Despite the importance of women living with HIV (WLHIV) engaging in fertility plan discussions with their healthcare providers (HCPs), little research exists. This study explored perceptions surrounding fertility plan discussions between WLHIV and their HCPs in western Ethiopia, from the perspectives of both women and providers.Thirty-one interviews (27 with WLHIV and 4 with HCPs) were conducted at four healthcare facilities in western Ethiopia in 2018. Data were transcribed verbatim and translated into English. Codes and themes were identified using inductive thematic analysis.There was a discordance between HCPs and WLHIV’s perception regarding the delivery of fertility plan discussions. Only nine of the 27 WLHIV reported they had discussed their personal fertility plans with their HCPs. When discussions did occur, safer conception and contraceptive use were the primary focus. Referrals to mother support groups, adherence counsellors as well as family planning clinics (where they can access reproductive counselling) facilitated fertility discussions. However, lack of initiating discussions by either HCPs or women, high client load and insufficient staffing, and a poor referral system were barriers to discussing fertility plans. Where discussions did occur, barriers to good quality interactions were: (a) lack of recognizing women’s fertility needs; (b) a lack of time and being overworked; (c) mismatched fertility desire among couples; (d) non-disclosure of HIV-positive status to a partner; (e) poor partner involvement; (f) fear of repercussions of disclosing fertility desires to a HCP; and (g) HCPs fear of seroconversion.Our findings highlight the need for policies and guidelines to support fertility plan discussions. Training of HCPs, provision of non-judgmental and client-centered fertility counselling, improving integration of services along with increased human resources are crucial to counselling provision. Enhancing partner involvement, and supporting and training mother support groups and adherence counsellors in providing fertility plan discussions are crucial to improving safer conception and effective contraceptive use, which helps in having healthy babies and reducing HIV transmission. This study explored perceptions surrounding fertility plan discussions between women living with HIV and healthcare providers in western Ethiopia, from the perspectives of both women and providers. Thirty-one interviews  were conducted at four healthcare facilities in western Ethiopia in 2018. There was a discordance between healthcare providers and HIV-positive women’s perception regarding the delivery of fertility plan discussions. Only nine of the 27 HIV-positive women reported they had discussed their personal fertility plans with their healthcare providers. When the discussions did occur, safer conception and contraceptive use were the primary focus. Referrals to mother support groups, adherence counsellors as well as family planning clinics facilitated fertility discussions. However, a lack of initiating discussions by either healthcare providers or women, high client load and insufficient staffing, and a poor referral system were barriers to discussing fertility plans. Where discussions did occur, barriers to good quality interactions were: (a) lack of recognizing women’s fertility needs; (b) a lack of time and overworked; (c) mismatched fertility desire among couples; (d) non-disclosure of HIV-positive status to a partner; (e) poor partner involvement; (f) fear of repercussions of disclosing fertility desires to a healthcare provider; and (g) healthcare provider’s fear of seroconversion. Training of healthcare providers, provision of non-judgmental and client-centered fertility counselling along with increased human resources are crucial to counselling provision. Enhancing partner involvement and supporting and training mother support groups and adherence counsellors in providing fertility plan discussions are crucial to improving safer conception and effective contraceptive use.Human immunodeficiency virus (HIV) remains one of the main public health problems globally, with 25.7 million people living with the disease in sub-Saharan Africa (SSA) in 2017. Women accounted for 56% of the HIV-positive population in SSA . The recPreconception care refers to pregnancy planning, reducing unplanned pregnancies, and optimizing maternal and child health prior to pregnancy. Importantly, the reproductive autonomy of WLHIV has received increasing recognition recently , despiteDiscussing reproductive plans with HCPs can be general, which refers to discussing general issues regarding pregnancy and HIV. Discussing reproductive plans with HCPs can also be personalized, which refers to discussing the client’s own fertility plan . A studyThis study employed a phenomenological approach to explore fertility plan discussions between WLHIV and their HCPs in western Ethiopia in August 2018. This framework allowed for the generation of new information about fertility plan discussions between WLHIV and their HCPs . The stuWomen for this study were identified during the main survey that was conducted prior to the qualitative interviews . On the Ethical approval was granted by the Human Research Ethics Committee (HREC) of The University of Newcastle, Australia (H-2017-0289), and the Oromia Regional State Health Bureau Research Ethics Committee, Ethiopia (BEFO/HBISH/1–16/257). All participants provided informed consent for the interview and for their interviews to be audio recorded. Further information regarding the ethical considerations for this project can be found in Feyissa et al. .Because of the sensitive nature of the information, two female nurses with master’s degrees were trained by the first author (TF) to conduct the interviews with the WLHIV. This was done to help participants feel more comfortable in discussing their fertility needs. Thus, female interviewers who had previous experience in HIV training conducted the face-to-face interviews with WLHIV after 5 days of training and pilot testing to reduce the possible impact of gender. Interviews with HCPs were conducted by the first author (TF), a PhD candidate with a background in nursing and postgraduate training in public health. None of the interviewers had ever worked at the selected health facilities. The motivation for this research arose from the desire to understand women’s fertility plan discussions with a focus on personalized fertility plan discussions. The researchers used open-ended questions to enrich the depth of information. The interviewers followed the direction of the participants to get a deep understanding of their fertility discussions. No one else was present during the interviews besides the participant and the researcher. All interviews were conducted in a private room in the HIV clinic in the local language, Oromo, using semi-structured guides. The guide for the interviews with the HCPs addressed perceptions regarding providing fertility plan discussions with WLHIV, what they discuss about women’s fertility plans, and what stops them from having or initiating such discussions. The interview guide for WLHIV contained questions regarding plans to have a child, discussions about fertility plans, pregnancy planning experiences and prevention of HIV to a child and/or a partner. All interviews were audio recorded. The interviews with HCPs took between 33 and 50 min (median 41 min), while the interviews with WLHIV took between 15 and 57 min (median 29 min).personal fertility plans with their HCPs (discussed/not discussed). HCPs were identified by code (HCP#1-HCP#4) to protect their privacy  for analysis. Thematic analysis was used as it is not wedded to a particular epistemology. Braun and Clarke’s (2006) thematic analysis principles  were appcy Table .Thirty-one interviews (27 with WLHIV and 4 with HCPs) were conducted in western Ethiopia. One HCP from each healthcare facility providing routine HIV-care at each HIV clinic  was interviewed. No women or HCPs declined to take part in the interviews. All WLHIV were using ART at the time of the interview. Participating WLHIV included five women who were pregnant, six who were breastfeeding and four who were attempting pregnancy as well as seven who were using contraception and three who were not using contraception. Nine women had three or more children. Five women desired one child in the future, while seven women desired two or more children in the future. The age of the WLHIV ranged from 18 to 38 years, with a mean of 28 years  Facilitators to discussing fertility plans presented contexts that enabled fertility plan discussions to occur; (c) Barriers to discussions highlighted factors that prevented fertility plan discussions from occurring; and (d) Barriers to good quality discussions identified the factors that hindered the quality of discussions.Four major themes were identified: (a) This theme contextualized the providers and women’s perceptions of discussing WLHIV’s fertility plans. Reflecting on fertility plan discussion, this theme also described the primary focus of fertility plan discussions.They [HCP] counselled me that I can have a child” . HCPs reported that there were more frequent fertility discussions compared to the women . Importantly, being free of opportunistic infections particularly not being at AIDS stage 3 or 4 were also mentioned as criteria to attempt pregnancy by a HCP . Meanwhile, another participant stated that timed condomless sex was after 10 days of menstrual bleeding through checking both viral load and CD4 count: “By checking CD4 [count] and what is called viral load, then, we [women] can have sex without condoms after 10 days of menstrual bleeding until fertilization of prepared eggs” . Although timed unprotected sex was one strategy for safer conception as noted by the women, HCPs reported that they do not advise this strategy. HCPs emphasized HIV transmission risks associated with timed condomless sex. However, HCPs reported that the final decision to attempt pregnancy through timed condomless sex was in the hands of HIV-positive people . This was confirmed by another woman who said: “I was taking Depo Provera. I discontinued [Depo] to have a child” .Some WLHIV reported their recent pregnancy was intended and they stopped using condoms to have a child. One participant explained: “Both of my children are checked and they are free” . Furthermore, a HCP explained that using ART for a longer period and high CD4 was associated with having a HIV-free child .Safer conception strategies for WLHIV who intended to have children focused on optimal CD4 count, viral load and timed unprotected sex. WLHIV who did not intend to have children need contraceptive discussions.Finally, the WLHIV wanted children and it was a priority that their newborn children are HIV free. This contextualized the demand for safer conception. A woman who did not discuss her personal plans said: ““They advise us on contraception. There is [are] contraception of 5 years, 3 years, 12 years, pills and injection types. Because it [injectable] can soften bone, the injection type of contraceptive method is not advised. We [women] are using the 3 years and 5 years types mostly. They [HCPs] also select which is suitable for us” .This sub-theme focused on contraceptive discussions, the roles HCPs played in contraceptive choice, availability of contraceptive methods and demand for contraceptive discussions. Both WLHIV and HCPs reported multiple contraceptive methods were available. Long-acting reversible contraception (LARC) and condoms were preferred methods and there seemed to be a lack of contraceptive decision-making autonomy for the women, with contraceptive choice dictated by the HCPs. According to WLHIV, contraceptive methods such as injectables were not recommended because it was thought that it ‘softens their bones’. A woman said: In contrast, the HCPs reported that there was shared decision-making regarding contraception and that they informed women of all available contraceptive types except for some that might interfere with HIV-drugs. In particular, HCPs did not recommend the use of oral contraceptives due to the possibility of drug-drug interaction with HIV-drugs and if adverse side effects were identified with their previous use Table , quote 8. However, another HCP reported a preference for hormone lacking contraception, such as Intrauterine Devices (IUD) and condoms .A lack of access to effective contraception was noted by HCPs with LARC only available at family planning clinics (not available at HIV-clinics) although these methods were recommended by HCPs  while another participant reported being hypertensive and was advised by a HCP to shift to tubal ligation.They advised me. It is risky to have additional children with this blood pressure [hypertension] and I decided to have ligation [tubal] … I did so [tubal ligation] .The importance of personalized client-provider discussions regarding the choice of contraception was reported by the women: “We discussed, but I said it is enough for me … I am using contraception that is for five years [implant]” . Some other participants expressed frustration regarding their current method of contraception and need for more effective methods. In particular, a woman expressed: “I am using the injectable which is for three months. Condoms may break” . However, there were women who were confident in using methods such as condoms: “There is no nagging; we use condoms” . The partner being unsupportive of using condoms was reported as a reason not to use condoms: “We rarely use condoms because he does not say ok . Gaps regarding availability and concerns with current contraceptive methods might have resulted in unintended pregnancies.According to the HCPs, it seemed that there was no restriction among long-acting contraception Table , quote 9Yes, it is without plan. I didn't want to get pregnant. I, myself, is like this [HIV-positive] … it was difficult to terminate after the pregnancy was big. Hence, I had this child .I know but I took and was taught to use condoms. However, one day I had sexual intercourse without a condom, then conceived .HCPs reported their frustration with the unintended pregnancies of their clients and counselled on the effectiveness of contraception  of WLHIV reported that they hadn’t discussed their personal fertility plans with their HCPs. According to the perspectives of both WLHIV and HCPs, lack of initiating discussions, high client load and insufficient staffing, and a poor referral system were barriers to fertility discussions.“They haven’t asked me. I haven’t told them either” . Women also reported that there was a delay in having discussions with their providers (though attending ART for more than a year), but the HCPs said they were going to talk to them: “… we are going to talk to a doctor on this issue since I don’t have knowledge about this” . Although the timeline to discuss their fertility plans was not clear, a woman reported readiness to accept the recommendations of HCPs: “If they say you can have, we will have an additional child” .This sub-theme identified that the majority of women reported there was no discussion regarding their personal fertility plans due to the lack of initiating conversations by either HCPs or the women themselves on the issues of childbearing. There were uncertainties about who should initiate the conversations. One woman’s statement summarized this: . This was also supported by a woman’s report that there was a lack of access to have discussions because their provider was occupied most of the time: “The healthcare workers are occupied most of the time” .This sub-theme contextualized high client load and insufficient staffing as a barrier to discussing fertility plans. Despite the ability to provide referrals to other units, high client load, mainly explained in relation to insufficient staffing, was noted as a critical barrier to discussing personal fertility plans. This resulted in an inability to translate policy into action . The HCPs also stated that discussions about having children were made if the WLHIV insisted on having children  of women expressed a desire to have a child in the future and the potential to transfer the disease was at the forefront of their concerns. The lack of recognizing fertility needs by HCPs was contextualized in that HCPs reported that only a few women (5–10 women out of a hundred women) desired a child (Table The differences in the rate of fertility desire reported by HCPs and WLHIV might have resulted in poor (inaccurate) information based on client’s own fertility needs and caused women fear to disclose their fertility needs.This sub-theme contextualized not allowing enough time for discussions because providers were rushing during discussions on different topics and focused on general fertility discussions as opposed to personalized. To reduce waiting for the next patient, booking times were too short, discussions were shallow and did not allow for consults beyond HIV management. According to the provider’s perspectives, the reasons for these discussions that were shallow was related to a shortage of human power and high client load. Service integrations  also demanded more time (time-consuming and creates longer waiting time). Parallel activities of the clinic, like recording and preparing reports added more working time on HCPs so that they rushed . In the context of HIV-positive people, a HCP explained that there was a pressure mainly from men that women bear more children , which hindered full disclosure of concerns, and prevented them from discussing their fertility plans fully. In the context of mismatched fertility desires, a woman expressed that her partner was not concerned about fertility issues and was just pushing her to have more children: ““I did not tell this to my husband. It is just one year since he tested but I have known my status for the past six years. I started using medications six years ago but it is just one year for him” . Due to ethical reasons, HCPs found it challenging to disclose their client’s HIV-status to their client’s partner when their clients did not want their partner to know about it  in some cases. Discussing fertility plans with a couple in relation to HIV was difficult during a longer nondisclosure period. A woman said: The providers advise me but he [partner] wants without protection. We use as such [without condoms though HIV-negative]” . HCPs also expressed that some partners were not coming to health facilities when invited for counselling . A HCP echoed the perception regarding the myth about HIV infections .Misinformation among discordant couples in which the HIV-negative person said they could not acquire HIV due to their blood type might lead to HIV infections .Prevention of HIV transmission to a partner was practised by using drugs and condoms. However, seroconversions were reported by both WLHIV and HCPs. A WLHIV who was HIV-positive at marriage while her husband was HIV-negative but acquired it later explained the situation.“They advise me to care for these children ... Health providers do not allow having children” . This was echoed by another woman: “Health care providers advised me not to have an additional child” .This sub-theme encompassed fear of repercussions due to disclosing their fertility desire purposely because fertility counselling by HCPs is mainly directed towards not having children. Fear of repercussions did not stop at the consequences of disclosing fertility desire before a pregnancy, but also extended to fear of the consequences of disclosing a pregnancy during its early stages. The HCPs wanted WLHIV to raise their existing children. In fear of HCP’s judgement, WLHIV preferred not to disclose their desires. A woman who discussed her fertility plan but was advised to care for the children she had said: . The concern of HCPs was mainly HIV-related, such as risk of transmission of HIV to the child  might be directed towards them . Importantly, referral to family planning clinics facilitated fertility discussions. However, our study found there were missed opportunities during the referral system as HCPs reported that a number of women failed to take up the referrals to other organisations such as Family Planning. Improving the quality of the referral system through an appropriate regular tracking system for the missed opportunities and dropouts and acquisition of feedback of referrals might have a better yield for contraception discussion and use. Furthermore, the recent unintended pregnancies were related to non-use, misuse of contraceptives , and parImportantly, we identified that referrals to peer educators (both mother support groups and adherence supporters) facilitated fertility plan discussions. A discrete choice experiment among WLHIV receiving HIV-care at hospitals in Ethiopia and Mozambique also showed peer educators were valued by WLHIV . TraininThe lack of recognizing women’s fertility needs by HCPs as well as the lack of initiating conversation by either women or HCPs were barriers to client-centered fertility discussions. This fear may be due to a lack of knowledge regarding effective safer conception strategies and/or stigma towards the fertility of WLHIV. Studies from South Africa and Uganda also documented that reproductive counselling environments were found to be unsupportive of open discussions , 51. TheDespite men’s positive roles in reproductive counselling , inadequAccording to the provider’s accounts, HCPs counsel discordant couples to avoid having a child because they fear seroconversion and want to avoid complaints. The blame towards the HCPs was related to seroconversion happening during attempting conception while following provider’s advice. In the current study, seroconversions were reported. Given a third of sexually active WLHIV were in discordant relationships , effectiThe rigour of this study was established using the criteria of Kitto et al. 2008  and by iThere is a need to provide client-centered reproductive healthcare through discussing the personalized fertility plans of HIV-positive people. Policies and guidelines supporting fertility discussions should be available. In particular, safer conception strategies are helpful in reducing the risks associated with having children. Training HCPs regarding safer conception (strategies and effectiveness), improving integration of services along with improved human resources and improving partner involvement to improve reproductive counselling are important. HCPs also need to avoid delaying discussions regarding the fertility issues of their clients. Redirecting tasks such as fertility plan counselling to mother support groups and adherence counsellors could be used as a strategy for HIV-positive people to work towards effective contraceptive use and to achieve safer conception. Improving content and quality of client-centered fertility plan discussions can significantly contribute to having healthy babies and a reduction in HIV transmission. Providing relevant information regarding safer conception strategies and effective contraceptive use in general, according to the fertility needs in particular can help clients retain the information they were given.Additional file 1: Supplementary file 1. Data processing and analysis.Additional file 2: Supplementary file 2. The rigour of this study.


F-box protein 21 (Fbxo21), a member of the F-box family proteins, constitutes one of the four subunits of an E3 ubiquitin ligase complex called SCFs (SKP1-Cullin-F-box). Despite the effect on antivirus immune response and ubiquitination regulation of a few oncoproteins, such as EID1 and P-gp, little is known about the Fbxo21 function in tumors, including gastric cancer. In our study, we confirmed that Fbxo21 expression was decreased in gastric cancer tissues. Decreased expression of Fbxo21 was significantly associated with poor prognosis in gastric cancer. Fbxo21 inhibited gastric cancer progression by inducing growth arrest and inhibiting migration and invasion. The expression of various EMT markers, such as E-cadherin, N-cadherin and Vimentin were altered after Fbxo21 knockdown or overexpression. Moreover, we demonstrated that Fbxo21 inhibited the EMT via the down-regulation of Nr2f2. Fbxo21 expression was negatively correlated with Nr2f2 protein expression in gastric cancer tissues and cell lines. And the Nr2f2 protein abundance was regulated by Fbxo21 via ubiquitination and proteasomal degradation. At last, we demonstrated the effects of Nr2f2 re-expression and inhibition on stable Fbxo21-overexpression or Fbxo21-silenced cell lines. These results suggested that Fbxo21 inhibited the proliferation and EMT in part through down-regulating the Nr2f2. Gastric cancer is one of the most common malignant tumors with high mortality Fbxo21 (F-box only 21), a new-found member of the F-box protein family, is one of components of the SKP1-CUL1-F-box protein (SCF) E3 ubiquitin ligase complex, which can recognize and bind to target proteins, leading to their ubiquitination and proteasomal degradation Nr2f2 , also known as COUP-TF II, encodes a steroid/thyroid hormone receptor superfamily protein, and plays an important role in tumor metastasis, especially in EMT transformation In this study, we demonstrated that Fbxo21 acted as a tumor suppressor by inhibiting proliferation and anti-EMT activities. Its expression was inversely correlated with Nr2f2 protein expression in gastric cancer, and Fbxo21 regulated Nr2f2 status by inducing Nr2f2 ubiquitination and proteasomal degradation. Importantly, we further showed that Fbxo21 regulated proliferation and the EMT process through the Nr2f2/Snail pathway.Gastric cancer samples and paired normal mucosa samples were obtained from the The Affiliated Suzhou Hospital of Nanjing Medical University between 2017 and 2018. None of the patients had received chemotherapy before the operation. The samples were immediately stored in RNAlater at -80℃ and formaldehyde at room temperature after collection. Immunohistochemistry were performed as described using antibodies against Fbxo21  and Nr2f2 . This study was approved by the Medical Ethics Committee of the The Affiliated Suzhou Hospital of Nanjing Medical University.2 at 37℃. The Fbxo21-siRNA, Flag-Fbxo21, Nr2f2-siRNA, HA-Nr2f2 and the negative control vector were purchased from Genepharma Co. . The Fbxo21-siRNA target sequence: si1, GCCTTCCCTTATGAAACACTA, si2, CCTGGACATCTTTGACTACAT, si3, CGGCAACAGAAGATCTTAAAT. The Nr2f2-siRNA target sequence: TGCAAGCGGTTTGGGACCTTGAACA.The SGC-7901, BGC-823, MGC-803, MKN-45, MKN-28 and AGS human gastric cancer cell lines, the GES-1 human gastric mucosa epithelial cell line were purchased from the cell resources of the Shanghai Institute of Digestive Surgery. The cell lines were cultured in DMEM  with 10% NBS  and 1% penicillin/streptomycin  in these media. All cells were cultured in an incubator containing with 95% air and 5% CO3 cells per well in 100μl of DMEM. In the following week, we assessed cell numbers using the Cell Counting Kit-8 (CCK8) assay  daily. For colony formation assay, the resuspended cells were seeded in 6-well plates in triplicate at a density of 800 cells per well in 2ml of DMEM and cultured for 2 weeks. Finally, the cells were stained with crystal violet, pictured and counted.The cells were resuspended and seeded in 96-well plates in triplicate at a density of 1×105 cells in the serum-free DMEM medium, and medium supplemented with 10% NBS was added to the bottom chamber. After 24h, the cells were fixed with 4% paraformaldehyde for 30min and then stained for 10 min in 0.1% crystal violet, pictured and counted. For invasion assay, the inserts were coated with Matrigel  before adding the cells. Six visual fields were chosen to calculate the number of migrated cells. For the wound healing assay, the cells were cultured in 6-well plates and scratched with a 20μl tip when the plate was filled. Cells were observed at 0, 24 and 48h after the scratches were made.The cells were resuspended and added to the top chamber of a transwell insert  at a concentration of 1×10We obtain the total protein lysates using RIPA cell lysis buffer first, then added 1μg of primary antibody into total protein lysates, and shook the mixture on a rotating shaker at 4℃, overnight. The next day, we added the protein A+G beads  to the mixture and rotated sample at 4℃ for 1h. The sediments were separated using instantaneous high-speed centrifugation, washed three times with PBS, and resuspended with 1× sample loading buffer. Then the agarose beads-antigen-antibody complexes were boiled for 10 min and used for western blot assays subsequently.We obtain the total RNA using RNA Purification Kit  and assess the concentration and purity of the total RNA samples in a UV spectrophotometer. The primers for Fbxo21, Nr2f2 and Gapdh were as follows: Fbxo21 -3'; Nr2f2 ; Gapdh . All experimental procedures were performed according to the instructions in the RT-PCR Kit .The cells were incubated in RIPA cell lysis buffer  with phosphatase and protease inhibitor Cocktail . The concentration of protein was quatified using a bicinchoninic acid protein assay kit. A total of 30μg of protein was resolved via SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk in TBST for 2h and incubated with the corresponding primary antibodies at 4℃, overnight. The antibodies were Fbxo21 ; Nr2f2 ; E-cadherin ; N-cadherin ; Vimentin ; Snail ; Zeb1 ; ubiquitin  and Gapdh . After the incubation with secondary antibodies for 2h, membranes were visualized with an enhanced chemiluminescence kit  using a Tanon system .6 cells were injected into caudal vena. All the mice were sacrificed after 2 months. The pulmonary metastatic tissues and transplanted tumors were removed and fixed with 10% formalin for subsequent studies.All animal experiments were performed with approval of the Local Medical Experimental Animal Care Commission of The Affiliated Suzhou Hospital of Nanjing Medical University. Four-week-old male nude BALB/C mice were injected with SGC-7901-shRNA or control cells. Tumor length and width were measured every three days. All the mice were sacrificed after a month. For lung metastasis experiments, 2ⅹ10All experimental results were repeated at least three times and presented as mean and standard errors. Single comparisons between two groups were evaluated via Student's t-test using SPSS 20.0. The association between Fbxo21 expression and Nr2f2 expression was determined using the Spearman's rank correlation analysis. P<0.05 was considered statistically significant.http://merav.wi.mit.edu/SearchByGenes.html). Then we performed Kaplan-Meier survival analysis using overall survival data from TCGA. The results showed that the overall survival rate in Fbxo21-high group was significantly better than that in Fbxo21-low group (P<0.01) . We detected Fbxo21 expression in gastric cancer in MERAV website and found that Fbxo21 expression was much lower in gastric cancer tissues than normal mucosa. Then we examined the Fbxo21 expression via western blot and immunohistochemistry in specimens from gastric cancer patients. The results were consistent with mRNA expression data from MERAV. Moreover, the data from TCGA database showed that the decrease of Fbxo21 expression in gastric cancer patients was related to the reduction of 5-year survival rate. Therefore, we have confirmed that the decreased expression of Fbxo21 indicates the poor prognosis of human gastric cancer.Fbxo21, a novel-estimated member of F-box family proteins, functions as a subunit of E3 ubiquitin ligase in proteasomal process. Fbxo21 was reported to participate in the development of a few human malignances in vivo and in vitro. We used CCK8 and colony formation assays to detect the cell proliferation, and transwell assays for migration and invasion. Our results indicated that overexpression of Fbxo21 inhibited the proliferation of MGC-803 and MKN28 cells, but the knockdown of Fbxo21 stimulated the proliferation of SGC-7901 and BGC-823 cells. Moreover, we found that increasing Fbxo21 expression inhibited the migration and invasion of MGC-803 and MKN28 cells, while reducing Fbxo21 expression had the opposite effect on SGC-7901 and BGC-823 cells. We also detected the role of Fbxo21 in the EMT of gastric cancer. The EMT is a process of cadherin conversion that is generally considered to enhance the motility and invasiveness of cancer cells, which is critical to tumor metastasis Then we examined the effect of different Fbxo21 expression levels on the biological function of gastric cancer cells As a new-found and estimated F-box family protein member, Fbxo21 showed its function mainly in immunology, such as EBV infection In conclusion, we confirmed that Fbxo21 played a crucial role in tumor growth, metastasis and prognosis via Nr2f2/Snail pathway Figure G. The st


Proviral load was significantly higher among individuals with CT/TT genotypes and CC genotype carriers had lower proviral load and higher levels of proinflammatory cytokines. ANAs were present only in the HAM/TSP group. TREX1 531C>T polymorphism seems to be associated with TREX-1 regulation and HTLV-1 infection.Human T-lymphotropic virus type 1 (HTLV-1) deregulates the immune system and cell cycle, resulting in loss of immune tolerance and disease, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Three prime repair exonuclease 1 (TREX1) maintains innate immune tolerance of the host and host-cell permissiveness to retroviral infections. TREX1 polymorphisms may influence the course of infection and autoimmune manifestations. The influence of TREX1 531C/T polymorphism was investigated in HTLV-1 infection and development of symptoms among 151 persons infected with HTLV-1  and 100 uninfected persons in the control group. Polymorphism genotyping and proviral load quantification were performed by real-time polymerase chain reaction (PCR) and antinuclear antibodies (ANAs) were screened by an indirect immunofluorescence assay. No statistically significant difference was found in polymorphism genotype and allele frequencies between the infected and control groups. HAM/TSP patients showed higher frequency of TT genotype than asymptomatic persons ( Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus responsible for the development of different types of diseases, including adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), in addition to other inflammatory syndromes ,3,4. VirThe pathogenesis of HAM/TSP is a result of the interaction between the virus and the host immune system, including innate and adaptive immunity mechanisms . Innate TREX-1 is able to recognize and degrade dsDNA and ssDNA molecules present in cytosol, contributing to innate immune tolerance of the host and inhibiting activation of autoimmune mechanisms ,11. On tTREX1 gene are associated with functional alterations of the encoded protein, and are directly related to the development of inflammatory autoimmune diseases and the susceptibility to and progression of HIV-1-related diseases [Polymorphisms in the diseases ,13.TREX1 531C/T) has been investigated in the development of autoimmune diseases [TREX1 531C>T polymorphism with the susceptibility to HTLV-1 infection, the development of infection-related symptoms, and the presence of ANAs.Among the polymorphisms studied, rs11797 , who were treated at the outpatient clinic of the Tropical Medicine Center of the Federal University of Pará. The patients were of both sexes, older than 18 years of age, and not treated with glucocorticoids.Chlamydia trachomatis, or syphilis; these samples were used to compare the frequencies of polymorphisms. In order to avoid statistical bias due to population stratification, the control group included individuals residing in the city of Belém, State of Pará, who presented a similar ethnic background to that of the HTLV-1-infected group.A control group included samples from 100 sex workers of both sexes, exposed to the risk of infection but not infected with HTLV-1/2, HIV-1, hepatitis B or C viruses, A sample containing 10 mL of blood was collected by intravenous puncture using a vacuum collection system containing ethylenediaminetetraacetic acid as an anticoagulant. Samples were centrifuged and separated into plasma and a leukocyte mass. The leukocyte samples were used to extract genomic DNA for analysis of TREX1 531C/T polymorphism and to quantify proviral load. Plasma samples were used for the detection of ANAs. Samples were stored in a freezer at −70 °C until use.DNA was extracted from peripheral blood leukocytes using the Puregene kit  according to the manufacturer’s protocol, which included cell lysis, protein precipitation, and DNA precipitation and rehydration. DNA was quantified using a Qubit® 2.0 fluorometer  and Qubit™ DNA assay kit reagents , following the protocol recommended by the manufacturer.3, and expressed as proviral DNA copies/mm3.Proviral load was quantified using a quantitative real-time PCR using three target sequences, synthesized through the TaqMan® system , according to a previously described protocol . Samples2O, 20× assay C_11537906_20, and 50 ng of DNA. The PCR cycling conditions were as follows: 10 min at 95 °C and 40 cycles of 15 s at 95 °C and 1 min at 60 °C.TREX1 531C/T polymorphism, located in exon 16 of the gene, was analyzed by real-time PCR using a StepOnePLUS™ real-time PCR system . The reaction used a commercial assay (C11537906-20) containing primers and probes specific for the amplification of the target sequence. The reaction contained 1× master mix, HThe detection of autoantibodies against cellular antigens was performed by indirect immunofluorescence, a technique that identifies antibodies which bind to their substrate antigens, using anti-immunoglobulin G (anti-IgG) molecules conjugated to fluorescein, according to V Brazilian Consensus Guidelines for detection of anti-cell autoantibodies in HEp-2 cells . SamplesPlasma cytokines  were measured by the Ready-SET-Go® enzyme-linked immunosorbent assay (ELISA) , which uses specific monoclonal antibodies to detect the cytokine. Procedures followed the manufacturer’s instructions.2 (chi-squared) test and the G-test. The Hardy–Weinberg equilibrium was calculated to assess whether the distribution of the observed genotype frequencies matched the expected frequencies. Sample sizes of proviral load were small and the statistical analyses were performed using a bootstrap test, that is, a hypothesis t-test with 1000 resamples, and the p-value nearest to the value of real data was considered. Comparison of cytokine levels was performed using the Mann–Whitney test. All tests were performed using the BioEstat 5.3 software  [p-values < 0.05 were considered statistically significant.The genotype and allele frequencies were estimated by direct counting, and the significance of differences between groups was calculated using the χ Brasil) , and p-vThe project was approved by the Research Ethics Committee of the Health Sciences Institute of the Federal University of Pará . All study participants were informed about the research objectives, and those who agreed to participate signed an informed consent form.p > 0.05). The frequency of the wild-type genotype was high in all groups investigated. No significant differences were observed in the genotypes and alleles between the HTLV-1-infected and the uninfected persons . However, the analyses were performed comparing individuals with wild genotype with those with at least one polymorphic allele, since it was observed that CT and TT individuals presented higher levels of proviral load, and in order to avoid stratification of the sample number of the analyzed groups. Proviral load was significantly higher among those individuals with the CT/TT genotypes, in the group of asymptomatic persons  and Asian (0.31) populations . The allIn the present study, the association of TREX1 531C>T polymorphism with susceptibility to HTLV-1 infection was not observed, as previously reported of other polymorphisms in HIV-1 infection ,26. HoweThe investigation of genotype frequencies and the presence of symptoms showed individuals with a higher frequency of TT genotype with HAM/TSP. In addition, HTLV-1 proviral load was higher in both symptomatic and asymptomatic persons carrying polymorphic T alleles (CT/TT genotypes). These results suggest that TREX1 531C>T polymorphism seems to influence TREX-1 regulation and HTLV-1 infection. A possible mechanism could be related to the reduction of degradation of viral dsDNA and ssDNA molecules in the cytosol ,11 that The increase of proviral load is a consolidated risk factor associated with the development of HAM/TSP and for rheumatoid diseases, such as rheumatoid arthritis and connective tissue diseases ,27.Therefore, identification of genetic factors that may influence the evolution of the infection are important for a better understanding of the pathogenesis and the establishment of improved follow-up strategies, allowing the detection of early identification biomarkers on the onset of symptoms and reducing the risk of disease development.Retroviruses are well-known etiologic agents of autoimmune and rheumatic disorders. The investigation of serum autoimmune markers  among HTLV-1-infected persons is a good strategy to assess this association, although a recent study has reported the low frequency of these markers among HTLV-1-infected individuals in Iran .In the present study, three patients with HAM/TSP were positive for ANAs, presenting a cytoplasmic dense fine pattern of fluorescence, but none of the asymptomatic individuals were. ANAs prevalence has been reported in patients with HAM/TSP , and a sThe pattern of cytoplasmic dense fine fluorescence is observed because of antibodies reacting with PL-7, PL-12, and antiribosomal P protein cell antigens. The same pattern is observed in systemic lupus erythematosus, polymyositis, and dermatomyositis .The ANA-positive patients showed reactivity up to a dilution of 1:160. Initial screening for ANAs is important for the diagnosis of autoimmune diseases, and individuals with a positive ANAs test should be further investigated for autoimmune diseases, and given appropriate treatment.The number of HTLV-1-infected persons in each geographical setting may be low, and this situation compromises the use of appropriate statistical testing. Because of the small sample size, the association of ANAs with TREX1 531C>T polymorphism genotypes limits the strength of conclusions but allows the suggestion of a possible influence of ANAs with the C wild allele in the development of HAM/TSP, since two of the individuals presented the CC genotype and one the CT genotype. However, these results are not in agreement with the information presenting the same polymorphism in relation to systemic lupus erythematosus in which the T allele was associated with the disease . In the The role of TREX1 531C>T polymorphism in the development of autoimmune diseases is not yet well understood. Although it is not associated with some autoimmune diseases , a higheThe possible role of TREX1 531C>T polymorphism has not yet been investigated in relation to the infection or progression of infectious diseases. Considering the unique characteristics of the genetics of the population investigated that result from interethnic crossings of Europeans, Indians, and Africans , this leAdditionally, even though this polymorphism was not associated with the presence of antinuclear antibodies, it seems to contribute, along with other factors, to increased proviral load and HAM/TSP pathogenesis in the population evaluated.INF type I induces the establishment of an antiviral state in the infected cells and also towards neighboring cells, and modulates innate immunity and activation of the adaptative immune response . The levIn summary, the TREX1 531C>T polymorphism was associated with HAM/TSP, which seems to alter the regulation of TREX-1, favoring HTLV-1 replication and higher proviral load. However, polymorphism was not related to the presence of autoantibodies, which appear to be influenced by other factors independent of polymorphism.


Akkermansia in iRBD.Twenty studies on gut microbiota in PD have been reported, whereas only one study has been reported on iRBD from Germany. iRBD has the highest likelihood ratio to develop PD. Our meta-analysis of iRBD in Japan and Germany revealed increased mucin-layer-degrading genus  Akkermansia and family Akkermansiaceae were consistently increased in both iRBD in two countries and PD in five countries. Short-chain fatty acid (SCFA)-producing bacteria were not significantly decreased in iRBD in two countries. In contrast, we previously reported that recognized or putative SCFA-producing genera Faecalibacterium, Roseburia, and Lachnospiraceae ND3007 group were consistently decreased in PD in five countries. In α-synucleinopathy, increase of mucin-layer-degrading genus Akkermansia is observed at the stage of iRBD, whereas decrease of SCFA-producing genera becomes obvious with development of PD.Gut dysbiosis has been repeatedly reported in Parkinson’s disease (PD) but only once in idiopathic rapid-eye-movement sleep behavior disorder (iRBD) from Germany. Abnormal aggregation of α-synuclein fibrils causing PD possibly starts from the intestine, although this is still currently under debate. iRBD patients frequently develop PD. Early-stage gut dysbiosis that is causally associated with PD is thus expected to be observed in iRBD. We analyzed gut microbiota in 26 iRBD patients and 137 controls by 16S rRNA sequencing (16S rRNA-seq). Our iRBD data set was meta-analyzed with the German iRBD data set and was compared with gut microbiota in 223 PD patients. Unsupervised clustering of gut microbiota by LIGER, a topic model-based tool for single-cell RNA sequencing (RNA-seq) analysis, revealed four enterotypes in controls, iRBD, and PD. Short-chain fatty acid (SCFA)-producing bacteria were conserved in an enterotype observed in controls and iRBD, whereas they were less conserved in enterotypes observed in PD. Genus IMPORTANCE Twenty studies on gut microbiota in PD have been reported, whereas only one study has been reported on iRBD from Germany. iRBD has the highest likelihood ratio to develop PD. Our meta-analysis of iRBD in Japan and Germany revealed increased mucin-layer-degrading genus Akkermansia in iRBD. Genus Akkermansia may increase the intestinal permeability, as we previously observed in PD patients, and may make the intestinal neural plexus exposed to oxidative stress, which can lead to abnormal aggregation of prion-like α-synuclein fibrils in the intestine. In contrast to PD, SCFA-producing bacteria were not decreased in iRBD. As SCFA induces regulatory T (Treg) cells, a decrease of SCFA-producing bacteria may be a prerequisite for the development of PD. We propose that prebiotic and/or probiotic therapeutic strategies to increase the intestinal mucin layer and to increase intestinal SCFA potentially retard the development of iRBD and PD. Akkermansia and decreased genera Roseburia and Faecalibacterium were shared in PD across countries. In addition, these taxonomic changes were independent of the confounding effects of constipation, body mass index (BMI), sex, age, and catechol-O-methyl transferase (COMT) inhibitor intake.Parkinson’s disease (PD) is a progressive neurodegenerative disease exhibiting four major motor deficits of tremor, slowness of movement, rigidity, and postural instability . PD also2–9–12–15–25–33–RBD is characterized by dream-enactment behaviors during the rapid-eye-movement sleep, when normal people lose muscle tone, called a state of atonia . RBD is Akkermansia and decreased short-chain fatty acid (SCFA)-producing taxa in PD in five countries including the German data set  compares the overall bacterial compositions between two groups by either taking into account or not the effects of possible confounding factors . PERMANOWe conducted principal-coordinate analysis (PCoA) of gut microbiota in controls and iRBD in our data set, as well as gut microbiota in our previously reported PD subjects  . The cen10.1128/mSystems.00797-20.2TABLE S1Lachnospiraceae ND3007 group is a putative SCFA producer, and the other nine genera are established SCFA producers. Download Table S1, DOCX file, 0.01 MB.The top 10 genera with the highest loadings in the first factor by LIGER analysis. Note that genus Copyright © 2020 Nishiwaki et al.2020Nishiwaki et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Lachnospiraceae ND3007 group). Scatterplots of the 10 genera in each enterotype showed that the abundances of these genera were also decreased in the order of enterotypes A to D  analysis  to make ountries . To summ10.1128/mSystems.00797-20.1FIG S1P values of Jonckheere-Terpstra trend test are indicated in the upper right corner to show whether the genus increases or decreases monotonically. Download FIG S1, EPS file, 2.8 MB.Relative abundances of 10 genera with the highest loadings in the first factor (Table S1) are plotted against enterotypes A to D generated by LIGER. Bar indicates the median value. Copyright © 2020 Nishiwaki et al.2020Nishiwaki et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Ruminococcus 2, Alistipes, Akkermansia, Ruminococcaceae UCG-005, Ruminococcaceae UCG-004, [Eubacterium] coprostanoligenes group, and Family XIII AD3011 group), two families were increased in iRBD (Rikenellaceae and Akkermansiaceae), and no genera or families were decreased in iRBD. To adjust for the environmental and dietary factors, we next examined taxonomic differences in eight pairs of iRBD patients and their spouses in our data set   and Wilc10.1128/mSystems.00797-20.3TABLE S2P value was calculated by Wilcoxon rank sum test. q value was calculated by the Benjamini-Hochberg method. We set the significance thresholds of W > 0.6 × N (N is the number of taxa tested) and q value < 0.05. Significant taxa are indicated in bold. (b) Families changed in RBD in our data set. W indicates a statistical measure generated by ANCOM. P value was calculated by Wilcoxon rank sum test. q value was calculated by the Benjamini-Hochberg method. We set the significance thresholds of W > 0.6 × N (N is the number of taxa tested) and q value < 0.05. Significant taxa are indicated in bold. Download Table S2, DOCX file, 0.04 MB.(a) Genera changed in RBD in our data set. W indicates a statistical measure generated by ANCOM. Copyright © 2020 Nishiwaki et al.2020Nishiwaki et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00797-20.4TABLE S3P value was calculated by Wilcoxon signed-rank sum test. q value was calculated by the Benjamini-Hochberg method. Increased or decreased taxa are marked as + or −, respectively, and unchanged taxa are marked as 0. Genera which are significant in our data set including all samples are indicated in bold letters. (b) Families changed in eight pairs of iRBD patients and their spouses in our data set. W indicates a statistical measure generated by ANCOM. P value was calculated by Wilcoxon signed-rank sum test. q value was calculated by the Benjamini-Hochberg method. Increased or decreased taxa are marked as + or −, respectively, and unchanged taxa are marked as 0. Families which are significant in our data set including all samples are indicated in bold letters. Download Table S3, DOCX file, 0.03 MB.(a) Genera changed in eight pairs of iRBD patients and their spouses in our data set. W indicates a statistical measure generated by ANCOM. Copyright © 2020 Nishiwaki et al.2020Nishiwaki et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the N . Among them, seven genera  were increased in both iRBD and PD. Similarly, two genera in iRBD and 27 genera in PD were decreased with W > 0.5 × N. No genera, however, were shared between iRBD and PD.We next compared the results of iRBD-ANCOM with those of previously reported PD-ANCOM . Nine geRuminococcus 2, Alistipes, Akkermansia, Ruminococcaceae UCG-004, and Family XIII AD3011 group) and two families (Rikenellaceae and Akkermansiaceae) were changed in iRBD after adjusting for constipation, BMI, sex, age, and PPI (Ruminococcaceae UCG-005 and Family XIII AD3011 group) were increased by age  and one family (Akkermansiaceae) were decreased by BMI (Ruminococcaceae UCG-004 and Family XIII AD3011 group) were increased by constipation  analysis with constipation, BMI, sex, age, and PPI. We found that five genera  analysis plotted in Fig. 2, as well as exact values of ANCOM analysis and Wilcoxon rank sum test. aSix taxa were also significantly changed in iRBD in the meta-analysis of the Japanese and German data sets. P values were obtained by GLMM analysis for each confounding factor. P values are plotted in Fig. 2. Taxa and their P values that were changed in iRBD after adjusting for the effects of age, sex, BMI, constipation, and PPI are indicated in bold . Taxa and their P values that were changed by age, BMI, and constipation are underlined . Note that no significantly decreased genus and family were identified in our data set. *, P < 0.05. Download Table S4, DOCX file, 0.01 MB.Exact Copyright © 2020 Nishiwaki et al.2020Nishiwaki et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the I2 < 25% and P values of both fixed-effects model [FEM] and random-effects model [REM] after Bonferroni correction of <0.05) showed that four genera  and two families (Rikenellaceae and Akkermansiaceae) were increased in iRBD  Effect sizes and relative abundances of all filtered genera in RBD in the meta-analysis of the Japanese and German data sets. (b) Effect sizes and relative abundances of all filtered families in RBD in the meta-analysis of the Japanese and German data sets. Download Copyright © 2020 Nishiwaki et al.2020Nishiwaki et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00797-20.7TABLE S6Table S6, DOCX file, 0.01 MB.Exact statistical measures of forest plots indicated in Fig. 3. FEM, fixed-effects model. REM, random-effects model. Download Copyright © 2020 Nishiwaki et al.2020Nishiwaki et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Faecalibacterium and Roseburia) and one putative SCFA-producing genus (Lachnospiraceae ND3007 group) were decreased in PD across countries  and two families (Rikenellaceae and Akkermansiaceae) were indeed accounted for by iRBD  and one family (Akkermansiaceae). Among the five genera and two families that were increased in iRBD, only genus Akkermansia and family Akkermansiaceae were also increased in PD in our meta-analysis of five countries  and two families (Rikenellaceae and Akkermansiaceae) were increased in iRBD  receptor encephalitis  were consistently decreased in PD across countries , Iwate Medical College Hospital (H28-123), Okayama Kyokuto Hospital (kyoIR-2016002), and Fukuoka University School of Medicine (2016M027). We obtained written informed consent from all patients and controls.We recruited 26 patients with iRBD and 137 healthy controls from four hospitals to participate in this study from September 2015 to February 2018. Among the 137 healthy controls, 8 were spouses of iRBD patients. All iRBD patients were diagnosed by International Classification of Sleep Disorders Criteria-Third Edition . The sevThe detailed procedures for transportation of a fecal sample from the participant’s home to the Nagoya University, freeze-drying of the fecal sample , and DNAt test or Fisher’s exact test. Subjects with the stool frequency of twice a week or less were defined to be constipated , five medications, and 17 kinds of foods were compared between iRBD and controls in our data set using either Student’s stipated .We examined lack of multicollinearity between iRBD, constipation, BMI, sex, age, and PPI by calculating the variance inflation factor (VIF) using the R package HH version 3.1-40. We verified that the VIFs were all less than 2, indicating that there was no multicollinearity between iRBD, constipation, BMI, sex, age, and PPI.Next, we analyzed the effects on the overall composition of gut microbiota of (i) iRBD versus controls and (ii) iRBD versus controls, age, sex, BMI, constipation, and PPI in our data set that was comprised of controls and iRBD using PERMANOVA . We simiFor the overall analysis of gut microbiota, PD samples in our previous report  were incWe next employed the Linked Inference of Genomic Experimental Relationships (LIGER) , which uTaxa were filtered at the genus and family levels using the following conditions. For each taxon, we counted the number of samples in which the relative abundance of the taxon of interest was greater than 1E−4. The number of such samples should be 17 or more . We thereby chose 50 families and 168 genera.https://github.com/antagomir/scripts/tree/master/R/ancom). The Wilcoxon rank sum test was performed with the mannwhitneyu functionality of scipy.stat on Python 3.6.5. The threshold of W calculated in ANCOM was set to more than 0.6 × N, where N is the number of taxa. The difference in the abundance of each taxon between iRBD and controls was also analyzed by the Wilcoxon rank sum test followed by calculation of the false-discovery rate (FDR) using the Benjamini-Hochberg procedure. The FDR threshold was set to 0.05. Bacterial taxa filtered for both W and FDR were assumed to be significant.The difference in the abundance of each taxon between iRBD and controls was analyzed by Analysis of Composition of Microbiomes (ANCOM) , as wellWe also analyzed the sub-data set of eight pairs of iRBD patients and their spouses by ANCOM and the Wilcoxon signed-rank sum test to adjust for the effects of diet and lifestyle.Seven genera and two families that were identified in our data set were subjected to GLMM  analysis using the function “glmer.nb” of the R package lme4 by setting an option to accept taxonomic variations from subject to subject.Our Japanese data set was comprised of 26 iRBD patients and 137 healthy controls, whereas the German data set was comprised of 20 iRBD patients and 38 healthy controls . We firs10.1128/mSystems.00797-20.8TABLE S7aMean and SD. n.a., not available. Download Table S7, DOCX file, 0.01 MB.Experimental methods and disease durations of the Japanese and German data sets. Copyright © 2020 Nishiwaki et al.2020Nishiwaki et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00797-20.9TABLE S8Table S8, DOCX file, 0.01 MB.The numbers of read counts in the Japanese and German data sets. Download Copyright © 2020 Nishiwaki et al.2020Nishiwaki et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the I2, representing heterogeneity in meta-analysis, was below 25% . FASTQ files of our PD data set were previously deposited with accession number DRA009229 (https://www.ncbi.nlm.nih.gov/sra/?term=DRA009229).FASTQ files of our iRBD data set are available with accession number DRA009322 (


Streptococcus mutans is a multi-virulent pathogen that can transform commensal oral microbial community to plaque biofilms. Major virulence factors that are associated with the cariogenicity of S. mutans include adhesion, acidogenicity and acidurity. All these pathogenic traits coordinate and alter the dental plaque ecology which provide room for interaction with other similar acidogenic and aciduric bacteria. This cariogenic flora increases the possibility of enamel demineralization which headway to caries development. The present study was aimed at evaluating the antimicrobial and antiinfective potential of a lichen secondary metabolite usnic acid (UA) against S. mutans. Minimum inhibitory concentration (MIC), Minimum bactericidal concentration (MBC) and growth kinetics were evaluated to determine the antimicrobial potential of UA against S. mutans. UA at 5 µg mL−1 and 10 µg mL−1 concentration were considered as MIC and MBC respectively. Effect on biofilm formation was microscopically assessed and found to be reduced in a concentration dependent manner. Gene expression of gtfB, gtfC, gtfD, vicR, ComDE and smu0630 was found to be downregulated upon treatment with sub-MIC of UA. Acidogenicity, acidurity, eDNA synthesis and response to oxidative stress were found to be attenuated by the influence of UA. It was also demonstrated to act on preformed mature biofilm of S. mutans. Moreover, UA was shown to possess very low frequency to acquire spontaneous resistance development in S. mutans. Besides, no morphological aberrations or toxic effect was instigated by UA in the human buccal epithelial cells as well as to the oral commensals. Altogether, these results demonstrate the therapeutic potential of usnic acid in the treatment of S. mutans infection.The principal etiological agent of human dental caries,  These oral diseases are not restricted to localized infections but possess the prospects enduring major complications due to invasion to neighbouring anatomical sites6. If left unattended, both the localized infections may transfigure to chronic diseases.Dental caries and periodontist are predominant human microbial infectious diseases often allied with high socioeconomic impactStreptococcus mutans has been implicated as the primary etiological agent due to its proficiency to sense and adapt to diverse environmental stress conditions during host colonization and biofilm formation7. As S. mutans resides exclusively in the oral cavity, its persistent survival in the oral region is significantly posed with major challenges due to counteraction of surplus immune and non-immune host defense systems such as antimicrobial peptides in the saliva, mechanical shear forces, antimicrobial attack, alterations in the nutrient availability, recurrent acidic condition and other environmental circumstances. To endure such complex harsh residential conditions, S. mutans have acquired multiple pathways which facilitate them to adhere, outcompete and interact with the microbiota through which they initiate and progress the infection8. Two-component Regulatory Systems (TCRS) are such response regulators which assist S. mutans to survive through environmental changes9. Whole genome of S. mutans has been sequenced completely from which existence of fourteen putative TCRSs have been identified11. One among these TCRSs is the VicRK signal transduction system which regulates various physiological processes and virulence phenotypes of S. mutans including the expression of gtfBCD, biofilm formation, oxidative stress response, acid tolerance, bacteriocin production, genetic competence and cell death14. ComDE is another quorum-sensing signalling system that is essential for genetic competence and biofilm formation15. These molecular circuits assist S. mutans to colonize on the tooth surface thereby altering the symbiotic oral ecosystem through shifting the non-pathogenic commensal flora to plaque microbiota which eventually progress the caries development16. Prime factors that contribute to plaque build-up and teeth erosion by S. mutans are acidogenicity, the ability to produce various organic acids through carbohydrate fermentation mechanisms; acidurity, proficiency to survive in low pH environment and biofilm formation18.In dental plaque formation, 19. In addition to decreased penetrance, biofilm cells can acquire resistance to antibiotics based on other phenomenon viz mutations, presence of drug efflux pumps and production of neutralizing enzymes and prolonged usage leading to modification in the drug targets etc.20. Hence biofilm cells are known to evade antibiotics and host defense mechanisms. Moreover, the use of antibiotics and antiseptic agents in the form of mouth washes and other oral care products against oral pathogens has been reported to result in their adverse side effects such as hypersensitivity, secondary infections and teeth staining22. Furthermore, due to rapid increase in the incidence of antibiotic resistance, it is imperative to identify novel antimicrobial agents with potential to inhibit the various virulence aspects of S. mutans.The architect of plaque biofilm restricts the penetrance of antimicrobial agents while in due course, planktonic cells develop novel phenotypes and become less sensitive to the antibiotics23 for the management of various virulence phenotypes of S. mutans. Usnic acid has been demonstrated to exhibit antiviral24, antiprotozoal25, antiproliferative26, analgesic27 and antiinflammatory activity28. Also, Usnic acid has been utilized in medical, cosmetic and perfumery applications. This study for the first time illustrates its added therapeutic potential against S. mutans.With this backdrop, the present study investigated the antimicrobial and antiinfective potential of usnic acid -dibenzo-furandione), one of the well-known lichen secondary metabolitesS. mutans was assessed for lower to higher concentration ranging from 2 to 1024 µg mL−1 in which the growth inhibition was found in the least concentration. Subsequently, lower concentration from 0.5 to 20 µg mL−1 was evaluated and found that at 5.0 µg mL−1 concentration of UA, visible inhibition in the growth of S. mutans was observed and hence the same concentration was considered as MIC  , MIC (5 µg mL−1) and MBC (10 µg mL−1) was assessed over a 24 h period. Significant inhibition in the proliferation of S. mutans was observed at sub-inhibitory concentration starting from around 5th hour of incubation and the proliferation was found to deteriorate drastically afterwards. At MIC and MBC, the proliferation of S. mutans cells was constantly supressed throughout the period of evaluation  and 1×, 5× and 10× MBCs  of UA. No resistant colonies were observed in the plates except the one added with sub-MIC of UA yet after 72 h of incubation. Accordingly, from the result it is contemplated that UA poses spontaneous resistance frequency of less than 108 . Considering the non-lethal effect of UA on both HBECs as well as oral commensals, the bioactive can be taken for use in dentifrices.S. mutans which has been well recognised as a pathogen that initiates the formation of dental caries. UA at MIC, i.e. 5 µg mL−1 concentration arrested the growth of bacterium whereas the bactericidal effect was found at 10 µg mL−1. MIC and MBC of UA against S. mutans was found to be at lower concentration which indicates that minimum concentration of drug is adequate to inhibit the growth of organism. Moreover, the proliferation of S. mutans was found to be substantially diminished even at sub-MIC of UA. Drugs with lower MIC values are generally considered as effective antimicrobial agents and thus UA can be regarded as an effective antimicrobial component to treat S. mutans infection. As S. mutans naturally resides in the human oral cavity and more specifically on the hard surfaces of tooth and in dental plaque, the bacterium often adopts a sessile lifestyle for the survival and insistence in its inherent ecosystem rather than free-living planktonic lifestyle31. Furthermore, cells that reside in biofilm state exhibit varied phenotypic attributes that are diverse from their planktonic counterparts which is usually accompanied with significant changes in the gene expression pattern33. Considering the role of biofilm in the virulence attributes of S. mutans, effect of UA on biofilm formation and the expression of genes involved in biofilm and its associated factors were analysed. While mentioned earlier, as S. mutans predominantly follows a sessile lifestyle it is known to form robust biofilm on dental surfaces34. In line with this fact, multi-layered, strong and entire surface covered thick biofilm was observed microscopically in the control S. mutans cells. Whereas, the biofilm of S. mutans grown in the presence of UA was found to be frail and moderately sparse in a concentration dependent manner. Quantification of biofilm biomass also evidenced that even at lowest concentration, UA could effectively reduce the number of biofilm cells. Influence of UA on biofilm inhibition of S. mutans was not due to the growth inhibitory effect as the concentration used to test the effect of UA on biofilm formation of S. mutans is analysed from a concentration which is less than 1/16 × MIC, where the growth inhibition was trivial and the biofilm formed was insubstantial. Gene expression analysis of major two-component signal transduction systems such as VicRK and ComDE; Smu0630, a gene which is critical for biofilm formation regardless of the carbohydrate source and extracellular glycosyl transferases (GTFs) such as gtfB, C and D revealed that UA at sub-MIC (2.5 µg mL−1) decreased the expression of these genes. vicR, a response regulator of the VicRK signal transduction system is reported to be essential for the viability of bacterium and the gene products of vic modulate the adherence, biofilm formation and genetic competence development in S. mutans12. In addition, they are demonstrated to regulate the expression of various virulence associated genes that are related with the synthesis of polysaccharides such as gtfBCD, ftf etc. Downregulation of vicR by UA influenced the expression of all three glycosyl transferases gtfBCD. Gene products of gtfB and gtfC results in the synthesis of water-insoluble glucans which are essential adhesive molecules that bridge bacteria to the acquired pellicle around tooth surface35. GtfD predominantly synthesizes water soluble glucans36. Reports have demonstrated that Gtfs are recognized as pivotal contributors for the alteration of biofilm architecture, disruption of homeostasis in the community of healthy microbiota and induction of caries progression37. Ren et al., in 2016 have pronounced that molecules that target the glycosyltransferase possess the ability to inhibit biofilm formation and cariogenicity of S. mutans38. Downregulation in the Gtfs B, C and D by the influence of UA demonstrates that the compound can be effectively expended for inhibiting S. mutans biofilm and cariogenicity. In addition to this, VicRK system has been demonstrated to respond to oxidative stress. Knockout mutant of vicK system has been proven to be sensitive to H2O213 and similar results were observed in the UA treatment. ComDE is a quorum sensing TCSTS which regulates the formation of biofilm in S. mutans39. This system also regulates the expression of various virulence factors in a cell density dependent manner and affects genetic competence and acid tolerance response40. UA treatment significantly reduced the expression of ComDE which allude that downstream targets and virulence attributes under the control of ComDE such as biofilm formation and acid tolerance are also influenced which is substantiated by the results of glycolytic pH drop assay, acid measurement and acidurity. Acidogenicity, the ability to produce acid from wide range of fermentable sugars and acidurity, the potential to survive in low acidic conditions are two remarkable capacity of S. mutans to initiate caries and progression of teeth erosion42. In the glycolytic pH drop assay, fermentation of glucose by S. mutans was found to be decreased upon UA treatment. From the acidurity results it has been corroborated that UA has the ability to act on survival of S. mutans cells that are adapted to survive under acidic condition. Kawari et al., have reported that under low pH conditions, S. mutans tend to form biofilm in an eDNA dependent manner43. Under Com-dependent biofilm formation at low pH conditions, as a stress response from the lysed cells, induction in eDNA release may be found. eDNA in the biofilm matrix acts as adhesives which further strengthen the biofilm45. Moreover, upregulation of biofilm formation is also correlated with the presence of eDNA46. Also, a mutant study reported that the deletion of vicR showed enhanced release of eDNA in the supernatant47. Remarkably, eDNA content in the UA treated biofilm conglomerate was completely repressed when compared to the eDNA from control. This could be due to the enhanced release of eDNA content to the external environment. Furthermore, UA precisely acted on the preformed mature biofilm of S. mutans.The present study demonstrates the antimicrobial and antiinfective potential of UA, a secondary metabolite of lichen species against 8. Downregulation of Smu0630 by UA was observed. Downregulation of key players in the biofilm formation and associated virulence factors by UA imply that probabilities of resistance development by S. mutans against this therapeutic is less likely. This has been further substantiated through spontaneous resistance assay where it is found that UA poses spontaneous resistance frequency of less than 108. UA was found to be safe against the HBECs as well as to the oral commensals, which completes its therapeutic potential in treating S. mutans infection. Schematic representation depicting the molecular targets of UA in S. mutans is portrayed in the Fig. S. mutans.Smu0630 gene was demonstrated to play a role in biofilm formation and it has been suggested as a potential target for novel therapeuticsHuman Buccal Epithelial Cells (HBECs) and saliva sample used in this study for toxicity analysis were collected from the healthy individuals. Written informed consents were obtained. For HBECs individuals were asked to gently rub the mucosal surface of cheeks. Protocol for experimentation and the use of HBECs and saliva were assessed and approved by the Institutional Ethical Committee, Alagappa University, Karaikudi (IEC Ref No: IEC/AU/2018/5). All methods were carried out in accordance with appropriate guidelines and regulations.Streptococcus mutans UA159 from American Type Culture Collection (ATCC 700610) was used in this study. Agar plates containing Todd Hewitt Broth (THB)  was used for routine maintenance of the culture. For subculture and performance of bioassays, S. mutans was cultured by incubating at static condition for 24 h at 37 °C on THB supplemented with 1% of yeast extract and sucrose (THYES).Reference strain −1 of Usnic acid (UA) was prepared in DMSO and stored at room temperature until further use.Usnic acid (PubChem CID: 5646) was procured from Sigma Aldrich, US. Stock solution of 5 mg mLS. mutans was determined by microbroth dilution assay in 24 well microtiter plate (MTP) according to the guidelines of Clinical and Laboratory Standards Institute48. Briefly, 1% of 24 h grown culture of S. mutans was used to inoculate 1 mL of THYES broth. Initial screening was assessed with increasing concentrations of UA ranging from 2 µg mL−1 to 1024 µg mL−1. Later, inhibitory effect of concentrations ranging between 0.5 µg mL−1 to 20 µg mL−1 was tested. Appropriate vehicle and negative controls were maintained parallelly. Following 24 h of incubation at 37 °C, visible difference in the growth was observed and the cell density was evaluated by measuring absorbance at 600 nm in multifunctional spectrophotometer .Growth inhibitory potential of UA against S. mutans, cfu and spot assay were performed. Briefly, 1% of 24 h grown culture of S. mutans was incubated in the absence and presence of various concentrations of UA which was predetermined through MIC assay. Following 24 h of incubation at 37 °C, cells were serially diluted and 5 µL from each dilution was spotted on THA plates and incubated for 24 h at 37 °C. Control  and treated groups (without dilution) were also spread plated on THA plates and incubated at 37 °C for 48 h. Further, the results were documented using Gel documentation system .To ensure the growth inhibitory potential and determine the MBC of UA against S. mutans over 24 h period of time with 1 h interval was assessed to evaluate the effect of UA on growth kinetics of S. mutans. Briefly, overnight grown culture of S. mutans was used to inoculate 50 mL of THYES broth. UA at various concentrations 0.5 µg mL−1 to 10 µg mL−1 were supplemented in the test samples whereas control received DMSO (vehicle control) and incubated at 37 °C. At every time point, 200 µL of culture was drawn from each tube for assessing the rate of growth by measuring the optical density (OD) at 600 nm using multifunctional UV–Vis spectrophotometer .Cell proliferation of S. mutans biofilm was allowed to form on 1 cm × 1 cm glass surface immersed in 1 mL of THYES broth in the absence and presence of various concentrations of UA (0.25 µg mL−1 to 5 µg mL−1). Subsequently, at the end of incubation period for 24 h at 37 °C, the glass slide pieces were taken out from the medium, rinsed twice in PBS and allowed to air dry. Later, the slides were stained with 0.4% crystal violet and incubated for 10 min. Surplus unbound stain was removed by a gentle rinse in distilled water and air dried. Biofilm cells stained with crystal violet was then visualized under light microscope  at a magnification of X400 and the images were documented with the accompanied digital camera.For microscopic visualization, S. mutans in the absence and presence of UA was quantitatively analysed through CFU analysis. Briefly, biofilm assay was performed in the absence and presence of various concentrations of UA in 24 well MTP. Following incubation at 37 °C for 24 h, the supernatant was discarded and the biofilm cells were washed with sterile PBS to remove unbound and loosely bound cells. Biofilm cells adhered to the surface was scrapped out and aspirated well in PBS. Cells were serially diluted in PBS and spread plated on THA plates, incubated at 37 °C for 24 h. Number of cells in the biofilms of control and treatment was enumerated49. Quantification was also performed through spectrophotometric analysis where the biofilm cells adhered to the surface of 24 well MTP in the absence and presence of UA was stained and destained with 0.4% crystal violet and 10% glacial acetic acid, respectively. Destained solution was read at 570 nm using multifunctional UV–Vis spectrophotometer .Number of viable cells in the biofilm of S. mutans cells cultured for 24 h at 37 °C in the absence and presence of UA at sub-MIC concentration by Trizol method of RNA isolation according to the manufacture’s protocol. Isolated RNA was dissolved in 30 µL of 0.1% diethylpyrocarbonate (DEPC)-treated water and the quality check was performed by agarose gel electrophoresis. Subsequently, extracted RNA samples were converted into cDNA using High-capacity cDNA Reverse Transcription Kit  in the thermal cycler . cDNA was quantified using Bionano Spectrophotometer .Total RNA was extracted from ΔΔ CT method50.Expression profile of candidate genes were examined through qPCR analysis. List of candidate gens, their function and primer sequences are mentioned in Table S. mutans through glycolysis was evaluated by glycolytic pH drop assay and acidity measurement. For glycolytic pH drop assay, S. mutans cells was harvested at mid-logarithmic phase, washed in PBS and resuspended in a salt solution containing 50 mM potassium chloride and 1 mM magnesium chloride in the absence and presence of various concentrations of UA . Glucose at 1% w/v final concentration was added and the initial pH of the mixtures was adjusted to pH 7.2–7.4 with 0.2 M potassium hydroxide. Alteration in the pH level by glycolytic activity of S. mutans and the impact of UA on the acidogenicity was monitored at 15 min intervals over a period of 120 min51.Effect of UA on acid production of S. mutans was added to 1 mL of THYES broth in the absence and presence of UA at different concentrations . pH was measured immediately after addition of UA and after 24 h of incubation.For acidity measurement, THYES broth was prepared with the pH of 7.2. In a sterile 24 well MTP, 1% overnight culture of S. mutans was assessed by measuring the viable count of bacteria after exposure to moderately acidic and extremely acidic pH. S. mutans cells cultivated in the absence and presence of various concentrations of UA  was pelleted by centrifugation at the mid-logarithmic phase. Pellet was equally split into two aliquots namely unadapted and adapted cells. Unadapted cells were directly resuspended into THYES broth of detrimental pH 3.5 and incubated at 37 °C for 2 h. Adapted cells were initially allowed to habituate in the moderately acidic THYES broth of pH 5.5 for 1 h at 37 °C and later exposed to THYES broth of lethal pH 3.5 and further incubated for 2 h at 37 °C. At the end of incubation period, viability of S. mutans cell from adapted and unadapted groups were assessed by plating on THB agar plates (pH – 7.5) for 48 h at 37°C52.Role of UA on acid tolerance of S. mutans culture grown in the absence and presence of various concentrations of UA was adjusted to OD 0.5 at 600 nm. Cells were then swabbed on THA plates and a sterile filter paper disk of 10 mm diameter was placed at the center of plate. Subsequently, the disk was loaded with 15 µL of 30% H2O2 and incubated for 24 h at 37 °C. After incubation, plates were documented and the zone of clearance was recorded53.S. mutans cells grown to ~ 0.3 OD600 nm was exposed to 50 mM H2O2 in the absence and presence of UA (5 and 10 µg mL−1) for 1 h at 37 °C. After incubation, cells were centrifuged, resuspended in PBS and serially diluted. Dilutions were spread plated on THA and incubated at 37 °C for 48 h54.Additionally S. mutans was qualitatively analysed by agarose gel electrophoresis. Briefly, S. mutans was allowed to form biofilm in the absence and presence of sub-MIC concentrations of UA  in a 6 well polystyrene plate for 24 h at 37 °C. Subsequently, the planktonic part was removed and loosely bound cells were eliminated by gentle rinse with PBS thrice. Further, the entire biofilm components were scraped from the wells, resuspended in TE buffer  and vigorously vortexed for 30 min to extricate the complex mixture. The tubes were then centrifuged to collect eDNA comprising supernatant. Eventually, amount of eDNA present in the supernatant was analysed through performing agarose gel electrophoresis (1.5% w/v) followed by ethidium bromide staining55.eDNA content in the biofilm mixture of S. mutans response regulator VicR domain (PDB ID: 5ZA3) and GtfC (PDB ID: 3AIC) was obtained from the PDB database. Chemical structure of usnic acid was retrieved from PubChem database (PubChem CID: 5646). Protein was prepared using the AutoDock Tools Version 1.5.6 and the molecular docking was performed through AutoDock Vina software56. Protein–ligand interactions were analysed and structural figures were prepared using Maestro 10 (Schrödinger) software.Crystal structures of S. mutans was determined as described by Min et al., 2017 with slight modifications57. Briefly, overnight cultures of S. mutans was washed and the cell culture was adjusted to 1 × 108 CFU/mL in THYES broth which was subsequently confronted with various concentrations of UA on THA plates and incubated for 72 h at 37 °C. After incubation period, number of colonies in control and UA challenged plates were observed and documented using Gel documentation system .Development of spontaneous resistance to UA by S. mutans was allowed to form mature biofilm. After 24 h, the planktonic part was removed and added with 1 mL of fresh medium together with various concentrations of UA and further incubated for 18 h. At the end of incubation period, cells from control and UA treatment were serially diluted and plated on THA plates which were incubated at 37 °C for 48 h. Plates were documented and variations in viability was noted in CFU/mL. Rest of the cell content from the assay was subjected to FTIR analysis. Briefly, the cells were pelleted down by centrifugation, dried completely under vacuum for 3 h at 50 °C . Samples were mixed with potassium bromide (KBr) and the spectral scan was taken from 4000 to 400 cm−1 with spectral resolution of 4 cm−1 .To evaluate the effect of biofilm disruptive potency of UA, 5 cells mL−1 was prepared by counting the pooled cell suspension through Automated cell counter Countess II FL, Invitrogen, United States). Further, HBECs were incubated with various concentrations of UA  for 20 min at 37 °C to assess detrimental effects, if any. Hydrogen peroxide was included as positive control. At the end of incubation period, cells were stained with 0.1% crystal violet and visualized under microscope  to assess morphology of the control and UA treated HBECs58.As the end application of this study is related to use in dentifrice, the safety evaluation of the compound was assessed with HBECs for which healthy individuals with proper oral hygiene was selected. To collect HBECs, individuals were asked to gently rub on their mucosal surface of cheeks with sterile cotton swab. Sequentially, the swabs were suspended in sterile phosphate buffered saline (PBS) which was used immediately. The suspended cells were pooled and centrifuged at 3000 rpm for 5 min and the pellet was washed thrice in sterile PBS to get rid of debris. A suspension with 5.0 × 1059 with slight modifications. Unstimulated saliva was collected from fourth volunteers with good oral health. Individuals were refrained from eating before 2 h from saliva collection. Saliva was pooled together and subjected to low speed centrifugation to eliminate epithelial cells. Supernatant was centrifuged at 4000 rpm for 15 min at 4 °C to collect saliva bacteria. Pellet containing the salivary commensal was washed thrice with sterile PBS and finally resuspended in the same. 250 µL of the bacterial suspension was added with UA at a final concentration of 10 µg mL−1. Appropriate vehicle control was maintained in parallel. Bacterial suspension exposed to 0.12% chlorhexidine was considered as positive control. After a brief exposure of 10 min  at 37 °C, the compounds were removed by three rounds of centrifugation. Finally, bacterial pellet suspended in the PBS was serially diluted and spread platted on Muller Hinton agar plates.Effect of UA on salivary bacteria was assessed through the procedure described by Concannon et al., 2003p value of < 0.05 by the SPSS statistical software package version 17.0 .All the experiments were carried out in at least three biological replicates with at least two technical replicates and values are presented as Mean ± standard deviation (SD). To analyse the significant difference between the value of control and treated samples, one-way analysis of variance (ANOVA) and Duncan’s post hoc test was performed with the significant Supplementary information.