 487 research paper submissions. The conference program committee selected 119 full research papers (acceptance ratio of 24.4%) and 23 short papers to be presented at the conference and published in the proceedings [Seven extended papers were selected from among all the accepted papers by the special issue guest editors Yingxia Shao, Yanyan Shen, Bin Cui, and Jeffrey Xu Yu, based on the relevance to the journal and the reviews of the conference version of the papers. The authors were asked to revise the conference paper for journal publication and in accordance with customary practice of adding 30% new materials. The revised papers again went through the review process in accordance with DSE guidelines and are finally presented to the readers in the present form.The seven extended papers cover a variety of topics related to database, data science and engineering. This issue includes four papers. The first “Exploiting Latent Semantic Subspaces to Derive Associations for Specific Pharmaceutical Semantics” presented an approach to extract interpretable latent semantic subspace from the neural-embedding models in the biomedical domain. The second paper “AGTR: Adversarial Generation of Target Review for Rating Prediction” introduced a recommendation model that can generate the unseen reviews for target users and items with adversarial training for rating prediction. The third paper “Show me the crowds! Revealing Cluster Structures through AMTICS” proposed a novel and efficient visualization approach to interactively and fast mine coarse insights in very dynamic and rapid changing applications. The fourth paper “Consensus-based Group Task Assignment with Social Impact in Spatial Crowdsourcing” proposed a novel framework for group task assignment based on two preference-related techniques—social impact-based preference modeling and preference-aware group task Assignment.In upcoming issues of DSE, three additional papers selected from DASFAA 2020 will be published. The first “Parrot: A Progressive Analysis System on Large Text Collections” presented a sample-based progressive query processing model that is based on an incremental execution engine for large text collections. The second paper “Blocking techniques for Entity Linkage: a semantics-based approach” presented two automatic blocking strategies that capture the semantic properties of data by means of recent Deep Learning frameworks. The last paper “Heterogeneous CPU-GPU Epsilon Grid Joins: Static and Dynamic Work Partitioning Strategies” presented a heterogeneous CPU-GPU distance similarity join algorithm via efficiently partitioning the work between CPU and GPU.We hope that the readers enjoy this special issue. We would like to acknowledge the work done by all the authors and their willingness to contribute their papers to this special issue. We thank all the reviewers for their expert comments and assistance in timely reviews. Finally, a note of thanks is to DSE Editors-in-Chief X. Sean Wang and Timos Sellis for their guidance and support in this process.


Conclusions: Compared to healthy and age-matched controls, adult individuals with OSAS had significantly higher serum/plasma TNF-α levels. For children with OSAS, significantly higher levels were observed for TNF-α in serum but not in plasma.Background: Obstructive sleep apnea syndrome (OSAS) is associated with a variety of inflammatory factors. Specifically, proinflammatory cytokines appear to be associated with the pathogenesis of OSAS. Methods: For the present meta-analysis and meta-regression on serum and plasma tumor necrosis factor alpha (TNF-α) levels in individuals with and without OSAS, we performed a systematic search without any restrictions of the PubMed/Medline, Scopus, Cochrane Library, and Web of Science databases to find relevant articles published up to 1 February 2020. Results: Fifty-five  were included and analyzed. Always compared to age-matched healthy controls, the pooled MDs were as follows: adults, serum: 10.22 pg/mL (95% CI = 8.86, 11.58;  Obstructive sleep apnea syndrome (OSAS) is a common sleep-related breathing disorder. It is estimated that 4% of both female and male adults suffer from OSAS . SpecifiPeople with objectively assessed OSAS are at increased risk to suffer from atherogenesis , vasculaAs regards the immune system, the tumor necrosis factor alpha (TNF-α) is a common cytokine secreted by adipocytes and the mononuclear-macrophage system , which rThe meta-analysis was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .One of the authors (M. Sadeghi) systematically searched four databases, namely PubMed/Medline, Scopus, Cochrane Library, and Web of Science, for articles published in English up to 1 February 2020, with no restrictions, and with the following expressions: “sleep apnea” or “OSA” or “obstructive sleep apnea” or “obstructive sleep apnea syndrome” or “OSAS“, with “TNF-alpha” or “TNF-α” or “TNF” or “tumor necrosis factor alpha”; and with “blood” or “plasma” or “serum”. We also manually inspected the reference lists of the articles  for publications related to our topic.Inclusion criteria were as follows: (1) studies evaluating the association between plasma or serum TNF-α levels and OSAS with case-control design without age (≥18 years as adults and <18 years as children), sex or BMI restrictions; (2) OSAS was defined as AHI > 5 events/h diagnosed by polysomnography; (3) participants with OSAS and controls had no other systematic diseases such as diabetes, asthma, neurological disorders such as multiple sclerosis, neurodegenerative disorders such as Alzheimer’s disease, or oral diseases; (4) studies reporting pretreatment morning serum and/or plasma levels of TNF-α (around 6:00–10:00 a.m.); (5) studies reporting sufficient data to compute the MD and 95% confidence interval (CI) in participants with OSAS and controls; (6) studies with more than 10 cases included as individuals with OSAS and controls.Exclusion criteria were: (1) studies with irrelevant or insufficient data or without clinical data; (2) Letter to the editors, review articles, conference papers, meeting abstracts, and book chapters; (3) studies without a control group; (4) studies reporting controls with AHI > 5 events/h; (5) studies with overlapped data with other studies; (6) studies without reporting cut-off AHI values for OSAS; (7) studies mixing pediatric and adult participants.Two authors (M.M.I. and M.E.) read independently the titles and abstracts of the retrieved studies. They then selected the relevant studies, while another author (M.S.) retrieved the full-texts of the articles and excluded several full-texts based on the exclusion criteria mentioned above. If two studies had overlapped data, we selected the study with the most recent publication year.Two authors (S.B. and M.S.) independently extracted the data from each study included in the meta-analysis. If there was a disagreement between the two authors, the third author (M.M.I.) helped to find a final decision. The data extracted for the meta-analysis included basic information including the first author, publication year, country of study, ethnicity of subjects in each study, age, BMI, and AHI of both groups, and mean and standard deviation (SD) of TNF-α levels in plasma and serum. Since some studies considered patients with mild OSAS (AHI: 1–5 events/h) as a control group condition, and since we had to use this protocol for other studies, reporting the quality of studies as for usual case-control studies did not seem to be reasonable.One author (M.S) evaluated the quality of the studies included in the meta-analysis using the Newcastle-Ottawa Scale (NOS) with a total score of nine for each study .p-value < 0.1 and I2 > 50% showed a statistically significant heterogeneity, the analysis was performed by the random-effect model to evaluate the pooled ORs and CI values. Otherwise, we used the fixed-effect model. The weight (%) represents the effect of an individual study on the pooled results obtained. In general, the larger the sample size and the narrower CI are, the higher is the percentage of weight.The data were analyzed by one author. Review Manager 5.3 software  was used to calculate the crude MD and 95% CI, which evaluated the significance of the pooled MD by Z-test. Heterogeneity across the studies was evaluated using both Cochran Q  and I2 mThe results of the Begg’s and Egger’s tests were analyzed by the Comprehensive Meta-Analysis version 2.0 software (CMA 2.0). The Begg’s funnel plot shows the standard error (SE) of the log (MD), and the precision of each study is plotted against its log (MD) . In addip-value (two-tailed) was less than 0.05, then there was a statistically significant difference. The meta-regression is a quantitative method used in meta-analyses to estimate the impact of moderator variables on study effect size. The trim-and-fill method was used to estimate potentially missing studies due to publication bias in the funnel plot and adjusting the overall effect estimate .Four studies compared serum TNF-α concentrations between children with OSAS and without OSAS  and founp = 0.35; I2 = 61% [Ph = 0.04]).Five studies compared serum TNF-α concentrations between children with OSAS and without OSAS  and founp < 0.00001), in Caucasians , and in mixed ethnicities  were significantly higher, compared to healthy controls. Serum TNF-α concentrations in Asians were 16.9 and 86.3 times higher than serum TNF-α concentrations in Caucasian and mixed ethnicities with OSAS, and always compared to controls.Subgroup analysis of serum TNF-α concentrations in adult samples are reported in p < 0.00001), in Caucasians ), and in mixed ethnicities  were significantly higher, compared to healthy controls. Next, in Asians, plasma TNF-α concentrations were 4 and 12.2 times higher than in Caucasian and mixed ethnicities with OSAS, and always compared to controls.For plasma TNF-α concentrations, for those adults with OSAS, plasma TNF-α concentrations in Asians . For adult participants with OSAS and a BMI ≤ 30 km/m2, the pooled MD was 23.07 pg/mL . Thus, results showed that serum TNF-α concentrations were 18.2 times higher participants with OSAS and a BMI > 30 kg/m2, when compared to those participants with a mean BMI ≤30 kg/m2.For serum TNF-α concentrations, and compared to healthy controls, adult participants with OSAS and a BMI > 30 km/m2 showed a pooled MD of 1.06 pg/mL . For adult participants with OSAS and a BMI ≤ 30 km/m2, the pooled MD was 10.82 pg/mL . Thus, results showed that plasma TNF-α concentrations were 10.2 times higher in adults with OSAS and a BMI > 30 kg/m2 than plasma TNF-α concentrations of adults with OSAS and a BMI ≤ 30 kg/m2 and always compared to healthy controls.For plasma TNF-α concentrations, and compared to healthy controls, adult participants with OSAS and a BMI > 30 km/m2 showed a pooled MD of 2.28 pg/mL ; while compared to controls, individuals with OSAS and with a BMI ≤ 30 kg/m2 showed a pooled MD of 18.64 pg/mL . Thus, serum TNF-α levels were 8.2 times higher in studies with participants with OSAS and a BMI >30 kg/m2 than serum TNF-α levels with participants with OSAS and a BMI ≤ 30 kg/m2.For serum TNF-α levels, compared to controls, individuals with OSAS and with a BMI > 30 kg/mp < 0.00001) for participants with OSAS and a BMI > 30 kg/m2; for participants with OSAS and a BMI ≤ 30 kg/m2, the pooled MD was not statistically significant .For plasma TNF-α levels, compared to controls, the pooled MD was 7.32 pg/mL  for studies with more than 100 cases, and 2.15 pg/mL  for studies with less than 100 cases. Thus, serum TNF-α levels were 25.8 times higher in the studies with more 100 cases, compared to studies with less than 100 cases.For serum TNF-α levels, the pooled MD was 55.47 pg/mL  for studies with more than 100 cases, while the pooled MD was 2.87 pg/mL  for studies with less than 100 cases. Thus, plasma TNF-α levels were 2.4 times higher in the studies with more than 100 cases, compared to studies with less than 100 cases.For plasma TNF-α levels, the pooled MD was 6.91 pg/mL , while the pooled MD was 127.01 pg/mL  in individuals with OSAS and a AHI ≤ 30 events/h. Thus, serum TNF-α levels were 22.2 times higher, when participants with OSAS had AHI ≤ 30 events/h, compared to AHI > 30 events/h.For serum TNF-α levels, in individuals with OSAS and a AHI > 30 events/h, the pooled MD was 5.72 pg/mL , while in individuals with OSAS and AHI ≤ 30 events/h, the pooled MD was 2.97 pg/mL . Thus, serum TNF-α levels were 2.7 times higher in participants with OSAS and AHI > 30 events/h, compared to serum TNF-α levels in participants with OSAS and AHI ≤ 30 events/h.For plasma TNF-α levels, in individuals with OSAS and AHI > 30 events/h, the pooled MD was 8.15 pg/mL , whereas for studies with participants with a BMI ≤ 30 kg/m2 serum TNF-α levels did not statistically differ between participants with or without OSAS , whereas no significant MDs were observed in studies including Caucasian participants with OSAS and a BMI > 30 kg/m2.For plasma TNF-α levels, studies with Caucasian participants with OSAS and a BMI ≤ 30 kg/m2, the pooled MD was 2.58 pg/mL . There was no statistically significant MD for serum TNF-α levels between Caucasians with or without OSAS and a mean ≤ 30 kg/m2.For studies with serum TNF-α levels and Caucasian controls with a mean BMI > 30 kg/m2, the pooled MD was 0.56 pg/mL . Studies with controls with a BMI ≤ 30 kg/m2 showed a pooled MD of 5.64 pg/mL . Thus, plasma TNF-α levels were 10.1 times higher in controls with a BMI ≤ 30 kg/m2, compared to plasma TNF-α levels of controls with a BMI > 30 kg/m2For studies with plasma TNF-α levels and controls with a BMI > 30 kg/mp = 0.0006), whereas there was no significant difference for studies with >100 cases.Serum TNF-α levels had a significant difference in studies with ≤ 100 cases , and plasma TNF-α levels were 7.80 pg/mL .In studies with individuals with OSAS and AHI of > 30 events/h, serum TNF-α levels were 1.40 pg/mL  and plasma  TNF-α levels differed statistically significantly  for studies including OSAS patients with a mean BMI of ≤30 kg/m2, and plasma TNF-α levels were 14.92 pg/mL  for studies including OSAS patients with a mean BMI of ≤30 kg/m2.For serum and plasma TNF-α levels, there were no studies including controls with a BMI > 30 kg/mp < 0.00001) for studies with 100 or more participants, and the pooled MD was 7.80 pg/mL  for studies less than 100 participants. Thus, serum TNF-α levels were 11.5 times higher in studies with more than 100 participants, compared to studies with less than 100 participants.For serum TNF-α levels, the pooled MD was 89.88 pg/mL  for studies with 100 or more participants, and the pooled MD was 21.51 pg/mL  for studies less than 100 participants. Thus, plasma TNF-α levels were 1.7 times higher in studies with less than 100 participants, compared to studies with more than 100 participants.For plasma TNF-α levels, the pooled MD was 12.34 pg/mL ; plasma TNF-α levels were 12.48 pg/mL .For studies including AHI > 30 events/h, mean serum TNF-α levels were 12.94 , compared to studies with controls with BMI of controls ≤ 30 kg/m2 (For studies with serum TNF-α levels and controls with BMI of controls >30 kg/m30 kg/m2 .p < 0.0001) and for plasma TNF-α levels , compared to studies with more than 100 participants.For studies with 100 or less participants, there was a significant difference for serum TNF-α levels ; the pooled MD for studies with participants with OSAS with AHI ≤ 30 events/h was 42.13 pg/mL . Thus, serum TNF-α levels were 42.9 times higher in studies with participants with OSAS and AHI ≤ 30 events/h, compared to the serum TNF-α levels in studies including participants with OSAS and AHI > 30 events/h.For serum TNF-α levels, the pooled MD for studies including participants with OSAS with AHI > 30 events/h was 0.96 pg/mL . When studies included participants with OSAS and an AHI ≤ 30 events/h, MDs between studies with participants with or without OSAS were not significant.For plasma TNF-α levels, the pooled MD for studies with participants with OSAS with AHI > 30 events/h was 1.69 pg/mL  for serum, and MD = 10.82 pg/mL  for plasma (For studies including BMI ≤ 30 kg/mr plasma .p < 0.00001), while in studies with ≤ 100 participants, the pooled MD was 4.42 pg/mL . Thus, serum TNF-α levels were 14.4 times higher in studies with more than 100 participants, compared to serum TNF-α levels in studies with ≤100 participants.For serum TNF-α levels and in studies with more than 100 participants, the pooled MD was 63.51 pg/mL  while in studies with ≤100 participants, the pooled MD 13.79 pg/mL . Thus, plasma TNF-α levels were 1.7 times higher in studies with more than 100 participants, compared to plasma TNF-α levels in studies with ≤100 participants.For plasma TNF-α levels and in studies with more than 100 participants, the pooled MD was 8.12 pg/mL ; the pooled MD for studies including participants with OSAS and an AHI ≤ 30 events/h was 173.00 pg/mL . Thus, serum TNF-α levels were 13.4 times higher in participants with OSAS and AHI ≤ 30 events/h, compared to the serum TNF-α levels in participants with OSAS and AHI > 30 events/h.For serum TNF-α levels, the pooled MD for studies including participants with OSAS and an AHI > 30 events/h was 12.94 pg/mL . When studies included participants with OSAS had an AHI ≤ 30 events/h, MDs between studies including participants with or without OSAS were not significant.For plasma TNF-α levels, the pooled MD for studies including participants with OSAS and an AHI > 30 events/h was 16.13 pg/mL  and plasma  TNF-α levels  revealed a publication bias. For plasma TNF-α levels, Begg’s test (p = 0.03943) revealed a publication bias, but Egger’s test (p = 0.19315) did not show any bias between and across the studies.For serum levels, Egger’s and Begg’s tests ; using the trim-fill method, the imputed point estimate was 0.323 . In addition, under the random-effects model, the point estimate and 95% CI for the combined studies was 8.245 ; using the trim-fill method, the imputed point estimate was 0.523 .For plasma TNF-α levels and 3 imputed studies, under the fixed-effects model, the point estimate and 95% CI for the combined studies was 2.463 , and using trim-fill method, the imputed point estimate was 2.294 . In addition, under the random-effects model, the point estimate and pseudo 95% CI for the combined studies was 5.452 ; using the trim-fill method, the imputed point estimate was 2.668 .The overall effect sizes on serum and plasma TNF-α levels reported in the forest plot appeared valid, with trivial publication bias effect based on fixed-effects model, because the observed estimates were similar to the adjusted estimates. In contrast, the overall effect sizes on serum and plasma TNF-α levels reported in the forest plot appeared invalid, with significant publication bias effect based on random-effects model, because the observed estimates had high difference with the adjusted estimates.The present meta-analysis with 55 studies evaluated the serum TNF-α levels (29 studies of adults and 4 studies of children) and plasma TNF-α levels (17 studies of adults and 5 studies of children) in individuals with OSAS, compared to controls. The results showed that plasma and serum TNF-α levels in adult individuals with OSAS were significantly higher than the corresponding levels of control. For children with OSAS serum TNF-α levels, but not plasma TNF-α levels, were statistically significantly higher than those of controls. The present results have clinical importance because elevated serum and plasma TNF-α levels can be a risk factor for the development of further systemic diseases in adults with OSAS. Further, more generally, inflammation may play a crucial role in the pathogenesis of OSAS, and there is evidence that individuals with OSAS have elevated interleukin TNF-α ,48,62,78Elevated TNF-α levels have been associated with the pathophysiology of reoxygenation injury, myocarditis, cardiac allograft vasculopathy, heart failure progression , arthritThe present pattern of results also sheds some light on further new findings: More specifically, for adults, it turned out that compared to healthy controls, TNF-α levels were higher both in blood serum and blood plasma. It follows that at least for adults with OSAS, assessing either blood plasma or blood serum does not appear to be of clinical importance. In contrast, for pediatric samples and compared to healthy controls, higher TNF-α levels were observed for blood serum but not for blood plasma. The quality of the data at hand does not allow a deeper understanding of the underlying physiological mechanisms. Thus, for want of such data, the following admittedly speculative assumptions are advanced. First, it is conceivable that for pediatric samples, the number of four studies on plasma TNF-α was too small, and therefore, the statistical variance of plasma TNF-α was blurred by sample size inconsistencies. Similarly, second, at least among adults, Hirotsu et al.  showed tThe pattern of results described in the present meta-analysis and systematic review consistently showed that higher TNF-α levels were associated with pathologically higher BMI scores as a proxy of obesity. Given that above all childhood overweight and childhood obesity is increasing worldwide and is a major concern of public health , the assThe results of one study  showed tp: 0.034) between higher BMI scores and higher TNF-α levels [Next, TNF-α appears to mediate both a somnogenic activity and fatigue related to excessive daytime somnolence in obese patients with OSAS ,28,30. Tα levels .As regards AHI and TNF-α levels, Kanbay et al.  and otheNext, Minoguchi et al.  reportedDespite the novelty of the results, the following limitations should be considered: (1) In all studies, results have not been adjusted for possible confounding factors such as obesity, smoking, or alcohol consumption. (2) The results of the funnel plots showed a publication bias across the studies; it follows that a systematic bias in the data presentation cannot be ruled out. (3) Studies with a small sample size (less than100 cases) had an inadequate power to detect possibly meaningful associations. (4) There was a high heterogeneity among studies in some analyses. (5) Studies reported different cut-off AHI values, which made comparisons between the studies difficult. (6) In some studies, TNF-α levels were considered as secondary outcome. (7) In some studies, the existence of mixed ethnicities might have blurred the associations between the ethnicity and TNF-α levels.By contrast, the strengths of the meta-analysis were as follows: (1) There were sufficient studies to allow the subgroup analyses. (2) The sensitivity analysis showed a stability of the results. (3) The studies written in other languages than English were included in the meta-analysis.The result of the meta-analysis and meta-regression confirmed that compared to healthy controls, individuals with OSAS (adults) had significant higher serum/plasma TNF-α levels, whereas for children with OSAS, this pattern of results was observed for serum TNF-α levels, but not for plasma TNF-α levels. Last, the results of the present meta-analysis showed that elevated TNF-α levels in individuals with OSAS appeared to be related to the severity of the disease. Future studies might investigate if and to what extent interventions on OSAS  favorably impact on TNF-α levels and possibly also on weight regulation.


ESR1 rs1643821 and TNF-α rs1800629 as potential genetic factors regulating anterior disc displacement without reduction-mediated inflammatory pathway.  The aim of this study was to investigate the role of  The temporomandibular joint is a complex synovial joint that allows mandibular movement in three directions. Although temporomandibular disorders are widespread, limited data is available on the biochemical characteristics of the displaced disc and quality of the surrounding soft tissue. Changes in degenerative tissue provoke disc displacement which involves secretion of inflammatory markers and sequential conversion of fibroblast-like cells into chondrocyte-like cells. Due to the high occurrence in female adolescents, the potential role of sex hormones in temporomandibular joint disorders has been speculated. Furthermore, anterior disc displacement without reduction severely affects the quality of life. ESR1) rs1643821, and tumor necrosis factor α (TNF-α) rs1800629 was performed.  124 Caucasian patients with a history of at least one anterior disc displacement without reduction within 3 months were enrolled. Anterior disc displacement without reduction was diagnosed based on clinical examination, diagnostic criteria (DC)/TMD, and cone-beam computed tomography/magnetic resonance imaging (CBCT/MRI). The control group consisted of 126 patients with no temporomandibular joint disorders. Genotyping of two single nucleotide polymorphisms, estrogen receptor 1  revealing the difference in anterior disc displacement without reduction frequencies while TNF-α rs1800629 polymorphism was found to be statistically insignificant when compared to the control group. Furthermore, patients with a genotype of ESR1 rs1643821 showed a decreased probability (OR = 0.412) against anterior disc displacement without reduction when compared to the GG genotype (OR = 1). ESR1 rs1643821 with A allele frequency was lower in patients with anterior disc displacement without reduction compared to the control group. Thus, the rs1643821 variant is significantly associated with susceptibility to the anterior disc displacement without a reduction in European Caucasians. Conversely, TNF-α rs1800629 was a statistically insignificant factor against anterior disc displacement without reduction when compared to the control group. It remains unclear and controversial whether it is the ADD or low serum estrogen expression that plays a significant role in ICR progression [α and ERβ) to mediate inflammation and central pain pathways [α (TNF-α) [β, and IL-6 have been detected in TMJ synovium during inflammation while IL-1 and TNF-α have been shown to inhibit proteoglycan synthesis and promote cartilage reabsorption as well as inflammation of joint structures and adjacent connective tissue [α, also known as NR3A1, is one of the two main types of estrogen-activated receptors. In humans, ERα is encoded by ESR1 and has been previously shown to be associated with distressed TMDs [α is located on chromosome 6:31,575,567-31,578,336. rs1800629 is a single nucleotide polymorphism (SNP) in the TNF-α, also known as TNFA-308. rs1800629(A) allele is often referred to as 308.2 or TNF2 while the conventional G allele is labelled as 308.1 or TNF1. Furthermore, A allele is associated with upregulation in TNF-α expression (ensemble database). On the other hand, TNF-β promotes cell apoptosis and inflammatory responses as it binds to TNF receptor type 1 and 2, respectively. TNF-β is produced by lymphocytes, and its structure resembles TNF-α. TNF-α and TNF-β have been shown to have similar structure and functions such as 30% amino acid sequence similarity and identical widely distributed cellular receptors [α polymorphism affects the production and expression of proinflammatory cytokines  in TMD patients. Thus, we hypothesized that ESR1 and TNF-α may be expressed in patients with ADDwoR due to disruption in TMJ disc and adjacent tissue homeostasis.Temporomandibular joint (TMJ) is a complex synovial joint that allows mandibular movement in three directions. The three components of TMJ that enable such complex motion include mandibular condyle, glenoid fossa of the temporal bone, and fibrocartilaginous articular disc that is surrounded by synovial fluid . The pregression . Subsequgression . Althougpathways . For exa (TNF-α) . Cytokine tissue . ERα, alsed TMDs . TNF-α ieceptors . ConsequESR1 rs1643821 and TNF-α rs1800629 as potential genetic factors regulating the ADDwoR-mediated inflammatory pathway. We focused on Caucasian patients as selected SNPs have not been investigated in European Caucasians for their role in TMD.In this study, we investigated the role of Pathological tooth mobility (grade 1 or more using Miller index)Have prior implemented occlusal splint therapyPresence of occlusal support only in limited areasCoexisting pathology or inflammation within jaws and muscles of the head and neckAny concomitant metabolic diseases or known connective tissue disordersIn this case-control study, we enrolled patients who were seeking treatment for TMD from 2014-2018 at the Department of Dental Prosthetics, Pomeranian Medical University, in Szczecin, Poland. Our study included 124 Caucasian patients from both the sexes. Patients with a history of ADDwoR within the last 3 months were included in the study. Prior informed formal consent was obtained from all patients enrolled in the study. ADDwoR was diagnosed based on clinical examination, diagnosed criteria for TMD (DC/TMD), and cone-beam computed tomography/magnetic resonance imaging (CBCT/MRI). The control group included 126 patients with no TMD based on DC/TMD analysis. Additional exclusion criteria for both the groups included the following:ESR1 rs1643821 and TNF-α rs1800629 as potential genetic factors mediating the signals of ADDwoR-induced inflammatory pathway. So far, the role of the selected SNPs has not been investigated in European Caucasians having TMD.In this study, we investigated the role of Genomic DNA was extracted from oral epithelial cells using the SWAB Genomic Extraction GPB Mini Kit  according to the manufacturer's instructions.ESR1 rs1643821 and TNF-α rs1800629, were analyzed using the predesigned Applied Biosystems TaqMan real-time PCR assays .Genotyping of selected SNPs was performed by real-time PCR using TaqMan probes. SNPs, The reaction mix for each sample consisted of GoTaq® Probe qPCR Master Mix , TaqMan real-time PCR assays , and nuclease-free, deionized water, according to the manufacturer's instructions.ESR1 and TNF-α are shown in Figures Reaction mix, DNA, and no template control (NTC) were pipetted into 384-well plates . Real-time PCR was performed on LightCycler® 480 . Genotyping data was analyzed using the LightCycler480 Basic Software Version 1.5 . Allelic discrimination plots with the results of TaqMan genotyping for U test was performed to determine the age difference between the groups. P < 0.05 was considered to be statistically significant. The calculations were made using MATLAB .A significant difference in the distribution of genotypes was analyzed using Pearson's chi-square test. The logistic regression modelling was performed to analyze the influence of the investigated SNPs on ADDwoR. The data is presented as allele frequencies and odds ratio (OR) with 95% confidence interval (CI). The Mann–Whitney No significant genotypic differences were found with respect to age or sex between groups. The complete demographic information and clinical parameters of the study population are illustrated in P value while comparing the genotypes of men and women that were enrolled in this case-control study. Thus, our study shows that gender has no influence on the frequency of TMJ ADDwoR.We did not find any significance based on the ESR1 rs1643821, revealed a statistically significant difference  in the frequency of developing TMJ ADDwoR. On the other hand, rs1800629 polymorphism in TNF-α was found to be statistically insignificant in comparison with the control group. Furthermore, patients with rs1643821 genotype AA showed a reduced probability (OR = 0.412) of developing TMJ ADDwoR in reference to genotype GG (OR = 1).On further analysis, polymorphism odds were evaluated with respect to most frequent allele combinations using 95% CI. The chi-square test at 5% confidence level was performed to determine the allele-based significant relationship between groups. The results of the OR analysis are shown in ESR1 rs1643821. None of the other statistical models showed any significance in the chi-square test vs. constant model. ESR1 rs1643821 and TNF-α rs1800629.Furthermore, the unconditional logistic regression analysis was performed to validate the findings. Consistent with our earlier data in the study, we did not find any additional significance in the experimental data, except for The data suggested that a variation in the rs1643821 allele combination is a significant factor that increases the probability of developing TMJ ADDwoR.TNFA-308 (rs1800629) and ESR1 rs1643821 in the pathogenesis of TMJ ADDwoR in European Caucasians. While we did not find significant changes in rs1800629 polymorphism, rs1643821 mutation with rare genotype AA was found to be lower in patients with ADDwoR compared to the control group. Furthermore, patients with rs1643821 genotype (AA+AG) showed a decreased probability (OR = 0.67) of developing ADDwoR compared to genotype GG allele carriers (OR = 1). Moreover, patients with genotype AA showed a decreased chance of developing TMJ ADDwoR. On the contrary to our findings, Furquim et al. have proved that TNFA-308 rs1800629 polymorphism is positively associated with TMD occurrence in general, while in their study, Brazilian patients with TMD had 2.87 times increased probability of having GA genotype compared to the controls.To the best of our knowledge, we are the first to investigate the role of β +252A/G variant was found to be significantly associated with susceptibility to TMD. However, they did not find a significant difference with respect to TNF-β +252A/G variant-related genotype (P = 0.010) or allele frequencies (P = 0.015) between the patient group and control group, respectively. A significant increase in TNF-β +252 AG genotype and G allele frequencies were observed in patients with TMD compared to healthy controls. The individuals with GG genotype and G allele were revealed to have an increased risk of developing TMD. Furthermore, a statistically significant association was observed when the patients were compared with the healthy controls based on the AA genotype vs. AG+GG genotypes . Additionally, the TNF-β +252A/G genotype distribution was shown to be associated with chewing problems (P = 0.046) [α is a proinflammatory cytokine that plays a vital role in the pathogenesis of joint osteoarthritis (OA) [β and TNF-α promote stiffness and impaired contractile function of articular chondrocytes [α-induced mRNA expression of inducible nitric oxide synthase (iNOS), IL-1β, and monocyte chemoattractant protein 1 (MCP-1) in fibroblast-like synoviocytes. Furthermore, iNOS has been described as calcium-insensitive which may be due to its tight noncovalent interaction with calmodulin (CaM) and Ca2+.Furthermore, using the pressure pain threshold (PPT) test, they showed that rare A-allele homozygotes have decreased pain sensitivity for TMJ and anterior fascicle of the temporal muscle compared to the ancestral allele homozygotes . Similar= 0.046) . TNF-α itis (OA) . A studyα-induced upregulation of the proinflammatory cytokines. Furthermore, histomorphometrically, the osteoclast number and receptor activator of nuclear factor kappa-Β ligand (RANKL) expression surrounding the subchondral bone in the tamoxifen-treated rats were significantly reduced in patients compared to the osteoclast number and RANKL expression in the control group. Remarkably, no significant difference in the expression of ERα and ERβ was detected in female and male synoviocytes by the real-time PCR and western blot analysis. These findings partly correlate with the data obtained in this study. However, there is a need for further investigation and confirmation in human trials [ESR1 rs1643821 with rare A-allele in patients with ADDwoR was found at a lower frequency compared to that in the control group. Furthermore, the variation is significantly associated with susceptibility to ADDwoR in European Caucasians. In addition, in this case-control study, TNF-α rs1800629 polymorphism was found to be statistically insignificant compared to the control group.Thus, tamoxifen (estrogen receptor antagonist) treatment can partially block TNF-n trials . This reESR1 rs1643821 variant with susceptibility to ADDwoR worldwide. Plus, the role of estrogen sensitization and its mutual relationship with the biomarkers of inflammation in TMD patients requires additional consideration in larger cohorts.Further insight into other ethnic populations is now essential to validate the association of ESR1 may be contributing to the pathogenesis of synovitis, and cartilage and bone degeneration in TMJ. This study is significant with regard to the pathogenesis of inflammatory joint diseases and provides a valuable reference for future investigations.To summarize,