Giardia duodenalis. It is prevalent in low-income countries in the context of inadequate access to water, sanitation and hygiene (WASH), and is frequently co-endemic with neglected tropical diseases such as soil-transmitted helminth (STH) infections. Large-scale periodic deworming programmes are often implemented in these settings; however, there is limited evidence for the impact of regular anthelminthic treatment on G. duodenalis infection. Additionally, few studies have examined the impact of WASH interventions on G. duodenalis.Giardiasis is a common diarrhoeal disease caused by the protozoan G. duodenalis infection in study participants every six months for two years, immediately prior to deworming, as a pre-specified secondary outcome of the trial. WASH access and behaviours were measured using questionnaires.The WASH for WORMS cluster randomised controlled trial was conducted in remote communities in Manufahi municipality, Timor-Leste, between 2012 and 2016. All study communities received four rounds of deworming with albendazole at six-monthly intervals. Half were randomised to additionally receive a community-level WASH intervention following study baseline. We measured G. duodenalis prevalence in either study arm between baseline and the final study follow-up. We found no additional benefit of the community-level WASH intervention on G. duodenalis infection . Risk factors for G. duodenalis infection included living in a household with a child under five years of age , living in a household with more than six people , and sampling during the rainy season . Individuals infected with the hookworm Necator americanus were less likely to have G. duodenalis infection .There was no significant change in G. duodenalis was not affected by a community WASH intervention or by two years of regular deworming with albendazole. Direct household contacts appear to play a dominant role in driving transmission. We found evidence of antagonistic effects between G. duodenalis and hookworm infection, which warrants further investigation in the context of global deworming efforts.Prevalence of Trial registration Australian New Zealand Clinical Trials Registry, ACTRN12614000680662. Registered 27 June 2014, retrospectively registered. https://anzctr.org.au/Trial/Registration/TrialReview.aspx?id=366540. Giardia duodenalis. It is one of the most frequent enteric protozoan infections worldwide, with a global disease burden of approximately 280 million cases annually. Giardia duodenalis is most prevalent in low-resource settings [Giardia to humans [Giardiasis is a parasitic enteric disease caused by the protozoan settings , where psettings –11. The settings , 12–15. o humans –19.Giardia duodenalis is frequently co-endemic with other parasitic diseases that are common in low-resource settings, of which soil-transmitted helminth (STH) infection is one of the most prevalent [Ascaris lumbricoides), hookworms , whipworm (Trichuris trichiura), and threadworm . While G. duodenalis control relies on treatment with antimicrobial agents following diagnosis, control of STH infections in endemic settings occurs mainly through large-scale mass deworming programmes, as recommended by the World Health Organization (WHO) [revalent , 21. STHon (WHO) . In the on (WHO) .G. duodenalis infection. Albendazole is efficacious in treating giardiasis when repeated doses are given over several days [G. duodenalis [G. duodenalis infection in the context of regular deworming with mebendazole [G. duodenalis infection [There is limited literature regarding the impact of regular deworming on ral days ; howeverral days –27. Seveodenalis , 8, and endazole , 29. Somnfection , while onfection .G. duodenalis transmission. A systematic review and meta-analysis of mainly observational studies found that latrine access, latrine use, and treating household drinking water were all associated with lower odds of G. duodenalis infection [G. duodenalis infection has been examined in small number of intervention studies, with mixed findings. In a longitudinal study in Ethiopia, a significant reduction in G. duodenalis prevalence was observed following a school-based WASH intervention [Giardia infections [Giardia prevalence among children who lived in villages where a community-based sanitation intervention was implemented [G. duodenalis was found following sanitation, handwashing, and combined WASH interventions [G. duodenalis prevalence [Given transmission of giardiasis by the faecal-oral route and the role of environmental contamination, interventions aimed at improving water, sanitation and hygiene (WASH) may play a role in reducing nfection . The effrvention , while ifections –36. An Rlemented . In the ventions . Howeverevalence .G. duodenalis, Entamoeba histolytica and Cryptosporidum spp. were pre-specified secondary outcomes of the trial [G. duodenalis infection was relatively common, with an overall prevalence of 13%, while E. histolytica and Cryptosporidium spp. were present in less than 0.1% of the study population [The WASH for WORMS cluster RCT was conducted in Timor-Leste to investigate the impact of a community WASH and deworming intervention, compared to deworming alone, on intestinal parasite infections. The impact of the study intervention on the primary outcome (STH infections) has been published separately . Prevalehe trial . Baselinpulation .G. duodenalis infection; examine the prevalence of G. duodenalis over time in the context of regular deworming; and identify risk factors for G. duodenalis infection.In this paper, we aimed to determine the impact of a community-based WASH intervention on Ascaris spp. infections), 24 communities were enrolled in the study and randomly allocated to a study arm. Five communities (two intervention and three control) were deemed ineligible prior to trial commencement and were replaced sequentially, rather than randomly, from a pre-generated list of replacement communities. It was not feasible to implement randomisation for the additional communities due to factors related to implementation of the WASH intervention by the partner organisation, including timing and community expectations. One community in the intervention arm was subsequently lost to follow-up after study baseline. Therefore, a total of 23 communities completed the study, and 18 communities (nine intervention and nine control) followed the randomisation protocol [WASH for WORMS was a two-arm cluster RCT conducted between 2012 and 2016 in the Manufahi municipality of Timor-Leste . Communiprotocol . A flow The deworming intervention, delivered in both study arms, involved distributing a single 400 mg dose of albendazole to all eligible community members . Children aged 12–23 months were given half a dose. Four deworming rounds, conducted at six-monthly intervals, were delivered, with a fifth round given following completion of data collection. Albendazole was delivered by members of the research team and taken under direct observation.The WASH intervention, implemented following study baseline and prior to the first deworming round, consisted of providing access to a protected community water supply, promoting improved sanitation through encouraging household latrine construction , and conG. duodenalis, Cryptosporidium spp., Entamoeba histolytica and Strongyloides spp., and the other for detection of Ascaris spp., N. americanus, Ancylostoma spp. and Trichuris spp. An Equine Herpes Virus target was spiked into each sample as an extraction and internal qPCR control [Sociodemographic data , clinical information (current and recent diarrhoea), and WASH information  were collected through individual and household-level questionnaires at baseline and four follow-up time points, at six-monthly intervals. Stool samples were collected from all willing community members over one year of age, at baseline and each of the four follow-up time points, immediately prior to deworming. Samples were preserved in 5% potassium dichromate and sent to the QIMR Berghofer Medical Research Institute  for analysis. DNA extractions were performed using the PowerSoil DNA Isolation Kit  and subjected to two multiplex real-time PCR reactions: one for detection of  control .G. duodenalis infection along with 95% confidence intervals (CI) was calculated for each study arm at each time point using logistic regression models that accounted for community-level clustering using robust standard errors. The impact of the community-level WASH intervention on G. duodenalis infection was evaluated in the 18 communities that followed the randomisation protocol, across all four follow-up time points. We constructed a generalised linear mixed model, with community, household, and individual entered as random effects, to account for repeated observations on the same individuals over time, nested within households and communities. We used Poisson regression to model relative risk (RR), incorporating an interaction term between study arm and follow-up time point, and adjusting for age group and sex. We calculated RR (and 95% CI) of infection for the study intervention, compared to the control arm, at each follow-up time point, by calculating a post-estimation linear combination of coefficients and standard errors, using Wald-type methods [Prevalence of  methods .G. duodenalis infection, we conducted an observational analysis of all individuals in the study cohort (i.e. in the 23 communities that completed the study), across all five study time points (baseline and four follow-ups). We again constructed generalised linear mixed models to account for clustering at the community, household, and individual level. We used Bernoulli logistic regression to model odds ratio (OR) for each predictor variable. Predictor variables included sociodemographic factors, WASH access and behaviours, household animal ownership, STH infections and season. Socioeconomic status was determined using principal component analysis as described previously [P < 0.2. Multivariable models were then constructed using a two-stage approach. “Within-domain” multivariable models were built for groups of related variables: demographic, individual socioeconomic, individual hygiene, individual sanitation, school sanitation, household socioeconomic, household hygiene, household water, household sanitation, environmental, and infection-related .Finally, we examined the impact of previous G. duodenalis infection. Baseline characteristics of this study population are shown in Additional file At study baseline, among the 23 communities that completed the trial, 2448 participants provided informed consent to participate in the study. Of these, 1971 (80.5%) provided a stool sample that was analysed for G. duodenalis infection across the study period in each arm, overall and disaggregated by age, is shown in Table G. duodenalis prevalence in the intervention arm was 14.5% (95% CI: 10.1–17.6%) and in the control arm was 12.3% (95% CI: 9.6–16.3%). At the end of the study, G. duodenalis prevalence in the intervention and control arms was 17.4% (95% CI: 12.9–21.6%) and 14.0% (95% CI: 10.3–18.0%), respectively. As depicted in Table G. duodenalis prevalence was highest among children aged 1–5 years of age, ranging between 23.7% (95% CI: 14.4–30.1%) and 34.3% (95% CI: 25.2–43.4%) in the intervention arm, and between 14.4% (95% CI: 5.5–23.1%) and 38.1% (95% CI: 27.7–48.5%) in the control arm. There was no significant difference in risk of G. duodenalis infection at the end of the study compared to baseline, in either the intervention arm  or the control arm .Prevalence of G. duodenalis was 11.5% (95% CI: 7.4–17.5%). This was not significantly different between the intervention arm  and the control arm , and was also not significantly different to the prevalence of diarrhoea reported among those not infected with G. duodenalis . Among those infected with G. duodenalis, the highest diarrhoea prevalence was observed in children aged 1–5 years in both the intervention arm  and the control arm .Across all age groups and study time points, the prevalence of self-reported diarrhoea (at the time of survey or within the previous 2 weeks) among those infected with G. duodenalis infection between the intervention or control arm at any time point. At the completion of the trial, the RR of G. duodenalis infection in the intervention arm, compared to the control arm, was 1.05 .The results of the generalised linear mixed model examining the impact of the study intervention are shown in Table G. duodenalis infection at P < 0.2; full results of univariable analyses are provided in Additional file G. duodenalis infection decreased significantly with increasing age. People who lived in a household with a child under five years of age had significantly increased odds of infection compared to those who did not . Similarly, those living in a household of more than six people had higher odds of infection compared to those living in households of six or fewer people . Odds of infection were higher in the wet season compared to the dry season . Infection with Necator americanus was associated with lower odds of G. duodenalis infection . No WASH variables remained as significant predictors of infection in the final model.Univariable analyses found various WASH, demographic and socioeconomic variables significantly associated with G. duodenalis infections at previous study time points on the risk of infection with G. duodenalis at the final follow-up. The risk of G. duodenalis infection at the end of the trial was significantly greater among those with a history of infection diagnosed at previous study time points, with relative risk of infection increasing with the number of time points.Table G. duodenalis infection in the context of a community-based WASH and deworming RCT. In our study setting in rural Timor-Leste, we observed a moderate overall prevalence of G. duodenalis in both study arms, ranging between 10.2 and 19.6% over the study period. Prevalence was highest (up to 38.1%) among children aged 1–5 years. This is consistent with the known epidemiology of Giardia infection that tends to predominate among young children [G. duodenalis reported diarrhoea, which was similar to the background diarrhoea prevalence among the study population. Over four rounds of community-wide deworming with albendazole, we observed no significant change in G. duodenalis prevalence in either study arm. This supports previous findings that a single 400 mg dose of albendazole is not efficacious against G. duodenalis [To our knowledge, this is the first study to examine children , 46. Mosodenalis .G. duodenalis prevalence, compared to the control arm that did not receive a WASH intervention. This is consistent with the findings of the primary outcome of the WASH for WORMS trial, where no additional impact of the WASH intervention on STH infections was identified [Giardia infection [G. duodenalis infection among participants who received WASH interventions [G. duodenalis. Additionally, although rates of reported handwashing with soap were high among study participants at the end of the trial, this was self-reported and may therefore overestimate true handwashing behaviours [G. duodenalis transmission following the WASH intervention include zoonotic transmission and contaminated drinking water. Given that protected water sources were provided as part of the study intervention, contamination occurring at the main source of community drinking water is unlikely. However, it is possible that individuals obtained drinking water from other (unprotected) sources, or that water contamination occurred during or after collection. WASH interventions may require higher coverage, over a longer duration of time, to achieve any impact on transmission of intestinal protozoa in areas with significant environmental contamination.We found no impact of the community-based WASH intervention in terms of reducing entified . Our finnfection , 38. Howventions , 37. A pventions . This wohaviours . Other pG. duodenalis infection. We examined a wide range of factors including WASH access and behaviours, animal ownership, STH infections, and socioeconomic variables. However, relatively few predictors remained in our final model. Age group was a major predictor of infection, and N. americanus infection was associated with reduced odds of G. duodenalis infection. This potential antagonism between hookworm and G. duodenalis has been identified previously [Our risk factor analysis provides further insight into the individual-level factors associated with eviously , with poeviously . Howevereviously , 31.G. duodenalis infection included living in a household with a child under five years of age, and living in a household with more than six people. This demonstrates that direct household contacts likely play a dominant role in transmission. Additionally, infection was significantly more likely during the wet season, consistent with previous findings of increased transmission during rainy seasons [Other factors that significantly affected the odds of  seasons , 48.G. duodenalis was associated with significantly increased odds of infection at the end of the study, with a dose-response observed with increasing number of previous infections. Although there is some evidence that people may develop a degree of acquired immunity to G. duodenalis [Giardia, potentially driven by persistent environmental exposure or repeated risk behaviours.We identified that previous infection with odenalis , it has odenalis . Furtherodenalis , 51. TheG. duodenalis infection, residual confounding due to unmeasured factors remains likely.This analysis was conducted in the context of a robustly-designed RCT, using a highly-sensitive diagnostic technique; however, there are several important limitations. The community-based WASH intervention, although delivered in accordance with national sanitation policy by local NGOs in a culturally-appropriate manner, did not achieve its aims of 100% household latrine coverage and “open-defecation free” communities. This made detecting any impact of the WASH intervention less likely. As mentioned above, data relating to WASH access and behaviours were derived predominantly from self-report, which could be biased towards reporting safer WASH behaviours , leadingG. duodenalis infection across the entire community should be undertaken. Identifying improved mechanisms to measure WASH behaviours in a research context is also required, to allow accurate determination of key risk factors and inform the optimal design of interventions. Food-borne transmission is an important source of G. duodenalis infection [Giardia also has potential for zoonotic transmission [A number of key priorities for ongoing research arise from this study. First, additional research examining the impact of WASH risk factors and WASH interventions on protozoan infections is required. Most previous studies on risk factors have focused on children . Given tnfection , 53 and smission –19; therG. duodenalis and hookworm infections, and to identify ways to mitigate this potentially antagonistic effect. This is particularly important given the high burden of both STH and Giardia infections in low-resource settings, especially among children, and the large-scale global deworming efforts currently underway. It may be necessary for STH control guidelines to include recommendations for undertaking diagnosis and treatment of G. duodenalis infections during monitoring efforts, and for educating communities about the symptoms of giardiasis and the importance of seeking additional treatment given that deworming is unlikely to be effective. Strengthening the understanding of the role of both WASH and deworming in the epidemiology and control of G. duodenalis is crucial, in order to inform the design and delivery of interventions tailored to the communities that will benefit from them most.Additional studies are required to investigate the potential antagonistic relationship between G. duodenalis across all age groups, but particularly among children, in communities receiving regular deworming for STH control. It also demonstrates some of the challenges involved in implementing and evaluating WASH interventions in low-income settings, and the complexities in generating evidence to demonstrate the impact of WASH on health outcomes. This study provides important evidence to inform the understanding of G. duodenalis epidemiology in the context of regular deworming and WASH interventions, and highlights important areas for future research, including further investigation of a potentially antagonistic relationship between hookworm and G. duodenalis infections.This study demonstrates the ongoing burden of Additional file 1: Text S1. Full list of variables examined as risk factors for Giardia infection.Additional file 2: Table S1. Baseline characteristics of study participants. Table S2. Participation rates in the study over time. Table S3. Intensity of infection with G. duodenalis over time, measured using qPCR cycle threshold (Cq) values among positive samples. Table S4. Results of univariable analyses for risk factors for G. duodenalis infection (n = 2333), based on generalised linear mixed models accounting for village, household and individual-level clustering.


Uric acid (UA) is a major antioxidant molecule that has been hypothesized to have a protective effect against cancer-induced oxidative damage. The aim of the present study was to investigate whether preoperative levels of serum UA are associated with the prognosis of laryngeal squamous cell cancer (LSCC).  A total of 814 male LSCC patients (followed up for five years) and 814 normal control subjects were enrolled from January 2007 to December 2011. The rates of total mortality and cancer mortality were 23.46% and 21.36%, respectively. The prevalence of overall survival (OS), disease-free survival (DFS), and cancer-specific survival (CSS) was analysed using the Kaplan-Meier method. Univariate and multivariate Cox regression models were evaluated to identify UA as a prognostic factor. P < 0.001 for both) in the LSCC group compared with the control group. The applied multivariate Cox regression model analysis found that low levels of UA and the UA/Cr ratio were independent poor prognostic factors for OS (UA (HR (95% CI) = 1.458 (1.095–1.942)), UA/Cr ratio (HR (95% CI) = 1.337 (1.004–1.780))), DFS (UA (HR (95% CI) = 1.504 (1.131–2.001)), UA/Cr ratio (HR (95% CI) = 1.376 (1.030–1.839))), and CSS (UA (HR (95% CI) = 1.494 (1.109–2.012)), UA/Cr ratio (HR (95% CI) = 1.420 (1.049–1.923))). The patients with high UA (>0.310 mmol/l) and UA/Cr ratio (>3.97) experienced five more years of OS, DFS, and CSS than did patients with low UA (<0.310 mmol/l) and UA/Cr ratio (<3.97) levels.  The serum UA and UA/Cr (creatinine) ratio levels were significantly reduced ( High preoperative UA serum levels were identified as an independent prognostic factor associated with improved clinical outcomes among LSCC patients. Laryngeal cancer is one of the most common cancers of the head and neck/respiratory tract, of which the estimated incidence and mortality in China in 2015 were 26,400 and 14,500, respectively . The pre60 months 40% vs. 66%, respectively; P = 0.006), which suggested that UA is an efficient predictor of survival in colon cancer patients; Strasak et al. [Q4 vs. Q1 (95% CI): 0.72 , Plinear trend = 0.043) and cancer mortality (HRQ4 vs. Q1 (95% CI): 0.75 , Plinear trend = 0.09) and indicated that higher levels of UA may be an efficient predictor of prognosis in patients with breast cancer. However, contradictory evidence from other studies does not support this hypothesis, suggesting instead that elevated levels of UA may raise the risk of cancer mortality and reduce life expectancy [Uric acid (UA), the naturally occurring product of purine metabolism, is a major water-soluble antioxidant molecule in human plasma. It has metal-chelating properties as well as the ability to scavenge nitrogen radicals and superoxide in the plasma, which helps block the generation of strong oxidant peroxynitrite , 14. In k et al.  found thk et al.  also reppectancy –26. In tpectancy . To the Although “omics”-based technology has enabled faster identification of their probable disease risk, prognosis, and/or response to treatment biomarkers, the validation of biomarkers is still stymied by high cost and poor output of results. Precision medicine relies on validated biomarkers with which to better classify patients by their probable prognosis . CurrentThis study was approved by the Ethics Committee of the Eye & ENT Hospital of Fudan University in Shanghai, China, and was conducted in accordance with the Declaration of Helsinki. Written informed consent for the use of any clinical data for research was obtained for all patients. Patients with LSCC were recruited from the Department of Laryngeal Medicine at the Eye & ENT Hospital of Fudan University using a primary cohort of consecutive patients who underwent partial or total laryngectomy between January 1, 2007, and December 31, 2011, as their first curative treatment option. All patients were followed up by telephone, and outpatient records were obtained every 3 months during the first 2 years and every 6 months thereafter until death to remain up-to-date on patient survival status, disease progress, and time of death. The last follow-up was September 30, 2016. DFS was recorded from the date of laryngectomy to the date of recurrence within the follow-up period. CSS was recorded from the date of surgery until death because of intercurrent disease within the follow-up period. OS was recorded from the date of surgery until death. Normal controls were recruited consecutively from subjects who participated in yearly health screenings during the study period. Written informed consent for the use of any clinical data for research was obtained for all normal controls. Medical examinations were performed by respective physicians for all subjects at the Eye & ENT Hospital of Fudan University.To select the LSCC group, 1295 LSCC patients who visited the Department of Laryngeal Medicine at the Eye & ENT Hospital of Fudan University between January 1, 2007, and December 31, 2011, were enrolled. The study cohort flow diagram is shown in A total of 485 normal control subjects were excluded based on the inclusion criteria. The exclusion rate for the control group was 30.69%. We performed propensity score matching analysis (1 : 1) to establish a control group from among the 2000 normal control subjects who met the inclusion criteria. The selected variable was age. A final total of 814 male control subjects were eligible for the study.All patients had histologically proven squamous cell carcinoma, confirmed by pathology and classified under the seventh edition of the TNM-UICC/AJCC stage classification (diagnostic criteria)Patients were a minimum of 18 years oldBlood samples were collected before the patients' pretreatmentComplete clinical, laboratory, imaging, and follow-up data were collectedLSCC subjects were selected from the population of inpatientsLSCC subjects voluntarily agreed to participate in this study, provided written informed consent, and were able to understand and comply with the research requirementsThe inclusion criteria and selection process for the included LSCC patients were as follows:Subjects were a minimum of 18 years of ageSubjects were maleSubjects had no LSCCComplete clinical and laboratory data were collectedSubjects voluntarily agreed to participate in this study, provided written informed consent, and were able to understand and comply with the research requirementsThe inclusion criteria for the control subjects were as follows:Subjects suffered from systemic diseases, such as infectious diseases , autoimmune disease, rapidly progressive visceral disease, and cancerSubjects were taking medications that could influence serum UA levels, for example, diuretic, benzbromarone, and losartanSubjects suffered from kidney disease or had a history of kidney diseaseSubjects suffered from hyperuricaemia or goutSubjects had undergone surgical intervention or an operationThe exclusion criteria for the LSCC patients and control subjects were as follows:Clinical and demographic information was obtained from the medical data platform of Eye & ENT Hospital by trained staff using standardized data collection and quality control procedures, which produced reliable data for analysis. For each patient, clinical and demographic information was collected from the medical data platform independently by two independent investigators. Moreover, the two independent investigators consulted the medical data platform again to resolve any disagreements. The data included the following demographic information: age, sex, UA levels, creatinine (Cr) levels, alcohol drinking habits, blood pressure, glucose levels, and smoking habits. The following clinical information was collected as well: medical history, date of diagnosis, tumour subsite, tumour size, local and regional extension category of the primary tumour, clinical stage, differentiation grade, surgical therapy experienced, and level of neck dissection. Follow-up data included date of primary resection, date and type of relapse, date of diagnosis of metastatic disease, and date of death.Patients' peripheral blood samples were collected at the first time of visits at our hospital. Meanwhile, blood samples were collected before the patients' pretreatment. Blood samples for biochemical measurements were obtained via standard venipuncture of the veins in the antecubital fossae (anterior elbow veins) the morning after subjects had fasted for 8 hours. Detection methods for UA and creatinine levels have been previously described in detail . Brieflyt-test and chi-square test were used to compare the characteristics of subjects between the groups. A one-way ANOVA test was used to compare UA, Cr, and UA/Cr ratio levels among the three groups. Receiver operating characteristic (ROC) analysis was performed to identify the sensitivity and specificity for UA and the UA/Cr ratio and the optimal cut-off values for predicting LSCC. Upon verification of the prediction value of UA and the UA/Cr ratio as a continuous variable, the relevance of the clinical stage of the categorized UA and UA/Cr ratio was evaluated by assigning subjects to two groups based on the optimal cut-off value. Logistic regression analyses were performed to identify the independent risk factors for LSCC patients. Moreover, the associations between UA, Cr, and the UA/Cr ratio and clinical stage of LSCC were examined using multiple linear regressions. Univariate Cox regression analyses were performed to determine the association between UA, UA/Cr ratios, and different clinicopathological parameters on OS, DFS, and CSS. After application of the univariate Cox regression analysis, a multivariate Cox regression analysis (adjusted for covariates) was used to analyse the association between the UA and UA/Cr ratio and the OS, DFS, and CSS of LSCC. The UA level and the UA/Cr ratio had a significance of P < 0.05 in the multivariate Cox regression analysis; thus, the regression models for OS, DFS, and CSS curves were plotted using the Kaplan-Meier method. A two-sided P < 0.05 was considered statistically significant.All analyses were performed using SPSS 13.0 software . All figures included in this study were created using GraphPad Prism 6 software . Patient demographics and clinical characteristics were displayed as frequency counts and percentages. The UA/Cr ratio equalled UA∗1000/Cr. The data are presented as the mean ± standard deviation (SD). Normality was assessed using the Kolmogorov-Smirnoff test. Independent Student's n = 191) and 21.36% (n = 174), respectively. Based on the TNM stage, the LSCC subjects were categorized into four stages, of which 195 (23.96%) were classified as stage I, 302 (37.10%) as stage II, 216 (26.54%) as stage III, and 101 (12.41%) as stage IV. Following initial entry into this study, 36.61% of the patients (n = 298) experienced tumour recurrence, of which the median recurrence interval was 23 months . The baseline characteristics and clinicopathological features of the study subjects are shown in A total of 814 male LSCC subjects and 814 male normal control subjects were eligible for inclusion in the study. The median follow-up period was 72 months (from a range of 3–116 months), and 623 (76.54%) patients were still living at the time of the last follow-up visit. The median subject survival time was 38 months . The rates of total mortality and cancer mortality were 23.46% (P < 0.001) in the control group than in the LSCC group. Logistic regression analysis showed that high levels of UA  and the UA/Cr ratio  were independent protective factors for the development of LSCC.The UA, UA/Cr ratio, and Cr levels in patients with LSCC are shown in P < 0.0001, 95% CI = 0.566–0.621), with a sensitivity of 70.27% and a specificity of 45.83% , with a sensitivity of 72.24% and a specificity of 53.20% . A similar trend was observed when UA and UA/Cr ratio levels among the four stages were compared with respect to T/N. Details are shown in Based on the TNM stage, the mean levels of serum UA and the UA/Cr ratio were the lowest among the stage IV patients, followed by stage III, stage II, and stage I patients  and the UA/Cr ratio  in the TNM stage of LSCC. Details are shown in Multiple linear regression models  for the associations between UA, Cr, and UA/Cr ratios and the overall stage of LSCC were used. The results showed a significant association between UA  and the UA/Cr ratio (UA/Cr ratio ≤ 3.98 vs. UA/Cr ratio > 3.98) in each subgroup. The distribution of the UA and UA/Cr ratio values differed significantly when the patients were stratified by T/N/TNM classification, and lower levels of UA (≤0.315) and the UA/Cr ratio (≤3.98) were significantly associated with LSCC severity. Details are shown in P = 0.008) and low UA/Cr ratio levels , P = 0.044) as poor prognostic factors for OS in this study cohort , hort see . Similarhort see .P = 0.0077), DFS , and CCS , compared with patients with UA > 0.315 , DFS , and CCS , compared with patients with a UA/Cr ratio > 3.98  was a key predictive risk factor for OS , DFS , and CSS  see . ReducedThe purpose of the present study, which involved 814 Chinese men with LSCC and 814 normal control subjects, was to evaluate the role of serum UA as an antioxidant molecule to protect against LSCC. It was found that serum UA levels were significantly lower among patients in the LSCC group compared to the sex- and age-matched control group without LSCC; in addition, the mean level of serum UA was lowest during stage IV, followed by stage III, stage II, and stage I. UA has been shown to be a biomarker for renal function; thus, UA/Cr ratios were used to reduce possible interference caused by differences in renal function , 29. UA/From the above results, the following two questions arise: First, why is the role of serum UA controversial in patients with cancer? Second, why was the concentration of serum UA lower in patients with LSCC? Limited data are available in the literature regarding the association of serum UA levels with LSCC. Two theories about the function of serum UA in cancer exist: one is that UA has a protective effect on cancer due to its antioxidant properties  and the In response to why serum UA concentrations are lower in patients with LSCC, high serum UA concentrations may have a protective effect against LSCC. Regarding this association, different factors might explain the prognostic value of serum UA: (1) a very obvious explanation could be that the patients with the most advanced disease  perhaps had changed their food intake to a lower purine-containing diet before their operations, (2) the lower levels are a result of decreased production and increased consumption of UA, and (3) UA secretion increases through the kidney . It was In addition to the role of UA as a simple biomarker, its independent prognostic role might also be explained by its antioxidant properties on tumour progression. UA is a well-known natural antioxidant present in fluids and tissues throughout the body and may contribute to up to two-thirds of the antioxidant capacity of human blood , 33. PreIn the present study, decreased UA levels (≤0.315) and UA/Cr ratios (≤3.98) were found to be key predictive risk factors for OS, DFS, and CSS. When the OS, DFS, and CSS curves were analysed via the Kaplan-Meier method and the differences assessed using a log-rank test, it was found that the UA and UA/Cr ratio levels were all positively associated with the prognosis of patients with LSCC in terms of OS, DFS, and CSS. Therefore, elevated UA levels could indicate better prognoses, a finding supported by Dziaman et al. who showAs this was the first study to focus on the evaluation of serum UA levels and UA/Cr ratios and their relationship with LSCC, it had some limitations. First, this was a single-center study, which limited the researchers' ability to explore the mechanisms underlying the associations between UA and LSCC. To compensate for this, further longitudinal studies are needed. Second, differences in dietary customs and their possible effects on serum UA concentration were not considered in the present study; thus, the UA results may not apply to populations outside of China. Only serum UA/Cr ratios were analysed with consideration for possible interference from diet customs, difference in the environment, and differences in renal function. Third, the weakness of this paper is the assumption that decreased serum UA results in increased cancer risk and not perhaps that more aggressive cancers result in decreases in uric acid levels. Fourth, the cancer cohort started with different pretreatment levels compared to the noncancer cohort, which may have introduced biases. Finally, education, liver function, total cholesterol, triglycerides, and income may be confounding factors, but we were not able to evaluate these factors in our study. Therefore, a forward-looking, multicenter study with a larger sample size should be conducted.Our study provides evidence to indicate that pretreatment UA and UA/Cr ratios have significant associations with cancer progression, OS, DFS, and CSS and that these parameters could be considered independent prognostic values for patients with LSCC.


The Cancer Attitude inventory (CAI) was developed to measure attitudes toward cancer. The aim of the present study was to describe the development of the Persian version of the CAI and to evaluate its psychometric properties in an Iranian sample.The forward–backward method was used to translate the CAI scale from English into Persian. After linguistic validation and a pilot check, a cross-sectional study was performed and psychometric properties of the Iranian version of the questionnaire were assessed.The scale validation was conducted with a convenience sample of 820 laypeople. Construct validity was assessed through both exploratory factor analysis (EFA) and confirmatory factor analysis (CFA). Internal consistency was assessed through Cronbach’s alpha analysis and test-retest analysis.p < .001), and the root mean square error of approximation (RMSEA) = .07 (90% CI = .06—.07). All comparative indices of the model had scores greater than .80, demonstrating a good fit to the data. Cronbach’s Alpha and the intra-class correlation coefficient (ICC) were .97, which is well above the acceptable threshold.Five factors were identified in CAI: isolation, helplessness, fear of consequence, belief of control and independence, and fear of death. The results achieved from the CFA displayed that the data fit the model: the relative chi-square (× 2/df) = 2.98 (The results indicate that the Persian version of the CAI is practical, reliable and valid. Consequently, the instrument could be used in plans to create positive attitudes about cancer control and treatment among Persian people. Over the past two decades, cancer incidence rates have increased, particularly in developing countries such as Iran . More thPublic attitudes toward cancer and cancer patients may significantly influence cancer control strategies and improve cancer patients’ quality of life and recovery. Furthermore, the effectiveness of cancer prevention plans is subject to public knowledge of cancer and cancer patients , 6. OverMore specifically, having a positive attitude is a supportive factor for one’s overall health, and optimism affects how one deals with cancer . ResearcIn addition, researchers have linked cultural beliefs to people’s cancer attitudes and screening behaviors , 15, 16.In Iran, obstacles to cancer prevention and screening include a deficiency of community-based events for some cancers—like cervical cancer screening, lack of knowledge, misunderstanding, and unsuitable health behaviors, as well the social and cultural issues stemming from customs, modernism, religious convictions, and bans . A systeCommon folk beliefs about cancer include that surgery spreads cancer and that a bruise or sore causes cancer . TherefoPeople who receive cancer diagnoses often feel rejected and isolated . HoweverAlthough attitudes are believed to play an important role in cancer management, surprisingly few studies have been devoted to measuring people’s attitudes toward cancer. Furthermore, the use of non-validated questionnaires makes comparison across studies difficult, if not impossible . As suchstrongly agree to 6 = strongly disagree [The CAI is a self-reported scale that consists of 41 items. Higher scores on the CAI indicate a more negative attitude toward cancer. Sample items include “A person is never really cured after cancer” and “Once you have had cancer, you can never be normal again.” Table disagree .First of all, permission was granted from the developer of CAI to translate the instrument from English into Persian by using a forward-backward translation method. First, two independent expert Persian translators who had no prior knowledge of the CAI translated the instrument into Persian. These translated versions were compared and integrated into a provisional Persian version of the CAI. This version was back-translated into English by two independent individuals who spoke fluent Persian and English and had never seen the English version of the questionnaire. These individuals translated the Persian version back into English, and an interim English version of the CAI was created. Finally, the original English version was compared with the latter version, and a number of additional minor revisions were made to make the Persian version more fluent.Thereafter, a professional team (four experts in cancer diagnoses and one epidemiologist) compared the original version of the CAI with the back-translated English version. After some cultural and linguistic adaptations, a preliminary Persian version of the CAI questionnaire was prepared. This Persian version was tested in a pilot study with 30 users who had no cancer, and thereafter, the final Persian version of the CAI was solidified.To test the psychometric properties of the Persian version of the CAI, we examined the face validity, content validity, construct validity, and reliability of the instrument.n = 10) was asked to evaluate each item of the CAI and indicate if they felt ambiguity or difficulty in replying to the items of the Iranian version of the CAI. In the quantitative phase, the item impact score (importance × frequency) was assessed to calculate the percentage of laypeople who considered each item of the CAI important or quite important on a 5-point Likert scale. Items with an impact score below 1.5 were considered inappropriate . Items that met such quantitative criteria were kept in the instrument; other items were deleted [The face validity of a scale determines the extent to which the scale seems valid. Thus, the establishment of face validity should be prioritized as the first step in the scale validation process . In this deleted . Generalnot relevant, not clear, and not simple to 4 = very relevant, very clear, and very simple. The CVI was calculated as the number of items that received a score of 3 or 4 from the experts [essential, 2 = useful but not essential, and 3 = not essential [Both qualitative and quantitative content validity of the CAI were examined after the face validity. For qualitative content validity, a panel of 10 scientific experts, including five health education experts, four cancer experts, and one epidemiologist, assessed the wording, grammar, and scaling of the CAI. For quantitative content validity, both the content validity index (CVI) and content validity ratio (CVR) were calculated. The experts were asked to indicate the relevance, simplicity, clarity, and ambiguity of each item  , 32. To  experts , 33. In  experts , 31–33. ssential . The CVRssential . Based oThe construct validity was tested using exploratory factor analysis (EFA), confirmatory factor analysis (CFA), and item-scale correlation for the 41 CAI items after testing their face and content validity.To test the psychometric properties of the CAI in an extensive setting, a cross-sectional study was conducted in Zanjan, Iran, in November 2017. The study population was healthy laypeople aged 20–50 years. The research environment was public places, including cultural centers, parks, universities, and so on, that provide easy access to research samples. With this aim, a convenience sampling method was used. People who were in public places were approached. If they could read and write in Persian, they were asked to participate. The sample size was estimated a priori. As concluded by Gable and Wolf (2012), a sample of five to ten persons per item is required to ensure a theoretically clear factor structure for EFA and CFA . The desAfter content validity, the construct validity of 41 items was examined by performing EFA to detect the main factors of the CAI. For this purpose, 410 lay people were selected in public places of Zanjan using a convenience sampling method.In this case, a principal component analysis (PCA) with varimax rotation was applied to extract the main factors of CAI. Furthermore, to assess the sampling adequacy for the factor analysis, the Kaiser-Meyer-Olkin (KMO) measure and Bartlett’s test of sphericity were applied . Any facIn order to assess the coherence between the data, a CFA was applied. Therefore, 410 lay people were selected using a convenience sampling method. The model fit was evaluated by using multiple fit indices. The model fit was evaluated using several fit indices, including relative chi-square, goodness-of-fit index (GFI), normed fit index (NFI), comparative fit index (CFI), root mean square error of approximation (RMSEA), and standardized root mean square residual (SRMR) . RelativLastly, item-scale correlation was applied to measure the degree to which each CAI item was correlated to its hypothesized subscale. Pearson correlation coefficient values equal to or above 0.4 were considered satisfactory .Cronbach’s’ alpha coefficient was applied to assess internal consistency of the whole scale and individual dimensions of the CAI questionnaire. The acceptable level of alpha values was 0.70 or higher , 44.n = 40) completed the CAI twice with a 2-week interval. ICC values of .40 or above were considered acceptable [Test-retest reliability was applied to examine the questionnaire’s stability by estimating the intra-class correlation coefficient (ICC). A separate sample of laypeople (ceptable . SPSS veceptable .p < .001), indicating adequacy of the sample for EFA. A five-factor solution for the 41-item instrument was revealed based on eigenvalues greater than 1 and factor loadings equal to 0.4 or above. The five-factor solution jointly explained 62.077% of the total observed variance . The RMSEA of the model was .07 (90% CI = .06–.07), and the SRMR was .050. All comparative indices of the model, including GFI, AGFI, CFI, RMR, NFI, and NNFI, were more than .80 , demonstrating a good fit to the data.A CFA was conducted on the 41-item scale in order to assess the fitness of the model obtained from the EFA. Covariance matrixes were used and fit indices were calculated. Figure The item-scale correlation for the CAI is shown in Table Cronbach’s alpha was calculated separately for CAI as well as for each factor of the CAI. Cronbach’s alpha coefficient for CAI was .97, which showed excellent and high internal reliability. Thus, no items of the instrument were removed in this step. In addition, test-retest analysis was performed to assess the stability of the CAI. The results showed satisfactory results. Intra-class correlation (ICC) was .97 for the CAI, providing support for the stability of the scale Table .Table 4In the present study, we described the translation and psychometric properties of the Iranian CAI, an instrument to measure attitudes toward cancer that might have an impact on the control and treatment behaviors for cancer. Overall, the results proved that the translated CAI is an appropriate and valid scale that can be used to assess attitudes toward cancer among individuals who speak Persian.Based on the results, the CVI and the CVR indicated good content validity. Furthermore, consistent with the original CAI , CronbacThe Farsi version of the CAI consisted of 41 items and resulted in five conceptual dimensions. The resulting five-factor solution  produced in the present study is sufficient for understanding attitudes toward cancer, although the solution is different from the original version of the CAI , which sThe relationships between attitudes toward cancer and fear of death, fear of consequence, helplessness, and isolation grow out of common sense. Still, providing factual knowledge about cancer is not sufficient to change people’s attitudes toward cancer. Fear of cancer and its consequences, such as severe illness or death, result in psychological anxiety and tension, which in turn affect people’s quality of life, decisions about whether to continue cancer treatment, and how they live the remaining days of their lives .Individuals respond in various ways when they have to deal with anxiety about death or consequences of disease. Some people may increase health-control behaviors, while others decrease these behaviors , 49. DesWe also conducted a CFA to determine whether there was coherence between the hypothetical construction of the CAI and data. The CFA delivered good fit indices for this model, and the convergent validity of the five factors of the CAI was suitable. These findings are consistent with the results of the original study , indicatThis study has some limitations. First, regarding the participants, we only engaged Iranian people without cancer, which possibly limited the external validity of this scale. Although healthy people vary both across and within countries as much as the general public , 46, we Other limitations related to the nature of survey designs is that respondents may not feel encouraged to provide accurate, honest answers or that they might not feel comfortable providing answers that present themselves in an unfavorable manner. Another limitation of the present research is linked to its sample size and its generalizability. The present sample was limited to a convenience sample of 820 laypeople, and whether we would achieve the same outcomes if we recruited a large representative group of both healthy people and cancer patients is unknown. As such, the present study is unable to measure differences in healthy people and cancer patients. Future studies should aim to include both groups to measure whether attitudes toward cancer and controlling behaviors are similar among healthy people and cancer patients and whether being healthy or having cancer influences individuals’ responses to treatment.Our study demonstrated that the psychometric properties of the Persian version of the CAI are appropriate. We believe the Persian version of the CAI may be helpful for healthcare groups to plan health methods that are practical and targeted to specific situations. Further studies in healthy persons and cancer patients are suggested to establish the psychometric properties of the Iranian version of the CAI.


To assess the prevalence of HTLV-1 and HTLV-2 infections in a cohort of immigrants living in southern Italy.We screened for antibody to HTLV-1/2 infection 1,498 consecutive immigrants born in endemic areas (sub-Saharan Africa or southern-Asia) by a commercial chemiluminescent microparticle immunoassay. If confirmed in a Western blot assay, which differentiates anti-HTLV-1 from anti-HTLV-2, the positive sera were tested for specific HTLV RNA by a home-made PCR. The immigrants investigated were more frequently males (89.05%), young (median age 26 years), with a low level of education (median schooling 6 years), born in sub-Saharan Africa (79.70%). They had been living in Italy for a median period of 5 months. Only one (0.07%) subject was anti-HTLV-1 -positive/HTLV-1 RNA-negative; he was an asymptomatic 27-year-old male from Nigeria with 6 years’ schooling who stated unsafe sexual habits and unsafe injection therapy.The data suggest screening for HTLV1 and HTLV-2 infections all blood donors to Italy from endemic countries at least on their first donation; however, a cost-effectiveness study is needed to clarify this topic. As in Italy the immigrants constitute one tenth of the whole population and its contribution to blood donation is strongly desired we believe that to start screening for HTLV-1 and HTLV-2 infections in all donors from endemic countries at least on their first donation is a practice useful for a correct cost-effectiveness analysis and for a conclusive decision on this topic. Human T cell leukemia virus type 1 (HTLV-1) or 2 (HTLV-2) infection has a worldwide distribution, with an estimate of up to 15–20 million people affected . EndemicOnce acquired, HTLV-1 infections persist life-long, most patients remaining asymptomatic viral reservoirs ensuring the transmission chain, but about 4% develop adult T-cell leukemia/lymphoma (ATLL), a highly aggressive CD4+ T-cell malignancy. Type 1 virus has been associated with other diseases, ranging from a mild non-specific dermatitis and uveitis to a disabling HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) affecting 2–3% of the infected people . HTLV-2 HTLV transmission is similar to that of human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV), but is less effective. The most important routes of HTLV-1/2 transmission are vertical transmission, unsafe sexual habits, transfusion of blood or blood products and needle sharing . In partDue to socio-economic and political crises in several countries in Africa, Eastern Europe and Central and Eastern Asia in recent decades, Italy has become a land of immigration from areas with an intermediate or high HTLV endemicity.In literature there are few data about the seroprevalence of HTLV-1 and HTLV-2 infection in immigrants from endemic areas for this infection ,12; moreAll procedures used were in accordance with the international guidelines and with the Helsinki Declaration of 1975 and revised in 1983. The Ethics Committee of the Azienda Ospedaliera Universitaria of the University of Campania “Luigi Vanvitelli” approved the study (214/2012). All patients signed an informed consent for the collection and storage of biological samples and for the anonymous use of their data for research purposes.Estimating a prevalence about 3% –4 for HTThe first-level clinical centers involved in the present study are clinical centers of international charity organizations empowered by the Italian National Healthcare System, with proven experience in the clinical, psychological and legal management of vulnerable groups, as previously specified –15 Each The study population consists of all the undocumented or refugee immigrants from sub-Saharan Africa and the India-Pakistan sub-continent consecutively seen for a clinical consultation. During the consultation, a physician from the clinical center and a cultural mediator explained to the immigrants the importance of testing for HBV, HCV, HIV and HTLV serum markers and offered them screening free of charge, in anonymity and in full accordance with the law on privacy.Informed consent to participate in a surveillance and monitoring program covering different viral infections, including HTLV-1 and HTLV-2, was obtained on a voluntary basis. Of the interviewed immigrants 94.7% agreed to participate and were included in the present study. Patients who did not consent to participation in the study received the same treatment if they had any medical problem.The participants were interviewed and relevant information was collected, including socio-demographic data and risk factors. More precisely, we recorded age, sex, geographical origin, time of immigration, level of education, religion, cohabitation details, sexual habits, a history of previous vaccination, surgery, dental care, tattooing, piercing, drug addiction, blood transfusion, tribal rituals (body scarification and/or infibulation) and abortion. In each case the clinical history was obtained with the help of a physician and a cultural mediator in the course of a prolonged, in-depth clinical consultation and counseling.The serum samples obtained were transported to the laboratory of infectious diseases of the University of Campania Luigi Vanvitelli—Naples, where they were tested for HBsAg, anti-HBc, anti-HCV, anti-HIV and anti- HTLV-I/II. All undocumented immigrants and refugees received the results of their serological screening and full instructions on the prevention and transmission of the infections.Serum samples were tested for HTLV by a commercial chemiluminescent microparticle immunoassay  and those positive by Western blot to confirm HTLV-1 or -2 infections (MP diagnostics HTLV- I/II blot 2.4). The Q6138 region was amplified by polymerase chain reaction (PCR) from whole blood sample  to confiThe demographic and serological data of the 1,498 immigrants obtained at the time of enrolment are shown in .These subjects, prevalently young males and with a low level of education, had been living in Italy for a median period of 5 months; 48.26% of them were undocumented immigrants, 45.86% low income refugees and 5.87% did not state their legal status . Most of shows thOf the 1,498 immigrants enrolled, only one (0.07%) was anti-HTLV-1 positive, 153 (10.2%) HBsAg-positive, 605 (40.39%) HBsAg-negative/anti-HBc-positive, 74 (4.9%) anti-HCV-positive, 24 (1.6%) anti-HIV-positive and 13 (0.87%) had a multiple infection  . No patiIn the present study, enrolling 1,498 African and Asian immigrants living in Italy, the prevalence of positive immunoezymatic tests for HTLV-1, confirmed by Western-blot test, was 0.07%. The only HTLV-1 subject infected was asymptomatic and HTLV-1-RNA-negative, in accordance with the literature data reporting a low prevalence of HTLV-1-RNA-positive patients among the anti-HTLV-1 positive ,6.In literature there are few data on the prevalence of HTLV-1/2 in immigrants  and, to The data on the prevalence of HTLV 1–2 infection in this population are useful to devise proper healthcare strategies of screening for immigrant populations from different countries and to evaluate the need for screening of blood donors, today not indicated for this infection in Italy. In fact, in high-income countries, considering the low prevalence of HTLV-1 infection and the low rate of disease onset (below 10%), cost-effectiveness of universal HTLV-1 screening of blood donors is debated. However, the identification of HTLV-1 carriers is nevertheless important to avoid transmission through blood transfusion. The World Health Organization global database on blood safety does not include HTLV testing as one of its data collected from some countries around the world. The following countries test all blood donations for HTLV-1 and -2 antibody: Argentina, Australia, Brazil, USA, Canada, China (some regions)(13), Colombia, French West Indies, Iran, Israel, Jamaica, Japan, New Zealand, Saudi Arabia, Peru, Sweden, Taiwan, Uruguay and Venezuela. In Europe, HTLV-1/2 antibody testing is currently performed on all blood donations in France, Greece, Ireland, Netherlands, Portugal, Romania, United Kingdom and on first time blood donations in Denmark, Finland, Norway and Sweden .Another important question is the high prevalence of HBV and HCV infection among immigrants living in Western countries ,14,20,21Worthy of mention, in Italy the immigrant population constitutes one tenth of the whole population and its contribution to blood donation is strongly desired. Although HTLV-1 and HTLV-2 infections were rarely detected in the immigrant population investigated in this study, we believe that every effort must be made to make all blood donations as safe as possible. It may be advisable for the Italian Healthcare Authorities to start screening for HTLV-1 and HTLV-2 infections all donors from endemic countries at least on their first donation, a practice useful for a correct cost-effectiveness analysis and for a conclusive decision on this topic. In fact, even if the prevalence of HTLV-1 and HTLV-2 is low, it is not negligible and, considering the transmissibility through blood and sexual route, could represent a significant problem in Western countries both in terms economic and public health. However, a cost-effectiveness study is needed to clarify this point.S1 Checklist(DOCX)Click here for additional data file.


O-methylated plant MIR167e-5p, followed by a treatment with 20 pmol of MIR167e-5p for 0, 24, 48, and 72 h. The cells were counted, and IPEC-J2 cell viability was determined by the MTT and EdU assays at different time points. The results showed that MIR167e-5p significantly inhibited the proliferation of enterocytes in a dose- and time-dependent manner. Bioinformatics prediction and a luciferase reporter assay indicated that MIR167e-5p targets β-catenin. In IPEC-J2 and Caco-2 cells, MIR167e-5p suppressed proliferation by downregulating β-catenin mRNA and protein levels. MIR167e-5p relieved this inhibition. Similar results were achieved for the β-catenin downstream target gene c-Myc and the proliferation-associated gene PCNA. This research demonstrates that plant MIR167e-5p can inhibit enterocyte proliferation by targeting the β-catenin pathway. More importantly, plant miRNAs may be a new class of bioactive molecules for epigenetic regulation in humans and animals.MicroRNAs (miRNAs) are important negative regulators of genes involved in physiological and pathological processes in plants and animals. It is worth exploring whether plant miRNAs play a cross-kingdom regulatory role in animals. Herein, we found that plant MIR167e-5p regulates the proliferation of enterocytes in vitro. A porcine jejunum epithelial cell line (IPEC-J2) and a human colon carcinoma cell line (Caco-2) were treated with 0, 10, 20, and 40 pmol of synthetic 2′- PB2 and NS1 genes encoded by influenza A viruses (IAVs) H1N1, inhibiting H1N1 viral replication [Cuscuta campestris miRNAs were induced through the haustorium to target and reduce the accumulation of mRNAs, such as those for F-box/RNI-like superfamily protein (TIR1) and sieve element occlusion amino-terminus protein 1 (SEOR1), in host plants [MicroRNAs (miRNAs) are critical biological molecules and have attracted much attention for more than a decade. The functions of miRNA are well established in plants and animals. These single-stranded molecules impact on fundamental biological processes, including proliferation, differentiation, immune responses, and metabolism in different species. The recent detection of plant miRNAs in body fluids, including serum , urine , saliva lication . MIR159 lication . MIR156alication . Plant Mt plants . These fLactobacillus rhamnosus (LGG) monooxygenase ycnE yielded increased indole-3-carboxaldehyde, inducing the production of interleukin (IL)-22 and ameliorating mouse colitis via IL-22-dependent mechanisms [Some researchers have identified all intestinal miRNAs and shown that miRNAs play a vital role in the differentiation and function of the intestine by using gene ablation of Dicer1 . A studychanisms . MiRNAs The intestinal tract not only is the first line of defense against harmful pathogens, but also is the main place for the digestion and absorption of nutrients. Ontogenetic development of the mammalian intestine is a topologically and temporally highly organized process. Intestinal stem cells (ISCs) constantly produce enterocytes and endocrine cells to maintain homeostasis in the intestinal epithelium . One of We hypothesized that plant miRNA might be an exogenous regulator influencing animal enterocytes’ functions. We found that plant MIR167e-5p could inhibit enterocyte proliferation and further identified β-catenin as a target of MIR167e-5p. This study demonstrates that plant miRNAs can influence the growth of enterocytes and thus serve as important bioactive molecules.5, 4 × 105, and 2 × 105 cells, respectively, in 12-well plates overnight (70–80% confluency) and transfected for 6 hours (h) the following day using Lipofectamine 2000 , according to the manufacturer’s instructions. The treatment groups were as follow: MIR167e-5p group , NC group , MIR167e-5p inhibitor group , i-NC group , MIR167e-5p+inhibitor group (20 pmol MIR167e-5p mimics +40 pmol MIR167e-5p inhibitor). The cells were harvested at 0, 24, 48, or 72h after transfection for cell counting, real-time quantitative PCR (qPCR), and western blot analysis.IPEC-J2, STC-1, and Caco-2 cells were seeded at a density of 1 × 10Total RNA was extracted from the cells using TRIzol Reagent (Invitrogen) according to the manufactures’ instruction. RNA concentration was determined by an ND-2000 Nanodrop spectrophotometer . RNA integrity was examined by gel electrophoresis using a 0.8% agarose gel. The RNA was used for further PCR and qPCR analyses.2O, and qPCR (20 μL) was performed on a Bio-Rad CFX Manager 3.1 instrument  with U6 (miRNA control) or β-actin (mRNA control) as internal controls. The reactions contained 2 μL cDNA, 10 μL 2× GoTaq Qpcr Master Mix (Promega), and 0.4 μL of each primer (10 μM). The thermal profile of real-time PCR involved an initial denaturation step at 95 °C for 5 min (min), followed by 40 cycles at 94 °C for 15 s, 15 s at the corresponding annealing temperature (Tm), extension at 72 °C for 30 s, followed by a quick denaturation at 95 °C for 10 s, plus a slow ramp from Tm to 95 °C to generate a melting curve to confirm the specificity of the amplified product. A no template (NT) negative control was included for each miRNA and mRNA, and all reactions were performed in triplicate. The 2−ΔCt method was employed to quantify and normalize the expression data. The miRNAs, mRNAs primers, and specific stem-loop RT primers were designed with Premier Primer 5.0   was reverse-transcribed to cDNA using M-MLV-R transcriptase  with OligodT18 (for mRNA) or a specific stem-loop RT primer (for miRNA). The samples were amplified by PCR using complementary primers. To quantify miRNAs and mRNAs, cDNA was diluted 5-fold with ddHCA, USA) .Total proteins were isolated from the cells using RIPA buffer with 1 nM phenylmethylsulphonyl fluoride (PMSF) and quantified with a BCA Total Protein Assay Kit . The protein suspension (30 μg) was supplemented with β-mercaptoethanol, boiled for 10 min, resolved by 5% and 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride membrane. β-catenin and c-Myc were detected by incubation with primary anti-human β-catenin  and anti-human c-Myc  antibodies, followed by incubation with rabbit HRP-linked IgG1 secondary antibodies . β-actin  served as a control. Band intensities were measured using Image J and were normalized against β-actin band intensity.XhoI and XbaI sites). HeLa cells were seeded in 96-well cell culture plates (3.5 × 104 cells per well) and cultured in RPMI 1640  with 10% fetal bovine serum (FBS). The next day, the cells were transfected with recombinant pmirGLO-3′UTR vector (100 ng/well) mixed with their corresponding MIR167e-5p mimics or NC  for 6 h using Lipofectamine 2000 (Invitrogen). The cells were harvested 24 h after transfection, and luciferase activity was detected by a dual luciferase reporter assay system (Promega) according to the manufacturer′s recommendations. The normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) of each construct was compared with that of the pmirGLO vector.On the basis of the predicted miRNA–mRNAs binding sequences, normal sequences bearing an MIR167e-5p target site or with MIR167e-5p target site-deleted β-catenin 3′-UTR were generated by two complementary chemically synthesized primers (Sangon Biotech) as follows: wt-β-catenin-3′UTR-sense (tcgagttgtatctaaagtccggtgttgccagcttcagttggttcctgt), wt-β-catenin-3′UTR-antisense (ctagaggaaccaactgaagctggcaacaccggactttagatacaac), del-β-catenin-3′UTR-sense (tcgagttgtatctaaagtccggtgttgcagttggttcctgt), and del-β-catenin-3′UTR-antisense (ctagacaggaaccaactgcaacaccggactttagatacaac). The complementary oligonucleotides were re-suspended at a 1:1 ratio (1 μg/μL each) in annealing buffer  and heated at 95 °C for 10 min to denature the secondary structures. The temperature was then gradually lowered to room temperature. Annealed products were cloned into the pmir-GLO vector (Promega) downstream from the firefly luciferase coding region  and used according to the manufacturer’s protocol. Briefly, IPEC-J2 cells were seeded in 96-well plates at a density of 4000 cells per well with 200 μL of complete culture medium. After being allowed to adhere and spread for 12 h, the cells were treated with 3 pmol of MIR167e-5p, NC, inhibitor, and i-NC for 0, 24, 48, and 72 h. MTT assays were performed by incubating the treated IPEC-J2 cells with 20 μL (5 mg/mL) of MTT labelling solution. After 4 h of incubation, IPEC-J2 were lysed with 150 μL of DMSO, and the purple formazan crystals were solubilized for detection at 570 nm.Cell proliferation was assessed by a Cell-Light EdU Apollo 488 In Vitro Imaging Kit (Ribobio), according to the manufacturer’s instructions. IPEC-J2 cells were incubated with 1:1000 EdU diluted in culture solution for 2 h at 37 °C before harvesting. After several washes, the cells were fixed with 4% paraformaldehyde (pH 7.4) for 30 min and incubated with glycine (2 mg/mL) for 5 min. The fixed cells were washed with 0.5% Triton-100 ) for 10 min, followed by staining with Apollo buffer (RiboBio) for 30 min at room temperature in the dark. The cells were incubated with Hoechst 33342 dye at room temperature for 30 min in the dark. Finally, images were captured using fluorescent microscopy and IR–DIC imaging on an Eclipse FN-1 upright microscope  at a magnification of 100×.t-tests were employed for pairwise comparisons . Data are presented as the mean ± standard error (S.E.), and all analyses were performed using GraphPad Prism software 6.0 .Least significant difference (LSD) tests were used for multiple comparisons, and O-methylated plant MIR167e-5p or 2′-O-methylated control nucleic acids (NC). The cell number in each sample was calculated 24 h after treatment with 0, 10, 20, and 40 pmol MIR167e-5p. We observed that 10 pmol MIR167e-5p determined a remarkable decrease of IPEC-J2 , there are 63 miRNAs sharing identical sequences (UGAAGCUGCCAGCAUGAUCUG). These miRNAs have been found in 27 plants, including soybean [Moringa oleifera, a widespread plant with substantial nutritional and medicinal value, and has a potential for cross-species control of human gene expression [Lactobacillus rhamnosus in the mouse intestine [MIR167e-5p is a conserved and highly expressed miRNA throughout the plant kingdom. In miRBase ( soybean , wheat [ soybean , and mai soybean , which tpression . It was pression , human bpression , porcinepression , and honpression . Maize Mpression . A studypression . Ginger ntestine . These sPB2 and NS1 genes and inhibit H1N1 viral replication [Plant miRNAs are of potential biological function in mammalian cells. Plant MIR2911 has been reported to target the lication . MIR159 lication . MIR156alication . Plant Mlication . In the β-catenin is a crucial component of the Wnt signaling pathway, which is crucial in many developmental processes , includiAs an essential organ for digestion and absorption, the intestine is the largest immune organ and plays a key role in maintaining animal health and growth. Recent studies have indicated that miRNAs can regulate gene expression in different species, and plant miRNAs regulate the intestinal flora and, thus, intestinal development. MiRNAs in EPDELNs have been detected and found to have the potential to regulate human mRNAs . GELNs cIn conclusion, this study provides new evidence supporting the idea that plant miRNAs can exert cross-kingdom regulatory functions in mammals. We provide the first evidence that plant MIR167e-5p suppresses intestinal cell proliferation by targeting β-catenin. We suggest that the large variety of plant miRNAs should be considered as a new class of nutrients, which has been neglected for centuries.


Jasmonic acid (JA) and its precursors and dervatives, referred as jasmonates (JAs) are important molecules in the regulation of many physiological processes in plant growth and development, and especially the mediation of plant responses to biotic and abiotic stresses. JAs biosynthesis, perception, transport, signal transduction and action have been extensively investigated. In this review, we will discuss the initiation of JA signaling with a focus on environmental signal perception and transduction, JA biosynthesis and metabolism, transport of signaling molecules , and biological function . JA and its methyl ester (MeJA) and isoleucine conjugate (JA-Ile) are derivatives of a class of fatty acids and are collectively known as jasmonates (JAs). Initially identified as a stress-related hormone, JAs are also involved in the regulation of important growth and developmental processes [Plants undergo many physiological changes to cope with biotic and abiotic stress. The survival of plants mainly depends on their ability to adapt in a varying environment through signaling networks . These nrocesses ,4. For erocesses . JAs andLycopersicon esculentum), Pearce et al. in 1991 found a systemin that responded to mechanical damage such as insect damage [In the last decades, a large number of studies have been conducted on how biotic and abiotic stress signals are perceived by plants and the biosynthesis of JA is initiated. In tomato  to AtPEP1, which binds to the receptor PEPR1 (an enzyme rich in leucine repeat units) on the plasma membrane, which ultimately activates the JA signaling pathway [Later, a polypeptide having the same function as a systemin, AtPEP1, consisting of 23 amino acids, was also found in  pathway .Arabidopsis, and these phospholipases have similar mechanisms of action [The process by which systemin and AtPEP1 bind to the receptor to activate the JA signaling pathway is complex. It is known that the most important process is to activate phospholipase on the plasma membrane, and then the phospholipase acts on the membrane to release linolenic acid (a precursor of JA synthesis) from the phospholipid . Howeverf action ,15,16.Arabidopsis thaliana and L. esculentum (tomato). So far, various enzymes in the JAs synthetic pathway have been identified, and our knowledge of the relationship between the JA synthesis pathway and other metabolic pathways is gradually improving  and the hexadecane pathway starting from hexadecatrienoic acid (16:3) [cis-jasmone (CJ), and 12-hydroxyjasmonic acid (12-OH-JA).Biosynthesis of JAs has been studied in a variety of monocotyledonous and dicotyledonous plants during the last decades. Most of the work is done in the model plants mproving . In Arabd (16:3) . All thrThe defense response triggered by a traumatic signal can result in a local defense response near the wound and/or a systemic acquired resistance (SAR) at the uninjured site, and/or even induced defense responses from adjacent plants. In these defense responses, short-distance transmission and long-distance transmission of JA signals are involved . With thIn plants, mechanical damage or insect feeding can cause rapid and transient accumulation of JA and JA-Ile at the site of injury, thereby activating the expression of defense genes surrounding the wound and producing a local defense response. In the local defense response, there are two ways of short-distance transmission of the JA signal. First, the systemin produced by the wounding acts as a signaling molecule, which is transmitted to the adjacent site through the apoplast and phloem to activate the JA cascade reaction pathway. Second, JA and JA-Ile induced by systemin act as signals and are transported to adjacent sites for defensive responses .So far, it is known that the long-distance transmission of JA signals is via vascular bundle transmission and/or airborne transmission.jai1), systemin-insensitive mutant (spr1), and the JAs biosynthesis deletion mutants spr2 and acx1A demonstrated that the systemin was not the systemically transmitted signal [Previously, many researchers believed that systemin functions directly in the long-distance signal transmission and is a mobile signal molecule. However, a series of grafting experiments using tomato jasmonate-insensitive mutant  mutant lost all responses to JA [COI1 gene encodes an F-box protein that is a component of E3 ubiquitin ligase [COI1, which targets the repressor proteins for degradation by ubiquitination [The understanding of JA receptors has undergone a complex process. In 1994, Feys first found that the es to JA , and furn ligase . In thistination ,35. The Arabidopsis, most of which have two conserved domains, Jas and ZIM [It was once thought that COI1 is the receptor for jasmonic acid signaling in cells, until the discovery of a jasmonate Zinc finger Inflorescence Meristem (ZIM)-domain (JAZ) protein family, which gave a new understanding of the jasmonic acid signal transduction pathway. In 2007, three research groups simultaneously found that JAZ proteins act as repressors in the JA signaling pathway ,37,38. T and ZIM . The JAZ and ZIM . Therefo and ZIM . It is cJA-Ile activates the MYC transcription factors by directly binding to JAZ and COI1, which results in the degradation of JAZ through the 26S proteasome pathway . Recent JIN1 gene. Most members of the JAZ protein family interact with MYC2 [The basic helix–loop–helix (bHLH) transcription factor MYC2 is a well-known regulatory protein encoded by the ith MYC2 . For a lith MYC2 . A closeith MYC2 . Besidesith MYC2 .Arabidopsis, MYB21 and MYB24 are key factors in stamen and pollen maturation [Most of the JAs-responsive MYB TFs belong to the R2R3-MYB family, which are widely distributed in plants and required for many processes. Schmiesing et al. showed that MYB51 and MYB34 regulate the synthesis of tryptophan and glucosinolates and act downstream of MYC2 . Howeverturation , and MYBturation . Recentlturation .Arabidopsis NAC family are both induced by JA signaling and involved in plant resistance to drought, salt stress, Botrytis cinerea, and other pathogens [Arabidopsis, ANAC019 and ANAC055, are also present downstream of the MYC2 protein and regulate seed germination, cell division, and the synthesis of secondary walls of cells [ATAF1 and ATAF2 TFs in the athogens . At the athogens . Two othof cells ,54,55.ERF genes. The first evidence for a link between AP2/ERF TFs and JA signaling was found in Catharanthus roseus. The JAs-induced ORCA proteins, ORCA2 and ORCA3, belong to the AP2/ERF-domain family and can activate the expression of monoterpenoid indole alkaloid biosynthesis genes [Arabidopsis ERF proteins, ERF1 and ORA59, function dependently on JAs and/or ET for the defenses against Botrytis cinerea [AOC2, indicating that ORA47 might act as an important regulator in the positive JAs-responsive feedback loop [Microarray experiments at the genetic level have confirmed that JA signaling can induce the transcription of many is genes . Based o cinerea ,58. Moreack loop . Moreoveack loop . InteresArabidopsis, there are 89 members in the WRKY transcription factor family. It has been shown that some WRKY TFs are regulated by the JA signaling pathway, such as WRKY70 [Nicotiana attenuata, two WRKY transcription factors, NaWRKY3 and NaWRKY6, regulate the expression of JAs biosynthesis-related genes  to increase the levels of JA and JA-Ile [Arabidopsis WRKY57 interacts with the inhibitor JAZ4/JAZ8 in the JA signaling pathway and the inhibitor IAA29 in the auxin signaling pathway, thereby regulating the interaction between JA and auxin-mediated signaling pathways and effects on plant leaf senescence [WRKY transcription factors play an important regulatory role in plant development, senescence, and coping with environmental stress. In s WRKY70 , WRKY22 s WRKY70 , WRKY50 s WRKY70 , WRKY57 s WRKY70 , and WRKs WRKY70 . These Wd JA-Ile . In addinescence .JA and its derivatives are plant signaling molecules closely related to plant defense and resistance to microbial pathogens, herbivorous insects, wounding, drought, salt stress, and low temperature. In addition to the traumatic signals, oligosaccharide signals induced by pathogens and fungal elicitors were also found in tomatoes, which ultimately activated the JA signaling pathway. Upon mechanical wounding in tomato, the prosystemin is hydrolyzed into a systemin, which can be transported to other cells via the apoplast and interacts with the cell surface receptor SR160 (a protein rich in leucine repeat units) to finally activate JAs responses. In this section, we will discuss the role of JA signaling in regulating plant responses in varying environments.Arabidopsis and rice to be affected by JA signaling [Arabidopsis and tomato [Nicotiana and Brassica genera to initiate the JA signaling pathway, and these processes are associated with chemical defenses of plants [To a large extent, the early development of plants is affected by light. It is observed that JA signaling mediates two aspects of the plant response to light: the photomorphogenesis of plants and the damage of plants by UVB. Red light/far-red light-mediated photomorphogenesis was observed in ignaling . The invd tomato ,70. Enhaf plants ,71.MaCBF1, MaCBF2, MaCOR1, MaKIN2, MaRD2, MaRD5, etc.) after cold storage, thereby reducing the damage of plants caused by cold [Musa acuminata) decreased slightly after low-temperature treatment, and the change was not significant [L. esculentum) [Punica granatum) [Eribotrya japonica) [Mangifera indica) [Psidium guajava) [Aquilaria sinensis) [The JA signaling pathway is involved in the response and adaptation process of plants to low temperatures. Recent studies on bananas have shown that the MeJA treatment can significantly induce the expression of MYC-family TFs and many cold-responsive genes ( by cold . It was nificant . Howeverulentum) , pomegraranatum) , loquat aponica) , mango ( indica) , and guaguajava)  have shoinensis) .Arabidopsis thaliana [Citrus paradisi × Poncirus trifoliate) [Arachis hypogaea) seedlings treated with MeJA showed enhanced drought resistance [Oryza sativa) [Glycine max) [Brassica oleracea) [Vicia faba) and barley (Hordeum vulgare) have shown that MeJA may regulate stomatal closure through K+ channels, thereby increasing the ability of plants to resist drought [Many studies have found that JA signaling pathways are involved in drought stress. In thaliana  and citrfoliate) , it was sistance . It was  sativa) , soybeanine max) , and broleracea)  by adjus drought ,86.Arabidopsis thaliana [L. esculentum) [Solanum tuberosum) [Capsicum annuum) [Rupestris riparia) [It was found that the endogenous JA content increased significantly after salt treatment in thaliana , tomato ulentum) , and potberosum) , among oberosum) . It has  annuum)  and verbriparia)  to salt 2  led to an increase in the release of JAs (MeJA and cis-JA) into the environment in lima bean (P. lunatus) [2 concentrations, and CO2-induced changes in plant resistance may result in genotypic-specific responses of plants to nematodes under elevated CO2 conditions [2.Ballhorn et al. found that high concentrations of COlunatus) . The indnditions . HoweverArabidopsis increased significantly after ozone treatment. Experiments with the ozone-sensitive mutants rcd1  [oji1 (ozone-sensitive and jasmonate-insensitive) [jar1 [Populus maximowizii × P. trichocarpa) [L. esculentum) [Gossypium hirsutum) have shown that MeJA exhibits inhibition of ozone damage diffusion only at high concentrations (volume fraction: 685) of ozone, accompanied by antagonism with ethylene [The endogenous JA content of wild-type death 1)  and oji1nsitive) ,95 and te) [jar1  have shohocarpa)  and tomaulentum)  to ozoneethylene .Arabidopsis using gene chip technology. Among them, 74 genes were upregulated, including JAs biosynthesis genes, various defense genes , oxidative stress genes , aging-related genes, cell wall modification-related genes, hormone metabolism-related  genes, and genes involved in storage, signal transduction, and primary and secondary metabolism. The 63 downregulated genes included photosynthesis-related genes , cold regulatory genes, drought-responsive genes, defense-responsive genes, plant growth and development-related genes, cell wall-modification -related genes, and some other unknown functional genes, etc. [Arabidopsis thaliana before and after MeJA treatment by proteomics and found 186 differentially expressed proteins. These proteins are involved in plant photosynthesis, carbohydrate metabolism, hormone metabolism, secondary metabolism, product transport, stress and defense, and gene transcription [RNA-seq and proteomics studies further confirm the involvement of the JA signaling pathway in regulating the physiological processes of plants. Systematic biological approaches such as using gene chip and proteomics can study various physiological processes of plants at the whole genome level, and thus analyze the relationship between various metabolic pathways. Jung et al. identified 137 genes with altered expression levels in MeJA-treated es, etc. . Chen etcription .In the last decades, the JA signaling pathway has been studied extensively, but our knowledge about the role of JA signaling in response to different environmental stimuli is limited. Environmental signals usually result in a complex response network regulated by multiple signaling pathways. The mechanism of action of JA signaling in plant–environment interaction is still not clear. A series of signal transduction pathways related to JAs biosynthesis and transmission are well known, but it has not been systematically studied how different environmental signals are perceived by plants and initiate JAs synthesis. Due to the large number of receptors and kinases on the cell membrane, different biotic and abiotic signals stimulate the activation of different enzymes, accompanied by a series of complex reactions, such as calcium channel and potassium channel opening. Therefore, the perception of environmental signals has still a lot of research space. The studies on JAs receptors has made great progress, and the JA signal transduction pathway has also been established, but there are still many questions regarding the regulatory process which need to be answered.