A small number of studies suggested that air pollution was associated with idiopathic pulmonary fibrosis (IPF) exacerbation, incidence and mortality. However, no studies to date were conducted in regions where air pollution is substantial. We aimed to investigate whether there are associations between acute increases in air pollution and hospitalization of patients with a confirmed primary diagnosis of IPF in Beijing.10, PM2.5, NO2, Ozone, SO2 were obtained from 35 municipal monitoring stations for the same period. The association between daily IPF hospitalization and average concentration of each pollutant was analyzed with a generalized additive model estimating Poisson distribution.Daily count of IPF hospitalizations  was obtained from an administrative database for 2013–2017 while daily city-wide average concentrations of PM2.5 concentration during 2013–2017 was 76.7 μg/m3. The relative risk (RR) of IPF hospitalization per interquartile range (IQR) higher (72 μg/m3) in PM2.5 was 1.049 (95% CI 1.024–1.074) and 1.031 (95% CI 1.007–1.056) for lag0 and moving averages 0–1 days respectively. No significant associations were observed for other lags. Statistically significant positive associations were also observed at lag0 with SO2, Ozone and NO2 (in men only). Positive associations were seen at moving averages 0–30 days for PM10 , NO2 , and SO2 (RR per 15 μg/m3: 1.060 (95% CI 1.025–1.097), but not with PM2.5 or Ozone.Daily 24-h mean PMDespite improvement in air quality since the implementation of clean air policy in 2013, acute exposure to higher levels of air pollution is significantly associated with IPF hospitalization in Beijing. Air quality policy should be continuously enforced to protect vulnerable IPF populations as well as the general public.The online version contains supplementary material available at 10.1186/s12931-022-01998-8. Idiopathic pulmonary fibrosis (IPF) is the most common type of interstitial lung diseases (ILD), characterized by inflammation and fibrosis of the pulmonary parenchyma . The exa3) were found to be associated with acute exacerbation of IPF six weeks after exposure in 436 and 192 patients in South Korea [2), Particulate Matter with a diameter less than or equal to 10 μm (PM10) or 2.5 μm (PM2.5) were high in Santiago, Chile [2.5 and nitric oxide in the previous 30 days were significantly associated with IPF acute exacerbation [2 and/or PM exposure was associated with IPF incidence and mortality. To date, no studies have been conducted in a region where ambient air pollution is substantial.Unlike major respiratory conditions (e.g. chronic obstructive pulmonary disease (COPD), bronchitis, asthma), for which evidence of harmful effects from ambient air pollution is well documented –8, evideth Korea  and Franerbation . Two stuerbation , or a faerbation , of forcerbation , 15. Whierbation  and Souterbation  reportedAir quality in Beijing has been progressively improving due to the implementation of the Clean Air Action Plan (CAAP) since 2013 , althoug3 is reported based on hourly data that are available for at least 20 h in every 24 h. For O3, a daily maximum 8-h moving mean concentration is reported based on hourly data that are available for at least 6 h in every 8 h.Since 2013, major air pollutants have been routinely measured at 35 monitoring stations across Beijing, following the new Chinese national standard (GB 3095-2012). These monitoring stations were set up at locations that best represent different sources, for example, sources from road vehicles, urban anthropogenic activities, rural background and wider regional transport or background sources , and O3 were obtained from Jan 18th, 2013 to Dec 31st, 2017 (1809 days) by averaging daily mean readings from all 35 stations. Of the 1809 days, five days with missing data on all air pollutants were excluded. In addition, for O3, daily mean concentration was missing for another 34 days. As a result, the analyses of PM10, PM2.5, NO2 and SO2 were based on the 1804-day dataset whilst the analysis of O3 were based on a 1770-day dataset. Daily mean temperature (°C) and relative humidity (%) were provided by the Beijing Meteorological Service website.Daily city-wide mean concentrations of PM2.5, as a general air quality indicator, was not routinely monitored in Beijing before 2013. In order to examine whether there is difference on the association between PM2.5 and IPF hospitalization before and after the CAAP policy, we further obtained daily mean concentration of PM2.5 data during Apr 1st, 2008 and Dec 31st, 2012 (1508 days) from a single monitor operated by the US Embassy in Beijing. It was previously reported that within a radius of 40 km, this monitor covers 79.2% of the total population, 97.8% tertiary hospital and 79.3% secondary hospitals in Beijing that are eligible to admit IPF cases [2.5 data from this single monitor have been widely used in previous studies [2.5 monitor, we correlated the daily mean concentrations of this monitor with a nearby 1.5 km-apart government monitoring station using data from 2013. Overall, observations from the two monitors showed a high correlation (r = 0.93).PMPF cases . The PM2 studies , 20. To Daily counts of IPF hospitalization for the periods 2008–2012 and 2013–2017 were obtained from a database compiled by the Beijing Public Health Information Center. In Beijing, every government or private hospital at secondary or tertiary level is eligible to provide specialised care and required to submit the front page of each electronic hospital discharge record to the database . DetaileWe defined same-day exposure as lag0 (e.g. the admission date) and examined a priori single-day exposure up to five days (single-day lag0 to lag4) and exposure of moving averages 0–1, 0–2, 0–3, and 0–4 before admission date.While our primary analysis aim was to determine the acute effects of air pollution on IPF hospitalization, we additionally investigated the moving averages 0–30 period to account for potential subacute effects of air pollution on development of worsening symptoms of IPF. The exact exposure time window during which IPF patients develop an episode of worsening symptoms remains debated, although by convention the onset should be within one month prior to diagnosis . ExposurE(Yt) represents the number of IPF cases on day t; ‘C’ is the city-averaged concentration of the pollutant and i is the day lag; β represents log-relative risk (RR) of IPF hospitalisation associated with a unit increase in the pollutant mean concentration; ps indicates penalised spline function to filter out long-term trends and seasonal patterns in daily IPF hospitalisations. temp and RH are the daily mean temperature (°C) and relative humidity (%) respectively at lag0; public holiday and day of week were included as categorical variables. Degrees of freedom (df) for calendar time, temperature and relative humidity were selected based on the parameters used in previous studies [The associations between daily IPF hospitalization and average concentration of each pollutant were analyzed with a generalized additive model (GAM) estimating Poisson distribution: studies –24.Each association was investigated in two-pollutant models if Spearman correlation ratios between these pollutants were less than 0.7. Subgroup analyses were conducted by age (18–64 years vs. ≥ 65 years), sex, season (warm vs. cool), asthma (yes vs. no) and COPD (yes vs. no). We conducted the sub-group analyses at lag0 only because for most studied pollutants, the association was found to be much stronger at lag0 in the main model. The Z-test was used to compare the two estimates derived from each sub-group.The smoothing function of the generalized additive model was used to graphically analyse the exposure–response relationships between the log-RR of IPF hospitalization and air pollutant concentrations at lag0.We did several sensitivity analyses by altering the generalized additive model: (1) to exclude calendar time, as long-term trends and seasonal patterns might also be partly related to pollutant concentrations; (2) to replace calendar time with an interaction term of exposure-by-season; (3) to increase the degrees of freedom of temperature and humidity to six; and (4) to model moving averages 0–4 of temperature and humidity instead of the current day (lag0). The latter two sensitivity analyses were to adjust potential non-linear and lagged confounding effects of weather conditions.All statistical analyses were conducted in R (version 3.0.2) using packages MGCV, DPLYR, and TTR. Relative risk (RR) of hospitalisation for IPF per interquartile range (IQR) increase for each air pollutant were calculated and presented.2.5 concentration during 2008–2012 and 2013–2017 was 88.9 and 76.7 μg/m3 respectively , except the high correlation between PM10 and PM2.5 (r = 0.93), PM10 and NO2 (r = 0.73), PM2.5 and NO2 (r = 0.78)  higher was 1.049 (95% CI 1.024–1.074) at lag0, 1.031 (95% CI 1.007–1.056) at moving averages 0–1 and 1.021 (95% CI 0.998–1.044) at moving averages 0–2. Expressed per 10 μg/m3 higher of PM2.5, the RRs are 1.007 (95% CI 1.003–1.010), 1.004 (95% CI 1.001–1.008) and 1.003 (95% CI 0.999–1.006) respectively. Results of PM10 were similar to those of PM2.5 except that for the moving averages 0–30, where there was a positive association with PM10 , compared to a null association with PM2.5 .Acute exposure to higher PM3) higher in PM2.5 was 1.062 (95% CI 1.025–1.101) .During 2008–2012, the RR of hospitalization for IPF at lag0 per interquartile range (IQR) (83 μg/m3) higher of NO2 on both lag0 and moving averages 0–30 . A significant positive association was observed for SO2 at lag0 only (RR: 1.023 (95% CI 1.002–1.045) per IQR (15 μg/m3) higher). Positive effect estimates of SO2 appeared to be in an increasing trend. For example, the RR at moving averages 0–1 and 0–30 was 1.019 (95% CI 0.996–1.043) and 1.060 (95% CI 1.025–1.097) respectively. A significant positive association was seen at lag0 for O3  higher). Similar associations were observed at least until lag3. Significant positive associations were seen for moving averages 0–1 up to 0–4 ; however, the association at moving averages 0–30 was positive but not statistically significant . The associations with O3 were statistically significant during cool season but not in warm season . While in women, per IQR higher exposure to O3 at lag0 was significantly associated with IPF hospitalization . There were no significant effect modifications by age, season, COPD or asthma with each pollutant, although there is a tendency that patients aged ≥ 65 years had a higher hospitalization risk with higher exposure to each pollutant except NO2. For IPF patients with COPD, hospitalization risk  was significantly higher as per IQR higher O3 exposure, as compared to patients without COPD .Stronger positive associations with all pollutants except OOur findings suggest that acute exposure to higher levels of air pollutants, both particulates and gases, was significantly associated with IPF hospitalization in Beijing during 2013–2017. The magnitude of the associated risk is comparable to what was previously observed in Beijing for COPD exacerbation requiring hospitalization .To date, only four studies reported associations between short-term air pollution exposure and acute exacerbation or hospitalization of IPF , 9–11, w2.5 exposure and IPF hospitalization is an important addition to current knowledge. Previously, the Korean study did not have PM2.5 data [2.5 and acute exacerbation of IPF among 192 patients [2.5 exposure in the previous four days was a significant risk factor of IPF hospitalization [2.5 was much higher in Beijing (77 μg/m3 2013–2017) than in Santiago (29 μg/m3 2001–2012), the risk estimate in our study was relatively smaller. This may reflect differences in statistical approaches used , PM2.5 toxicity and population characteristics.Our finding of an association between acute PMlization , which i2.5, as compared to more recent exposure (i.e. past 30 days), was relatively more important in leading to IPF hospitalization. This is in contrast to a Japanese study of 152 patients in which a significant positive association between IPF acute exacerbation and prior 30-day average PM2.5 exposure was found [10 exposure and IPF hospitalization risk. This finding highlighted the potential of coarse PM in triggering symptoms among IPF patients via direct deposition in the respiratory tract over a sub-acute period [10 mainly come from dust whilst for PM2.5, traffic is the largest source with coal combustion and biomass burning contributing in winter months [Our study has suggested that more acute exposure (i.e. past three days) to PMas found . We did e period . In Beijr months , 26. We r months , which er months . Both cor months . Collect2.5 observed in 2013–2017, the associations observed in 2008–2012 were relatively stronger and the lag time to hospitalization was longer (5 vs. 2 days). It should be noted that PM2.5 data from 2008 to 2012 were only based on a single monitor. Although this is not a direct assessment of health benefits resulting from the Clean Air Action Plan, our findings seem to echo previous reports that improvement in air quality has indeed improved several morbidity and mortality outcomes in the Chinese population [We found that, as compared to the associations with PMpulation , 29, 30.3 up to preceding five days was significantly associated with IPF hospitalization. Further stratifying the associations by season, only in cool season can the statistically significant positive associations be observed. This is somehow unexpected as previous studies in Beijing or the northern part of China found that short-term effects of O3 on COPD exacerbation [3 concentrations in summer months and reported a significant positive association with acute exacerbation of IPF [3. As ours is the only study to date that reported seasonal variations of O3 associations with IPF hospitalization, these patterns remain to be determined in future studies. An alternative explanation is that O3 exposure in cool season may represent a proxy of other more important factors, such as viral infection, which is an known trigger in worsening IPF that may require hospitalization [3 is known to have the capability to exacerbate chronic lung conditions including asthma and COPD. In addition, O3 can cause breathing difficulty by bronchoconstriction, thereby increasing the risk of hospitalization among individuals with existing respiratory diseases including IPF [3 can reach and accumulate at the lower lungs because of its low water solubility, leading to inflammation and impaired small airways function, and potential hospitalization risk [This study found that acute exposure to Oerbation  and dailerbation  were morn of IPF  whilst Sn of IPF . The stun of IPF  and Japan of IPF  did not lization . Being ading IPF . Animal ion risk , 34.2 exposure [2 concentrations by 70% was recorded in Beijing from 2013 to 2017 [2 concentration. Associations did not appear to be significantly affected by further adjustment of PM2.5. These results indicated that continued investment on renewable and clean energy, with a steady reduction of traditional fossil energy consumption, still remain pivotal to SO2 abatement in order to mitigate the harmful impacts on respiratory diseases. As with O3, patients with chronic respiratory diseases may be sensitive to the effects of SO2, which has the capacity to induce oxidative stress, airway inflammation and breathing difficulty [Previous studies either found no associations between acute exacerbation of IPF and SOexposure , or thatexposure . Despite to 2017 , we stilfficulty .2 exposure and IPF hospitalization in the overall population. Interestingly, we found the same effect size for both moving averages 0–30 and lag0. Only in the men-only sample, this positive association became statistically significant. This finding is partly in line with the Johannson study of 436 (80% of them were men) South Korean patients [2 was significantly associated with IPF exacerbation. It should be noted that, association with the most acute exposure period (i.e. the week before manifested IPF exacerbation) was weaker and not statistically significant in the Johannson study. Similarly, the Japanese study of 152 patients (70% of them were men) reported significant positive associations between prior 30-day average NO2 and IPF acute exacerbation. In contrast, the French study did not find any association with NO2 [2 in the previous four days and IPF hospitalization in all populations, with the association being stronger among women [2 has been commonly studied as an indicator of traffic emission and is usually highly correlated with PM2.5, therefore it remains difficult to study its independent effects. Nonetheless, some have argued that NO2, via the airway inflammation pathway, could have direct and adverse impacts on respiratory health [We observed a marginally positive association between concurrent-day NOpatients , in whicwith NO2 . The timng women . NO2 has3 on IPF hospitalization was more pronounced in women, in line with studies which generally found women had a higher respiratory hospitalization risk from O3 exposure, particularly during the cold season [2 effects, we only found significant associations in men. The sex-specific  and gender-specific  differences of air pollution effects on respiratory health outcomes still largely remain inconsistent across studies [We observed that the risk of Od season . And for studies , and futTo our best knowledge, this is by far the first and the largest time-series study that was conducted in a city with high levels of ambient air pollution, using a relatively large number of IPF hospitalizations over a 5-year period from a representative database. The study has limitations. First, it is an ecological study by design, and hence the modelled risk estimates should not be interpreted as predictive of individual hospitalisation probability. Given the time-series study design, we were unable to adjust for important individual-level factors of IPF such as smoking and socioeconomic status. However, the proportion of these risk factors are unlikely to change substantially in the same population which we were investigating over a short period of time. Notably, time-varying factors such as seasonal viral or bacterial infection will likely have a more important role to confound these relationships, but we did not have these data for further investigation. Second, the use of daily, outdoor, city-wide average air pollutant concentrations inevitably will have introduced bias to health estimates because we cannot take into account place of residence and time-activity patterns  from the study population. Due to privacy reasons, we were only provided area-level (urban district or township) residential address for each patient for this secondary analysis, therefore we cannot model air pollution exposure at individual residence. However, this time-series analysis is an important first step to inform our next immediate work to study the impact of modelled air pollution at residence on health outcomes in a cohort of IPF patients should consent of accessing address data was granted. In addition, personal monitoring of air pollution exposure among IPF patients will provide even more novel insights into the exposure patterns and the associated health risks. Third, as with the Chilean study , our out2.5 by one-third (from 87 μg/m3 in 2013 to 58 μg/m3 in 2017) in the first phase. Although the annual mean PM2.5 in Beijing decreased to around 40 μg/m3 by end of 2021, it is still eight times higher than the recently updated WHO target value of 5 μg/m3, and therefore remains a significant health threat as demonstrated in this study. The concentrations for nitrogen dioxide and ozone, both of which are known to be harmful for respiratory health [The implementation of Clean Air Action Plan in Beijing successfully reduced the annual mean concentration of PMy health  and the In conclusion, this study provides evidence that acute exposure to higher levels of air pollution is associated with IPF hospitalization in Beijing. Local air quality policy should be stringently enforced, and carefully monitored for the progress to protect vulnerable populations, the general public and the planet.Additional file 1.Figure S1. Air quality monitoring stations across Beijing. Table S1. Spearman correlation between air pollutants during 2013-2017. Table S2. Associations between PM2.5 and PM10 (per IQR increase) and IPF hospitalisation in Beijing at different lags during 2013-2017. Table S3. Associations between PM2.5 (per IQR increase of 83 μg/m3) and IPF hospitalisation in Beijing at different lags during 2008-2012. Figure S2. Comparisons of associations between PM2.5 and IPF hospitalization for periods 2008-2012 and 2013-2017. Table S4. Associations between gaseous pollutants (per IQR increase) and IPF hospitalisation in Beijing at different lags during 2013-2017. Table S5. Associations between ozone (per IQR increase) and IPF hospitalisation in Beijing at different lags during 2013-2017: by season. Table S6. Results of the sensitivity analyses. Table S7 Results of two-pollutant models. Figure S3. Exposure-response curves between air pollution exposure and IPF hospitalization at lag0.


Numerous studies have documented inconsistent reporting of sexual behaviour by adolescents. The validity and reliability of self-reported data on issues considered sensitive, incriminating or embarrassing, is prone to social-desirability bias. Some studies have found that Audio Computer-Assisted Self Interviewing (ACASI) that removes the personal interaction involved in face-to-face interviews, decreases item non-response and increases reporting of sensitive behaviours, but others have found inconsistent or contradictory results. To reduce social desirability bias in the reporting of sensitive behaviours, face-to-face interviews were combined with ACASI in a cluster randomized trial involving adolescents in Zambia.th time since recruitment. Young, female research assistants who had conducted interviews for the trial were also interviewed for this study.To explore adolescent girls’ experiences and opinions of being interviewed about sexual and reproductive health, we combined Focus Group Discussions with girl participants and individual semi-structured interviews with teachers. This study was done after the participants had been interviewed for the 6Respondents explained often feeling shy, embarrassed or uncomfortable when asked questions about sex, pregnancy and abortion face-to-face. Questions on sexual activity elicited feelings of shame, and teachers, research assistants and girls alike noted that direct questions about sexual activities limit what the participant girls may be willing to share. Responding to more indirect questions in relation to the context of a romantic relationship was slightly easier. Efforts by interviewers to signal that they did not judge the participants for their behavior and increased familiarity with the interviewer reduced discomfort over time. Although some appreciated the opportunity to respond to questions on their own, the privacy offered by ACASI also provided an opportunity to give false answers. Answering on tablets could be challenging, but participants were reluctant to ask for assistance for fear of being judged as not conversant with technology.Strategies to avoid using overly direct language and descriptive words, asking questions within the context of a romantic relationship and a focus on establishing familiarity and trust can reduce reporting bias. For the use of ACASI, considerations must be given to the context and characteristics of the study population. Numerous studies have found that adolescents may not answer truthfully or consistently when questioned about issues such as sexual activity, abortion and pregnancy. Such issues are considered sensitive, incriminating, or embarrassing, and answers may be influenced by a fear of being misjudged or sanctioned. In an attempt to collect more reliable data on sexual behaviour and childbearing, we combined face-to-face interviews with Audio Computer-Assisted Self Interviewing (ACASI) in a research trial involving adolescents in Zambia. In this study we aimed to explore adolescent girls’ experiences and opinions of being interviewed about sexual and reproductive health when participating in the trial. We combined group discussions with girl participants and individual interviews with teachers. Participants said they felt shy, embarrassed or uncomfortable when asked questions about sex, pregnancy and abortion face-to-face. Teachers, girls and interviewers alike noted that very direct questions limit what the participants may be willing to share. Efforts by interviewers to signal that they did not judge the participants for their behaviour, and repeated interviews with the same interviewer reduced discomfort over time. Some liked ACASI whereas some said it made it easier to give false answers, and answering on electronic tablets could be challenging. To reduce embarrassment and increase the likelihood of honest answers we recommend avoiding overly direct language and descriptive words, and to relate questions about sexual activity to a romantic relationship. Interviewers who are able to establish familiarity and trust can make participants more comfortable. Adolescent sexual and reproductive health is a global public health concern which has received growing interest and increased research attention over the last two decades . To addrThe Audio Computer-Assisted Self Interviewing (ACASI) technique has gained some popularity amongst researchers due to the privacy it affords the respondent. When using ACASI, respondents receive and respond to questions using a handheld computer or tablet, which removes the personal interaction involved in face-to-face interviews, and this has been found to reduce socially desirable responses. Encouraging evidence for increased reporting of sexual risk behaviour using ACASI was found in USA in the 90 s and 2000s –12, and In the Research Initiative to Support the Empowerment of Girls (RISE) we combined face-to-face interviews with ACASI to reduce social desirability bias in the reporting of sensitive behaviours. RISE was a cluster randomized controlled trial that aimed to measure the effect of economic support for adolescent girls, sexuality education and community dialogue on childbearing rates amongst adolescent girls in Zambia . ThroughControl Group, (2) Economic support group with cash transfers to girls and their families and payment of school fees or (3) Combined intervention group receiving the same economic support combined with sexuality education and community meetings targeting norms related to education and SRH amongst parents and community members  but the most embarrassing is the sex question, so it is difficult to answer.GirlSo why do you think this particular question is embarrassing? Because you are saying that asking if you have a boyfriend is not difficult to answer.Researcher: :Because they will start thinking of me as a girl who opens her legs (girls laugh) Ah! (FGD 2)GirlQuestions about sexual behaviour were perceived to be the most difficult to answer. Girls in a group elaborated a bit on this:Another group gave several reasons why girls would lie about ever having had sex: “[the girl’s]self-esteem is low”; “Some girls get offended if you ask them that how many men have you ever slept with”; “some fear that their parents may find out at home and tell the Rise teacher at school”; “fear of being laughed at”, “fear she may be reported to people”; “not just understanding the questions”, “maybe they think that when you meet them [RAs] in the streets, you will be looking at them with an eye that you know about them” (FGD 1).: So, are there any questions that make you feel uncomfortable? Maybe even embarrassed?Researcher: (laughs) Those ones whereby she is asking you if you have ever been pregnant, have you ever given birth? They are embarrassing but more embarrassing if you are actually pregnant.Girl 1Researcher 2: Can I ask why they are embarrassing?: If you have been pregnant it is embarrassing or shameful. (FGD 2)Girl 2Questions around pregnancy also made the respondents uncomfortable:“Some people who respond to these questions do not tell the truth. They lie mainly because they feel guilty and ashamed and don’t want to be discovered as a prostitute” . The girls reported that they are continuously reminded and ‘warned’ by parents, teachers and the community that it is wrong and dangerous to have sex before marriage. This may result in an internalization of shame, guilt and negative feelings, which may have direct implications for the way they respond to sex-related questions.: because these girls they know that when they are doing these things [sexual behaviours], they know it’s not right and they are advised not to be doing these things. So when they say that they do it, they feel guilty because they are doing the wrong thing or have been told not to be doing sex before marriage so that they don’t contract STIs and HIV and they don’t become pregnant. So, when they are asked to say ‘Have you ever had sex?’, they are going to deny because they will know now they are not trusted. (Interview with RISE teacher)TeacherQuestions on sexual activity elicited feelings of shame and teachers, RAs and girls alike noted that they limit what the participants may be willing to share: : you said with sex there was guilt and shame, is that the same with having a child? Is there shame?Researcher: sometimes, for having a child they are not shy, they are going to tell you straight away, that one is not very difficult. The most difficult is where you ask them, do you have a boyfriend, have you ever had sex? Are you pregnant? They hide! But if you ask them if they have a child, they don’t usually hide that.TeacherAnother teacher suggested that questions about having a child are less likely to create feelings of shame than questions about sexual behaviour or pregnancy.When FGD participants were asked why some respondents would report having a child in the face-to-face interview but respond ‘no’ to the questions referring to sex in ACASI, they explained that it is easier to say that they have a child because, “it’s like a lighter version” of saying sex. The shame, guilt and perceptions of promiscuity associated with ‘sex’ amongst these adolescent girls, made them want to distance themselves from it.: we have some situations where a girl will say she has a child and then if you ask her later if she’s ever had sex, she’ll say noResearcher: yes, she’ll say no. (laughs). Why? Because of sex! Aha!! Because of sex…. Just that word ‘sex’, it means ‘a man in me’, just that! She will feel shy, unless another term is used. (Interview with RISE teacher)TeacherTeachers and RAs would also prefer to avoid the word ‘sex’ since the word itself is source of embarrassment. It is rarely, if ever, used in the local public sexual and reproductive health discourse. Although the act of sex is implicit when talking about having a child or pregnancy, they considered that using the word itself creates a risk that participants alter their response.: But others are not fine with these questions. Some may end up slapping you over these questions [about miscarriage/stillbirths], it may be that she was once pregnant but had a stillborn, then you are asking her these questions repeatedly. They may not be in the mood or they may respond to you in a way that is not good just to offend you. (FGD 2)GirlQuestions about miscarriages and stillbirth were perceived as offensive and upsetting despite of RAs being trained to approach such sensitive topics with care and empathy. It appeared that this was partly worsened by the structure of the interview which meant that a number of these questions were repeated (they were asked face-to-face as well as in the ACASI portions).: Why do you think these questions [about abortion] are difficult to answer?ResearcherBecause they feel like if they say the truth, someone is going to tell someone and so on. Abortion is not acceptable in society. It’s actually embarrassing to be known as a person who has aborted. (FGD 4)Girl: Reactions to questions relating to induced abortion were particularly strong as there is a lot of shame associated with abortion.There is just one question that I was asked, and I thought to myself ‘what has made this person to ask me this question?’Girl 2: : Which question is that?Researcher: The questions ‘Have you ever aborted?’. I was disappointed because it is not allowed to abort and I thought the person asking me was looking at me as if we do not respect ourselves. (FGD 5)Girl 2Fears around confidentiality and anonymity of answers were frequently cited by RAs and teachers as a reason for participants not responding truthfully to sensitive and/or taboo questions. In contrast, RISE participants explained that the questions about sex and abortion made them feel judged by the interviewer. A girl who has a child and is asked whether she has ever had sex, may answer ‘no’ because she is afraid of being laughed at, according to participants in one of the groups (FGD 3). Questions about unacceptable behaviours made respondents believe they were suspected of engaging in that sort of behaviour, implying that the interviewer had made a negative evaluation of their character.The following section describes the various respondents’ suggestions for overcoming barriers in collecting self-reported SRH data. Four key strategies were mentioned by all three respondent groups.: At first I was uncomfortable and shy because I was not used to being asked these questions by a stranger, a person you don’t know completely, asking you these questionsGirl 1: What made you not be shy afterwards?Researcher: Because I became used to being asked such questions. You can only talk about these things to people you are close to. (FGD 3)Girl 1As outlined above, the sensitive nature of several of the questions meant that respondents required a great deal of confidence in the interviewer if she was to answer honestly. The girls claimed that they felt comfortable being interviewed by the trial RAs and emphasised the importance of ‘getting to know’ and developing a close relationship with them. This familiarity was created in the informal conversations prior to commencing the interview, throughout the course of each interview, and through repeated interviews (conducted every 6 months) by the same person. The respondents became gradually more comfortable with the interview process as well as the RA.: The thing is that we would like to be interviewed by the same person because we are now used to her as opposed to someone we haven’t met. We also feel like she cannot release our information to people and we are not scared of her. (FGD 6)GirlWe were curious whether changing RAs between interview rounds would increase the feeling of anonymity amongst the RISE participants, but this did not appear to be the case. As can be seen in the quote below, when the RAs returned for subsequent interviews, the girls felt they had already heard their secrets.“We’ve been coming here for a long time—have you ever heard of anyone being ‘told on’ or information being shared?”. Familiarity with the respondent also made it easier to judge the reliability of her answers and the appropriate approach to assume. For example, if the participant was pregnant in the previous interview, the RA could be more attentive when asking questions around childbirth or miscarriage.RAs often used their repeated visits to reinforce the confidentiality of the interviews. Several of them said things like: Teachers and FGD participants suggested that the school was considered the ‘safest’ location to conduct the interviews. The presence of community members at the clinic could create a risk of rumours and curiosity, according to the girls. At their homes, the presence of parents seemed to prevent speaking about sex and reproductive issues. Several of the RAs had experienced that the girls avoided being found in their homes when they arrived.: Another way to make us comfortable so that we can answer truthfully, is to tell the person who interviews us to open up freely. They can say that ‘Even I have slept with a man and know everything that happens between a man and a woman. So whatever girls go through, I have also gone through that, so feel comfortable.’ (FGD 1)GirlWhen RAs were able to normalize sex and pregnancy by giving examples from their own lives, it seemed to be an effective way of making a respondent comfortable enough to answer questions on this topic.RAs occasionally referred, without negative connotations, to a young family member who was pregnant, which was meant to signal to the respondent that they had no negative judgement towards that behaviour.: Ok, if you want to ask a friend if she had sex, how would you ask her?Researcher: I can ask her, ‘Do you have a boyfriend. Or are you dating?’ Then that’s when you ask her if she had slept with him. (FGD 6)Girl“In fact they should be asking these questions like do you have a boyfriend and do you have a child together, so as to avoid the awkwardness or just removing the sex question.” (FGD 2)When reviewing the questionnaire items with respondents, they noted that the sequencing and formulation of the questions may impact on the respondent’s reaction to the interview. The comments of the respondents suggested that in the follow up interviews, sex-related questions were not appropriately contextualised in the frame of a stable and/or romantic relationship. All three respondent groups emphasized the need to present questions around sexual activity with the assumption that it was with a boyfriend.Researcher: Is there some way to make it less direct?Head Teacher: Sometimes you start asking ‘Do you have a boyfriend?’ And from there ‘From the time that you met that boyfriend, have you done anything with him?’ Or maybe ‘What type of stories do you share with your boyfriend, and at what time do you meet your boyfriend?’ Such things. Maybe ‘Do you see your boyfriend in the presence of your mother? Or maybe you have got some places where you meet this boyfriend?’ Just like those minor questions before you hit to the real point of ‘Have you ever slept with your boyfriend?’. (Interview with head teacher)A head teacher also suggested that the interviewed girl should be eased into the question.Ok, the tablet is fine to those who are shy to talk face to face with the interviewer, even though there is a chance they can lie. For instance, if she is pregnant when answering on the tablet, she can say ‘no’. But when asked during face to face, she admits it because you have seen for yourself that she is pregnant. (FGD 3)Girl 1: When the girls shared their opinions about the ACASI interview, the privacy that it provided was often highlighted as a positive feature. However, this privacy also provided an opportunity to give false answers:: Which one do you prefer? The tablet or face to face?ResearcherI prefer being asked live [face to face]Girl 2: : We prefer face to face because we are interacting with a person who can read our expressions and use better judgement. And also if you did not really understand the questions, you can ask if you are not clear. (FGD 3)Girl 3The lack of support and contact with the interviewer was a clear drawback of ACASI.“Sometimes we feel shy because we feel like they will tell someone that we don’t know how to use the tablet” . Indeed, many of the informants—both the girls and RAs—suggested more support and training should be given to those RISE participants who struggled with the ACASI technique.RAs and FGD participants noted that the language and dialect of the audio recordings in ACASI could be challenging, but participants hesitated to ask for assistance for fear of being judged as not conversant with technology: : Ooho! It’s reading sometimes which is the problem. Sometimes you are reading and at the same time you are listening from the headphones, and then you have to press at the same time. You end up making a mistake pressing ‘Yes’ to the question when you meant ‘No’.Girl 1: But the tablet asks which language you understand. It’s just that others do not know or hear the instructions where to press. So, others find it difficult to use.Girl 2: So sometimes you put an answer you didn’t want to put?Researcher: Yes.Girl 2: So, what can we do to make it easier on the tablet?Researcher: Maybe they should come and teach those who find it difficult for some time, not just a short time. (FGD 1)Girl 1When the FGD participants were asked why some girls may prefer the face-to-face interview, they suggested that with the ACASI, being presented with a written text, a voice recording and being asked to select a response at the same time, could be confusing.Several RAs felt that some girls had rushed through the questions in the ACASI portion and gave examples of respondents who completed the ACASI portion of the interview in less than five minutes when on average it took 10 min to complete. FGD participants suspected that some girls may have rushed through the ACASI portion because they were uncomfortable with the method and/or technology. All respondents  emphasised the importance of offering continuous clarification and support throughout the interview even when not requested by the participant herself.This study found that questions about sex, pregnancy and abortion made the female adolescent trial participants uncomfortable and elicited feelings of shame. These reactions were common despite efforts to mitigate them by the use of female research assistants of similar age who were instructed and trained to normalize the questions and topics. Repeated questions about private, sensitive issues such as stillbirths could even elicit feelings of anger. The reasons given for underreporting sexual activity in this study were largely in line with findings from previous studies; namely shyness, embarrassment, social desirability, concerns around confidentiality and fear of penalties and social consequences , 9, 19. Throughout the FGDs and interviews it became apparent that sex-related questions which had been used in the follow-up interviews were perceived to lack any reference or association with love, trust, or intimate and stable relationships. Not surprisingly therefore, all the informants advised the researchers to introduce sexual activity questions in a context which assumed that they were performed with a boyfriend. Similar observations on the need for such contextualising have also been made by interviewed sex-workers in Malawi and South Africa who described their relationship with clients in terms which connoted premarital love and trust; not simply transactional relations , 21.Some participants highlighted that the use of the word ‘sex’ can affect how girls answer certain questions. This may be explained through other studies from Zambia which found concerns that the local terms related to sex emerged as more insulting than the English ones . This suggests that other ways of asking sex-related questions should be considered such as asking about ‘boyfriend’ instead of ‘sex partner’, ‘sleeping with’ instead of ‘having sex’, or ‘dating’ instead of ‘sexual relations’. Although the use of local euphemisms and culturally appropriate terminology may require additional preparation and adjustment of research tools, it can make the interview less awkward for both the researcher and the respondent. Alternative expressions such as ‘slept with’, not only distances the respondent from the act of sex but as found by Langhaug and colleagues (2010), it also avoids the embarrassment of hearing sexual terms being read out loud by the interviewer. Formatting of questions in a vague manner, however, contrast with typical survey design guidelines that recommend clear and unambiguous questions to avoid misunderstandings and to increase the validity of responses , 23.The RISE participants explained that the questions about sex and abortion made them feel that they were judged by the interviewer and suspected of engaging in socially unacceptable behaviour. They seemed most concerned about being associated with promiscuity and/or prostitution. This was also seen in a study of reporting of sexual behaviours amongst South African women, which found that 32% of respondents purposefully under- or overreported certain sensitive behaviors to avoid criticism or to seek praise . As poinThe participating girls stated that the school was the most preferred place for interviews. Having interviewers coming home to them created a risk of embarrassing situations with family members asking why they were talking to strangers about sexuality related issues. They also feared that being seen at the health clinic could cause negative rumours. These findings should be seen in relation to other studies in the region who have reported that adolescents rarely communicate with their parents about sexuality and most often seek to hide any sexual relations or interest in boys. Studies have also suggested that adolescents may avoid health clinics out of fear that fellow community members will suspect them of seeking contraception –30.Discussions around the audio computer assisted (ACASI) section of the interview seemed to confirm previous claims around the increased privacy and anonymity afforded by the technique. The use of ACASI was welcomed by some, but the participants did not believe that adolescent girls would necessarily be more truthful when left to respond to sensitive questions on their own. Some studies in low-income contexts have shown higher rates of self-reported sexual activity using ACASI , 31. Fordoes require in-depth details about adolescents’ sexual activities, careful piloting of alternative wording and combinations of data collection methods may increase the validity of the findings. Comparative studies suggest that culturally specific interactive interviews [Research on adolescent SRH may need more careful consideration of the value, relevance and wording of detailed questions about sexual behaviour. If a study terviews  and in-dterviews  yield hiA strength of this study is that we triangulated the responses from participants, teachers, and the RAs themselves. Their observations and suggestions were similar, thus giving credibility to the findings. Another strength was the use of two interviewers. One was a white, 37-year-old woman from Norway and the other interviewer was a Zambian 25-year-old female. The latter spoke the local languages the girls were comfortable with and her age was close to the participants. As for the former, her Scandinavian background with liberal and open views about SRHR issues may have enabled her to ask more direct questions than what a local person would feel comfortable doing, and it is possible that the presence of an outsider made it possible to talk about things that are normally regarded as too private. However, direct ways of questioning may be intimidating for participants who are less accustomed to talking about SRH. The identification of the researchers with the main study may have caused concern that any negative opinions about the RISE project would have implications for the support that it provided to the girl or the school, but explicit efforts were made to ensure the participants that they could be honest and that their responses would have no bearing on them or their participation in the RISE project.Two FGDs and interviews with RAs were not recorded, which may have resulted in some loss of detail or nuance although detailed notes were made as soon as this was discovered. In addition, the transcriber was not fully conversant with the language used in one of the FGDs and quotes may not have been completely accurate.Since we reached saturation, we believe the findings are transferable to the study population of the RISE project. Due to the special context of the study, the generalizability is uncertain but it is likely that the findings are relevant to other studies on sexual and reproductive health among adolescent girls in sub Saharan Africa.This study found that the female adolescent trial participants felt uncomfortable and embarrassed when asked questions about socially disapproved behaviour such as premarital sexual activity and pregnancy, despite repeated experiences of being asked the same questions. Strategies to avoid using overly direct language and descriptive words, asking questions within the context of a romantic relationship and a focus on establishing familiarity and trust can reduce reporting bias. Considering discrepancies in the research on ACASI, it is premature to make generalised claims about its suitability for research in low-income contexts. The findings of this and previous studies suggest that considerations must be given to the context and characteristics of the study population, as well as the time and resources available to familiarize the respondents with ACASI technology.


Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant tumor with an extremely poor prognosis in digestive tumors. Pyrroline-5-carboxylate reductase 1 (PYCR1) plays an important role in tumor development. Therefore, we aimed to explore the effect of PYCR1 on the growth of PDAC cells.Methods: Tumor tissues and adjacent normal pancreatic tissues were collected from 89 patients with PDAC. And immunohistochemistry (IHC) was used to analyze the expression level of PYCR1 in both. RNA interference was used to inhibit the expression of PYCR1 in PANC- 1 and AsPC-1 cells. After infection, the expression of PYCR1 protein was detected by Western blot. The proliferation and growth of PDAC cells were detected by Celigo analysis, MTT, and clone formation assay. Cell apoptosis was analyzed by flow cytometry. Furthermore, the effect of PYCR1 interference on tumor growth was evaluated in vivo through injecting tumor cells subcutaneously into nude mice.Results: The expression of PYCR1 in pancreatic cancer tissues was significantly higher than in paired adjacent normal pancreatic tissues (P <0.01). In vitro, the downregulation of PYCR1 expression significantly inhibited the cell proliferation and colony formation, and increased apoptosis in PANC-1 cells and AsPC-1 cells compared with the shCtrl group (P <0.01). And in vivo, PYCR1 interference also significantly inhibited tumor growth both in the tumor volume and weight.Conclusion: PYCR1 interference was able to inhibit cell proliferation and promote cell apoptosis of pancreatic cancer. The PYCR1 may serve as a potential therapeutic and prognostic biomarker for the treatment of pancreatic cancer. Pancreatic cancer is a highly malignant tumor with poor diagnosis in the digestive system. Pancreatic cancer is occult without specific symptoms, which means that many patients exhibit locally advanced disease or metastasis at the time of diagnosis Pyrroline-5-carboxylate reductase (PYCR) is mainly located in the mitochondria and plays an important role in the biosynthesis of proline As non-essential amino acids in the human body, proline has a unique secondary amine structure, and enzymes involved in the metabolism of proline play a key role in the control of tumor initiation and progression. According to previous reports, PYCR1, one of the most important enzymes in the process of proline metabolism, is closely associated with tumorigenesis and tumor progression, including prostate cancer, breast cancer, colon cancer, lung cancer, and liver cancer in vitro and in vivo.In this study, we aim to analyze the correlation between PYCR1 and PDAC. The expression level of PYCR1 was evaluated in pancreatic cancer. And we explored the biology behavior of PYCR1 both This study involved 89 tumor tissues and paired adjacent normal pancreatic tissues from patients who underwent curative surgical resection in Zhongshan Hospital, Fudan University from September 2012 to May 2016. Tissues were collected during surgery and frozen in liquid nitrogen. All specimens were pathologically confirmed to be ductal adenocarcinoma of the pancreas. And all the clinical pathological data and detailed follow-up data of the 89 patients were collected. Patients were followed up until May 1, 2020. Furthermore, the tissues were used for the analysis of immunohistochemical (IHC). This study was approved by the Ethics Committee of Zhongshan Hospital, Fudan University. All patients signed informed consent before the operation.All pancreatic tissues were fixed in 10% formalin and embedded in paraffin. The sections were dewaxed with xylene and dehydrated with ethanol. After deparaffinization, the sections were treated with 3% hydrogen peroxide for 10 minutes. For antigen retrieval, the sections were heated in sodium citrate buffer (pH 6.0) for 20 minutes. After that, sections were cooled to room temperature naturally and dipped in distilled water for 10 min. Sections were blocked with 10% fetal bovine serum (FBS) for 30minutes and incubated overnight with primary rabbit polyclonal anti-PYCR1 antibody . Then, the second antibody and Vulcan Fast Red Chromogen kit2 was added. Sections were stained with diaminobenzidine (DAB) and were counterstained with hematoxylin. The results were observed under an optical microscope after dehydration and sealing.The brown staining in the cytoplasm indicated positive staining for PYCR1. The tissues were evaluated by two different pathologists. The expression of PYCR 1 staining was scored according to the following criteria independently.(1) The percentage of positive staining was scored as follows: 0, 0-5% positively stained cells; 1, 6-20% positively stained cells; 2, 21-50% positively stained cells; 3, 51-100% positively stained cells.(2) The intensity of staining was scored as follows: 0, negative; 1, weakly positive; 2, moderately positive; 3, strongly positivity.(3) The histochemistry score  was calculated by multiplying the percentage of positive staining score with the staining intensity score. Then, the tissues were divided into two groups: low expression (score≤3) and high expression (score >3).2.The pancreatic cancer cell lines used in this study include SW1990, PANC-1, AsPC-1, BxPc-3, which were purchased from the Chinese Academy of Sciences Cell Bank . These cells were cultured in DMEM media containing 10% FBS at 37 °C with 5% COSmall-hairpin RNA (shRNA) interfering sequences targeting PYCR1 (shPYCR1: 5'-CAGTTTCTGCTCTCAGGAA-3') and negative shRNA sequences (shCtrl: 5'-TTCTCCGAACGTGTCACGT-3') were designed and synthesized. After that, the single-stranded DNA oligo containing the interference sequence was synthesized and converted to double-stranded DNA by annealing. And GV115 vector  was linearized with Age I and EcoR I restriction enzymes. After digestion, the double-stranded DNA oligo was ligated to the GV115 vector using T4 DNA Ligase (Fermentas) according to the manufacturer's instructions. Then, the products were transformed into the E. coli TOP10 strain . The positive clones were identified and selected by sequencing. According to the manufacturer's instructions of EndoFree Maxi Plasmid Kit , the plasmid was extracted from the previous positive clones for further lentiviral packaging.The AsPC-1 and PANC-1 cells were seeded in a 6-well plate at a density of 2×10^5 per well and infected with recombinant PYCR1 (shPYCR1) or negative control (shCtrl) lentivirus separately (MOI of 20). At 72 hours post infection, the fluorescence signals of green fluorescent protein (GFP) were observed with a fluorescence microscope . Only cells, in a well-growing state and positive for GFP fluorescence over 70%, were collected for further detection. And fluorescence-activated cell sorting  was used to sort for GFP-positive cells.For the examination of PYCR1 expression, total RNA was extracted from the cell lines using TRIzol reagent . And total RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase kit (Promega). The RT-qPCR was performed using SYBR® Premix Ex Taq™  in a volume of 12μL on LightCycler 480 II Real-Time PCR System (Roche). PCR cycling conditions were as follows: 95 °C for 30s, 40 cycles at 95 °C for 5 s, 60 °C for 30s. GAPDH was used as an internal control to calculate the relative expression levels of genes. The relative mRNA expression levels were calculated by the 2-ΔΔCT method. All samples were assayed in triplicate.The primers used in this study were listed as follows.PYCR1-forward (F): 5'-GGCTGCCCACAAGATAATGGC-3';PYCR1-reverse (R): 5'-CAATGGAGCTGATGGTGACGC-3';shPYCR1-F: 5'-TTGGCTGCCCACAAGATAAT-3';shPYCR1-R: 5'-ATCACTGTGCTGCACCGTCT-3';GAPDH-F: 5'-TGACTTCAACAGCGACACCCA-3';GAPDH-R: 5'-CACCCTGTTGCTGTAGCCAAA-3'.The AsPC-1 and PANC-1 cells infected by shPYCR1 or shCtrl lentivirus were seeded in a 96-well plate in triplicate at a density of 1.5×10^3 per well. From the second day after seeding, the cells with GFP fluorescence were counted by the Celigo Imaging Cytometer  for 5 days. And the cell proliferation assay is performed at specific times.Further, MTT assays were performed to examine the effect of PYCR1 on cell viability and proliferation. MTT assay began from the second day after seeding. MTT solution  was added to each well and incubated for 4 hours at 37 °C. After that, the MTT solution was removed. 100 μl dimethyl sulfoxide (DMSO) was added to dissolve the formed formazan crystals. The absorbance at 490 nm was measured by a microplate reader . Three independent sets of experiments were performed at least.Cell apoptosis assay was performed by the Annexin V-APC Apoptosis Detection Kit  following the manufacturer's instructions. The PANC-1 and AsPC-1 cells infected by shPYCR1 or shCtrl lentivirus were seeded in 6-well culture plates and cultured for 5 days. The adherent cells were digested with trypsin, washed with PBS, and centrifuged. The cell suspension was mixed with Annexin V-APC and incubated for 10 minutes protected from light. The data were collected and analyzed using a flow cytometer with CellQuest software . Three independent sets of experiments were performed at least.The colony formation assay was performed to assess the effect of PYCR1 on cell survival. The cells infected by the lentivirus were cultured for 5 days and then digested into cell suspension with trypsin. After digestion, the cells were seeded in 6-well culture plates at a density of 2,000 per well. After cultured for 14 days, the cells were fixed with 4% paraformaldehyde and stained with Giemsa solution . The colonies were counted on a fluorescence microscope. Three independent sets of experiments were performed at least.The infected cells were collected, washed twice with PBS, and lysed in lysis buffer. After lysed on ice for 15 min, the cells were broken by sonication. Then, the samples were centrifuged at 10000 g for 15 min at 4 °C to collect the supernatant. The protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit . The proteins were separated by 10% SDS-polyacrylamide gel and electro-transferred onto a polyvinylidene fluoride (PVDF) membrane . The membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature and incubated with primary antibody overnight at 4 °C. And then the samples were incubated with the respective second antibody  for 1.5 hours. The protein signal was visualized using the enhanced chemiluminescence kit . GAPDH was used as an internal control.The antibodies used in this study were as follows: Anti-PYCR1 , Anti-GAPDH , Anti-Mouse IgG , Anti-Rabbit IgG .in vivo. All mice were randomly divided into two groups, normal control group  and knockdown group . AsPC-1 cells (5×10^6 cells) infected with shPYCR1 or shCtrl lentivirus were subcutaneously inoculated into the right flank of the nude mice. Data collection started 1 week after the injection. The mice were weighed and tumor volumes were measured twice per week. The tumor volume was calculated by the formula: Tumor volume = π/6 × Length × Width × Width. After 28 days, all mice were euthanized, and the tumors were removed and weighed. All experimental protocols involving animal experiments were approved by the Ethics Committee of Zhongshan Hospital, Fudan University.Twenty Balb/c nude mice  were purchased from Shanghai Lingchang Biotechnology to assess the effect of PYCR1 In this study, all statistical analyses were performed by the SPSS software version 26.0  and the GraphPad Prism software version 8.0 . The comparison between the means of two groups was analyzed by the F-test to test the homogeneity of variance, followed by paired-samples t-test or Student's t-test when appropriate. A Pearson's chi-square test, Fisher's exact test were used to compare the proportions. Survival analyses were performed by the Kaplan-Meier survival analysis and the overall survival (OS) was calculated from the time of diagnosis until tumor-related death. The log-rank test was used to compare the survival curves. Univariable and multivariable Cox regression analysis were used to identify independent factors.Risk factors are expressed as the hazard ratio . p < 0.05 was considered statistically significant . All experiments were conducted three times and the error bars indicate the standard deviation from the mean of triplicate measurements.To evaluate PYCR1 protein expression in PDAC patients, immunohistochemical staining was performed to measure the expression of PYCR1 in 89 paraffin-embedded PDCA tissues and paired normal adjacent tissues. Our results revealed that PYCR1 expression was higher in tumor tissues compared with that in the adjacent normal tissues . Relative abundances are shown as average ΔCt for low abundance (ΔCt ≥ 16), moderate abundance (12 < ΔCt < 16), and high abundance (ΔCt ≤ 12). The result showed that the mRNA expression abundance of PYCR1 was high in all pancreatic cell lines  and low expression group (n=35) according to the IHC scores of PYCR1 expression. The representative IHC staining of PYCR1 in PDAC tissues and normal pancreatic tissues is shown in Figure And the Kaplan-Meier survival curve was plotted to evaluate the prognostic value of PYCR1. The results revealed that high expression of PYCR1 had an important effect on the OS of PDAC patients. Patients with high PYCR1 levels had significantly longer OS than the low counterparts (Low expression vs High expression = 45.74±5.47 months vs 27.90±3.82 months, P=0.013; Fig. in vitro, we treated the PANC-1 and AsPC-1 cells with shCtrl or shPYCR1 lentivirus. Then the level of mRNA and protein was assayed for PYCR1 using RT-qPCR and Western blot 3 days after the lentivirus infection. Compared with those in the shCtrl group, the mRNA expression of PYCR1 in the shPYCR1 group was significantly inhibited (AsPC-1, 92.6% knockdown, shCtrl vs shPYCR1 = 1.003±0.101 vs 0.074±0.005, P=0.004; PANC-1, 78.6% knockdown, shCtrl vs shPYCR1 = 1.020±0.254 vs 0.214±0.043, P=0.006; Fig. Before proceeding to explore the effect of PYCR1 After infection, the proliferation of PDAC cells was analyzed by Celigo and MTT assays. The results were plotted into line graphs and the results of first day were used as controls. The results showed that the inhibition of cell growth and proliferation was significant in the shPYCR1 group, respectively. (Celigo/Cell count/Day5: AsPC-1, shCtrl vs shPYCR1 = 2757±157 vs 1467±68, P<0.001; PANC-1, shCtrl vs shPYCR1 = 8183±397 vs 1661±162, P<0.001; Fig. in vivo. The AsPC-1 cells were treated with shPYCR1 or shCtrl lentivirus. The average tumor volume in the NC group was significantly larger than that in the KD group (NC vs KD = 748.07±104.83 mm3 vs 578.25±98.43 mm3, P=0.002; Fig. In the final part of the study, the AsPC-1 cells were injected subcutaneously into nude mice to explore the effect of PYCR1 Glutamine is an important energy source for cancer cells and the metabolic disorders of glutamine are closely related to tumorigenesis and cancer progression. Glutamine converses into glutamate in the first step of glutamine metabolism. In particular, glutamate, a key role both in glutamine metabolism and tumorigenesis, is one of the most significant sources for the synthesis of proline in vitro and in vivo. Celigo assay, MTT assay, and colony formation assay showed that the pancreatic cells infected by lentivirus grew slowly. And the results of the flow cytometry showed the percentage of apoptotic cells was decreased in the infected cells. Additionally, PYCR1 interference significantly suppressed tumor growth in nude mice. Taken together, all data demonstrated that PYCR1 plays an essential role in tumorigenesis and progression of PDAC cells.The study found that the expression of PYCR1 in 89 tumor tissues was significantly higher than that in the paired adjacent normal tissues. And the further analysis confirmed that the overexpression of PYCR1 may be associated with pancreatic cancer occurrence and progression. The results showed that the median OS of the high PYCR1 expression group was only 27.9 months, while the median OS of the low PYCR1 expression group was 45.7 months. Multivariate Cox regression analysis in 89 patients revealed that the high PYCR1 expression level was an independent prognostic factor predicting worse OS for PDAC patients. Based on the above study, we hypothesized that there may be a close relationship between PDAC tumorigenesis, progression, and PYCR1 expression. To confirm our discovery, we further investigated the role of PYCR1 both These results corroborate the findings of a great deal of the previous work on the effect of PYCR1 on tumors. A previous study, which collected about 2500 cases from 13 independent microarray datasets, found that PYCR1 was overexpressed in breast cancer. The PYCR1 interference significantly suppressed the breast cancer cell growth and invasion by regulating AKT/ERK signaling pathway. And it enhanced the cytotoxicity of doxorubicin in breast cancer cell lines In summary, according to our preliminary results of the relationship between PYCR1 and PDAC, PYCR1 interference leads to inhibition of proliferation and promotion of apoptosis. It will provide a potential biomarker of diagnosis, prognosis, and therapy for pancreatic cancer. However, more detailed studies are needed to confirm the mechanism of PYCR1 on pancreatic cancer. This remains a goal of our further research.


CD42a was lower in IPF subjects than in controls (p = 0.0153), while CD209, Cd133/1, MCSP, and ROR1 were higher in IPF patients than in controls . Kaplan-Meier survival analysis for IPF patients: for median values and a cut-off of 0.48 for CD25, the two subgroups showed a significant difference in survival rate ; the same was true for CD8 . Conclusion: Our multicenter study showed for the first time the expression of surface epitopes on EVs from IPF patients, providing interesting data on the communication signatures/exosomal profile in serum from IPF patients and new insights into the pathogenesis of the disease and a promising reliability in predicting mid-term survival of IPF patients.Background: Extracellular vesicles (EVs) are secreted by cells from their membrane within circulation and body fluids. Knowledge of the involvement of EVs in pathogenesis of lung diseases is increasing. The present study aimed to evaluate the expression of exosomal surface epitopes in a cohort of idiopathic pulmonary fibrosis (IPF) patients followed in two Italian Referral Centres for Interstitial Lung Diseases, comparing them with a group of healthy volunteers. Materials and Methods: Ninety IPF patients (median age and interquartile range (IQR) 71 (66–75) years; 69 males) were selected retrospectively. Blood samples were obtained from patients before starting antifibrotic therapy. A MACSPlex Exosome Kit, human, , to detect 37 exosomal surface epitopes, was used. Results: CD19, CD69, CD8, and CD86 were significantly higher in IPF patients than in controls ( Extracellular vesicles (EVs) are secreted by the cell membrane into the circulation and body fluids. They contain cytoplasmic components, such as proteins and miRNA. Since EVs express surface proteins linked to their origin, they can adhere and fuse with circulating or distant resident cells. This capturing property of distant cells enables EVs to shuttle functional nucleotides or proteins, providing horizontal transfer of a variety of biological molecules . EVs incIn respiratory medicine, there is increasing evidence of the involvement of EVs in the pathogenesis of lung diseases including lung cancer, chronic obstructive pulmonary disease, and pulmonary fibrosis ,5,6.In the field of lung cancer, studies on EVs are extensively growing, especially about the potential of EVs as diagnostic biomarkers of lung cancer ,7. ExosoA few reports have investigated the role of miRNA in a murine bleomycin-induced lung fibrosis model and in IPF patients. Their results suggest that miRNAs could be useful prognostic indicators of disease .Unlike conventional biomarkers of IPF, such as Krebs von den Lungen-6 (KL-6) ,11,12,13The aim of the present study was to compare the expression of exosomal surface epitopes in a cohort of IPF patients monitored in two Italian referral centres for interstitial lung diseases (ILDs) and in a control group of healthy volunteers.Ninety IPF patients (median age and interquartile range (IQR) 71 (66–75) years; 69 males), monitored at the Siena Regional Referral Centre for Interstitial Lung Diseases and Foggia Regional Referral Centre for Rare Pulmonary Diseases, were selected retrospectively. Patients with clinical evidence of concomitant infections, malignancies, and/or acute exacerbations of the disease were excluded, as well as IPF patients with a radiological pattern of indeterminate usual interstitial pneumonia (UIP). The diagnosis of IPF was confirmed by multidisciplinary discussion according to international guidelines . Only fiBlood samples were obtained from patients before starting antifibrotic therapy ,17. LungDemographic and clinical data, comorbidities, family history, lung function parameters, including diffusion capacity of carbon monoxide (DLCO), and radiological features were obtained from the medical records and entered in an electronic database for statistical analysis.The description of the study design with a flow chart is reported in Healthy controls and patients gave their written informed consent to participate in the study, which was approved by our local ethics committee .g for 10 min. Samples were then frozen at −80 °C.Serum samples were processed as follows: 6 mL blood samples from IPF patients and healthy volunteers were isolated in serum-separating tubes. The blood was spun at 2500× g for 30 min and 10,000× g for 45 min at 4 °C. The pellets were resuspended and filtered through a 0.22 μm membrane. EVs were isolated by ultracentrifuging the supernatant at 100,000× g for 2 h at 4 °C. The EVs were resuspended in 100 μL PBS and stored at −80 °C.According to the manufacturer’s instructions , serum wThe exosome protein concentration was measured by the Bradford protein assay . Equivalent amounts of protein (80 µg) from exosomes were separated by SDS-polyacrylamide 12% gel and transferred to PVDF (Biorad). The membrane was blocked with 5% milk in Tris-buffered saline with 0.1% Tween-20 and incubated overnight at 4 °C with primary antibodies: CD81 , Alix , and β-actin  in blocking solution. After three washes with Tris-buffered saline/0.1% Tween-20, the membrane was incubated for 1 h with HRP-conjugated secondary antibodies. Immunoreactive bands were detected with ECL substrate .The Bradford protein assay was used to measure protein concentrations in BAL fluid samples, as previously reported ,21,22. Tg for 5 min. The supernatant was aspirated and 5 μL MACSPlex Exosome Detection Reagent CD9, CD63, and CD81 were added to each tube followed by incubation for 1 h at room temperature protected from light in an orbital shaker (450 rpm). The washing step was repeated, and 500 μL MACSPlex Buffer was added to each tube followed by incubation for 15 min at room temperature protected from light in an orbital shaker (450 rpm). EV-containing samples were centrifuged and the supernatant aspirated, leaving about 150 μL in the tube. Flow cytometric analysis was carried out on a Facs CantoII flow cytometer followed by Kaluza Analysis 2.1  , which detects 37 exosomal surface epitopes plus two isotype controls . After iCA, USA) a,b. For p value less than 0.05 was considered statistically significant. Statistical analysis was performed by GraphPad Prism 9 software.The results were expressed as means and standard deviations (SD) or medians and quartiles (25th and 75th percentiles) for continuous variables, as appropriate. One-way ANOVA non-parametric test  and Dunn test were performed for multiple comparisons. The Chi-squared test was used for categorical variables. The Kaplan-Meier estimator test was used for survival analysis, and the hazard ratio (Mantel-Haenszel) was calculated. A Clinical, demographic, and immunological data are reported in The purity of serum-derived extracellular vesicle preparations was evaluated by western blot analysis, testing for ALIX and CD81 in three random samples . EVs arep = 0.0023, p = 0.0471, p = 0.0082, and p = 0.0143, respectively) (p = 0.0153), while CD209, Cd133/1, MCSP, and ROR1 were higher in IPF patients than in controls   c. CD42a ctively) c.p = 0.0243, hazard ratio: 0.52 (95%CI 0.29–0.92)  (29–0.92) a; the sa75–2.53) b.The present multicentre study is the first to examine the expression of 37 exosomal surface epitopes in IPF patients. To our knowledge, no data are currently available in the literature on protein expression in EVs from serum of IPF patients. Only one study has investigated miRNA expression and did so in a murine bleomycin-induced lung fibrosis model and in IPF patients: in the murine model, miR-21-5p in EVs was found to be significantly upregulated in acute inflammatory phase and the later fibrotic phase. This increase in miR-21-5p expression in circulating EVs was also observed in IPF patients and showed a significant correlation with disease progression .Our study identified increased expression of many exosomal surface epitopes  in IPF patients with respect to controls, suggesting that an alteration of these markers may be associated with lung fibrosis. From a mechanistic point of view, overexpression of these epitopes in our population is probably related to adaptive immune system dysregulation, which has been described in IPF patients ,25,26,27CD69 is a human transmembrane C-type lectin, initially detected on the surface of activated lymphocytes and known as a very early lymphocyte activation marker antigen . It has Unfortunately, no data are available on NK-CD69-expressing cells in interstitial lung diseases, although increased NK (CD56+) cells were reported in peripheral blood from IPF patients by Esposito et al. . InteresOur study showed high CD42a (known as platelet glycoprotein IX (GP9)) expression in EVs from IPF patients. CD42a is expressed on platelets and megakaryocytes ,41. TranCD209 (known as dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)) was abundantly expressed in our IPF patients. CD209 is a C-type lectin receptor present on the surface of macrophages  and dendOn the other hand, CD133/1 , is a recognized marker of hematopoietic stem cells and committed progenitors  and is aNo data are available in the literature on MCSP  or chondroitin sulphate proteoglycan 4 (CSPG4)) in pulmonary fibrosis. MCSP is a surface type I transmembrane core proteoglycan that is crucially involved in cell survival, migration, and angiogenesis ,59. HoweThe last marker abundantly expressed in EVs of our IPF patients was ROR1 (receptor tyrosine kinase like orphan receptor (1)). This is not surprising since ROR1 and ROR2 are key mediators of WNT5a signalling . WNT5a, Mesenchymal stem-cell-derived EVs have a strong therapeutic effect in cases of organ damage, including acute lung injury . Recent This is a novel and intriguing research study contributing to the definition of EVs’ potential role in the pathogenesis of IPF. Although preliminary, the results of this study were validated in two different cohorts of IPF patients recruited from two Italian Regional Referral Centres for ILD. The main limitations of study include the retrospective design of the research, the lack of data from EVs in relation to IPF treatments and the need to compare our results in different biological fluids.In conclusion, our study shows the expression of surface epitopes on EVs from IPF patients for the first time. It provides interesting data on their communication signatures/exosomal profile in serum from IPF patients, offering new insights into the pathogenesis of the disease. Some of these epitopes proved to reliably predict mid-term survival of IPF patients treated with antifibrotic drugs, suggesting their possible use as prognostic indicators in the management of these patients.