Insufficient recruitment of participants remains a critical roadblock to successful clinical research, particularly clinical trials. Social media provide new ways for connecting potential participants with research opportunities. Researchers suggest that the social network Twitter may serve as a rich avenue for exploring how patients communicate about their health issues and increasing enrollment in cancer clinical trials. However, there is a lack of evidence that Twitter offers practical utility and impact.This pilot study aimed to examine the feasibility and impact of using Twitter monitoring data  as a tool for enhancing clinical trial recruitment at a comprehensive cancer center.We will conduct a mixed-methods interrupted time series study design with a before-and-after social media recruitment intervention. On the basis of a preliminary analysis of eligible trials, we plan to onboard at least 84 clinical trials across 6 disease categories: breast cancer, colon cancer, kidney cancer, lymphoma, non-small cell lung cancer, and prostate cancer that are open to accrual at the University of Southern California (USC) Norris Comprehensive Cancer Center. We will monitor messages about these 6 cancer conditions posted by Twitter users in Los Angeles County. Recruitment for the trials will occur through the Twitter account . Primary study outcomes—feasibility and acceptance of the social media intervention among targeted Twitter users and the study teams of the onboarded trials—will be assessed using qualitative interviews and the 4-point Likert scale and by calculating the proportion of targeted Twitter users who engaged with outreach messages. Second, impact of the social media intervention will be measured by calculating the proportion of enrollees in trials. The enrollment rate will be compared between the active intervention period and the prior 10 months as historical control for each disease trial group. This study has been funded by the National Center for Advancing Translational Science through a Clinical and Translational Science Award. Study approval was obtained from the clinical investigations committee at USC Norris and the institutional review board at USC.Recruitment on Twitter started in February 2018. Data collection will be completed in November 2018.This pilot project will provide preliminary data and practical insight into the application of publicly available Twitter data to identify and recruit clinical trial participants across 6 cancer disease types. We will shed light on the acceptance of the social media intervention among Twitter users and study team members of the onboarded trials. If successful, the findings will inform a multisite randomized controlled trial to determine the efficacy of the social media intervention across different locations and populations.ClinicalTrials.gov NCT03408561; https://clinicaltrials.gov/ct2/show/NCT03408561 (Archived by WebCite at http://www.webcitation.org/72LihauzW)RR1-10.2196/9762 Recruitment of study participants in clinical research, particularly clinical trials, remains a critical roadblock to successful clinical research -5. A recsocial media describes widely accessible Web-based and mobile technologies that allow users to view, create, and share information and to participate in social networking [With billions of users, social media provides new venues to better connect potential participants with research opportunities in a variety of disease and health contexts ,13. The tworking ,15. Usertworking ,17. Neartworking -20. Socitworking  as well tworking -24.digital footprint [surveillance or listening) describes the use of social media data  to gain insights into their interests, attitudes, and behaviors. In this study, we explore Twitter monitoring as a tool to examine cancer-related conditions and concerns expressed by Twitter users in Los Angeles County.The data that social media users generate by creating and interacting with Web-based information, also referred to as their ootprint -28, provootprint -33 and tootprint -38. SociUsing social media monitoring data for targeted clinical trial outreach is considered “active recruitment that occurs when research staff members approach and interact with specific individuals with the aim of enrolling them in research, usually on the basis of knowledge of characteristics that would make them suitable candidates for particular trials” . To datelike, reply, and retweet (share) them. Twitter is primarily a public social network. By default, basic Twitter account information such as the profile name, description, and location is public unless a user decides to opt out and make a private account [Nearly 25% of US adults use the social media platform Twitter with billions of users across the world ,19. Twit account ,45.Due to the more public nature of Twitter, previous research suggested that Twitter provides a “rich and promising avenue for exploring how patients conceptualize and communicate about their specific health issues” . The incMore investigators are incorporating social media in their study recruitment strategy for human subjects’ research in general and clinical trials with varying results -66. In sThe objective of this pilot study is to examine the feasibility and impact of using targeted Twitter monitoring as a tool for enhancing and complementing clinical trial recruitment among Twitter users in Los Angeles County at a comprehensive cancer center. In collaboration with the USC Norris Comprehensive Cancer Center (USC Norris) at the University of Southern California (USC), where the study will be implemented, we will conduct a mixed-methods interrupted time series study design with a before-and-after social media recruitment intervention. We plan to onboard all clinical trials open for accrual, at least 84 based on a preliminary analysis of eligible trials for this study in 6 cancer disease categories: breast cancer, colon cancer, kidney cancer, lymphoma, non-small cell lung cancer, and prostate cancer , page 24likes, retweets, direct messages, following, and contact form use on the trial webpage). Second, the impact of the social media intervention will be measured by calculating the proportion of people who consented and enrolled in trials . The enrollment rate will be compared between the active intervention period and the prior 10 months as historical control for each disease trial group. To aid in the design of larger and more definitive studies, we also intend to estimate the effect size of the number of people enrolled associated with the use of targeted social media monitoring on Twitter as a tool for enhancing cancer trial recruitment. Finally, we aim to establish a method for implementing a social media–driven centralized clinical trial recruitment approach at a comprehensive cancer center, taking into account their internal workflows and processes. The study has 2 primary outcomes. First, the feasibility and acceptance of the social media intervention among targeted Twitter users and the study teams of the onboarded trials, which will be assessed through qualitative interviews using a 4-point Likert scale and a number of quantitative measures to calculate the proportion of targeted Twitter users who engaged with outreach messages  at a time?How does the proposed social media monitoring and recruitment intervention affect the workflow of the study team?How much time and effort does it take to respond to the resulting inquiries from Twitter, to decide whether or not to follow up with a potential participant, and to bring the patient in for an evaluation?How many targeted Twitter users engaged with the outreach message ?How does the social media intervention affect potential participants’ satisfaction and their level of privacy concern?How many targeted Twitter users were prescreened for eligibility?How many targeted Twitter users were eligible based on prescreening?How many targeted Twitter users were screened for eligibility?How many targeted Twitter users were eligible based on screening?How diverse are Twitter users that were targeted for outreach?How diverse are Twitter users that were prescreened?How diverse are Twitter users that were eligible based on prescreening?How diverse are enrolled trial participants ?How effective is the application of social media monitoring to enhance enrollment for clinical trials?What is the enrollment rate  that results from social media monitoring on Twitter ?Study approval was obtained from the clinical investigations committee (CIC) at USC Norris  monitoring disease-specific conversations by Twitter users in Los Angeles County with a focus on 6 cancer topics: breast cancer, colon cancer, kidney cancer, lymphoma, non-small cell lung cancer, and prostate cancer , and (2)To access public Twitter user data, we will use Symplur Signals , a healttrue random number generator [Several aspects of this study will be randomized to reduce selection bias. First, the order in which the cancer trial disease groups will be onboarded in this study will be shuffled randomly using a Fisher-Yates shuffle . Second,enerator . Third, Inclusion criteriaFocus on one of the 6 cancer disease types: breast cancer, colon cancer, kidney cancer, lymphoma, non-small cell lung cancer, or prostate cancerBe institutional review board–approved and open to accrual at the USC Norris Comprehensive Cancer CenterBe a phase 1 trial in expansion, phase 2 or 3Be an interventional trialRecruit in EnglishRecruit for at least 9 months at the point of enrollmentSet a monthly accrual target ≥1 and annual accrual target ≥12Exclusion criteriaPhase 1 trials in dose escalationInclusion criteriaBe located in Los Angeles County based on the self-reported description provided on user’s Twitter profile Mention in any of their Twitter messages at least one word or hashtag related to the 6 cancer disease types Message is an original Twitter message or reply to another user’s messageMessage indicates that Twitter user has been diagnosed with the cancer disease or that they know someone who has been diagnosed with the cancer diseaseExclusion criteriaCancer patients in remission Cancer survivors Persons younger than 18 yearsPersons who note that a relative or friend has died of the diseaseRetweets Clinical trials will be required to meet the eligibility criteria outlined in Participant recruitment for the onboarded clinical trials will occur on the social network Twitter. The study will be limited to those Twitter users who meet the eligibility criteria outlined in Prescreening of Twitter users to determine if they should be triaged to the USC Norris team will occur over the phone. See Study teams of the onboarded cancer trials will not receive monetary or any other compensation for enrolling their clinical trials in this study. We will work closely with the CISO team at USC Norris to recruit all clinical trials in the select cancer disease areas that are open for accrual.Participants  will not receive monetary compensation for participating in the social media study but may receive compensation if they consent to participate in one of the clinical trials depending on the trial. Participants will be recruited using public replies to their Twitter messages that mention words related to the selected cancer disease types . We willinvestigator transparency that “demands investigator truthfulness and honesty when interacting with research volunteers” and promoting “public trust in the research enterprise”  messaging consist of three types of messages, which we refer to as the “outreach message package” . An initerprise”  as suggeerprise” . Via theerprise”  and 2 thInclusion criteriaHave active cancer or recently underwent surgical resection for cancerCancer is visible on scans  unless recently resectedAble to do activities of daily life independently Exclusion criteriaHave completed curative cancer therapy more than 12 months agoInitial outreach messages :We noticed your interest in #disease and wanted to share the latest open clinical research opportunities @KeckMedUSC. You can find more information here: URL #ClinicalTrialWe noticed your mention of #disease and wanted to reach out. Did you know about these open #disease studies @KeckMedUSC? You can find more information here: URL #ClinicalTrialWe noticed your interest in #disease and wanted to share the latest open clinical research opportunities @KeckMedUSC. You can find more information here: URL #ClinicalTrialWe noticed your interest in #disease and wanted to share that the following #disease clinical trials @KeckMedUSC are looking for participants. More information is available here: URL #ClinicalTrialWe noticed your interest in #disease and thought you might be interested in open #disease clinical trials @KeckMedUSC that are looking for participants. More information is available here: URL #ClinicalTrialProject-related message: We’re reaching out to you as part of a research project trying to understand if Twitter can be used to better connect patients with clinical research opportunities.Privacy and security disclaimer: The security of social media is not guaranteed. Contact us about the study. Don’t post if concerned about privacy.follow each targeted Twitter user to whom the outreach message package was sent. This adds the respective person (Twitter account) to the network of @USCTrials and allows them to send us private, direct messages to the @USCTrials account, which some Twitter users may prefer. By default, Twitter only allows direct messages to be sent to followers to prevent misuse.The trial disease group webpage  and 2 inStudy teams of the onboarded cancer trials will not receive monetary or any other compensation for participating in the study team interviews. We will work closely with the CISO team at USC Norris to invite and recruit (via email using USC’s email system) study team members of the enrolled clinical trials to participate in an interview .Brief survey interviews with targeted Twitter users  will be conducted by the study team during prescreening over the phone. The goal of the prescreening questionnaire  is to coPostqualitative interviews with study team members of the onboarded clinical trials  will be undertaken to explore their views of and acceptance of the social media intervention. The interview guide will be based on the research questions to assess feasibility and acceptance . The intlikes, retweets, direct messages, following, and contact form use on the trial webpage). The second outcome will be the impact of the social media intervention, which we will measure by calculating the proportion of people who consented and enrolled in trials . The enrollment rate will be compared between the active intervention period and the prior 10 months as historical control for each disease trial group Diversity of Twitter users targeted for outreach Number of targeted Twitter users who engaged with outreach message Effect of social media intervention on potential participants’ satisfaction and their level of privacy concernNumber of targeted Twitter users who were prescreened for eligibilityDiversity of prescreened Twitter users Number of targeted Twitter users who were eligible based on prescreeningDiversity of Twitter users who were eligible based on prescreening Number of targeted Twitter users who were screened for eligibilityNumber of targeted Twitter users who were eligible based on screeningDiversity of Twitter users who were eligible based on screening Diversity of enrolled participants Outcome: ImpactEnrollment rate per month: number of people enrolled per month divided by number of people targeted on Twitter per trial disease group per month Finally, there are few research studies that report the use of Twitter for participant recruitment. However, these studies focused on other diseases or health conditions . We decided not to use the recruitment results reported by these studies as baseline data or estimates, as we believe that the disease or health condition of a study as well as the type of “ask”  influences the engagement and enrollment rate among potential participants. Hence, we will use the preliminary data from this study to estimate the effect size of the number of people enrolled associated with the use of targeted social media monitoring on Twitter as a tool for enhancing recruitment to cancer trials.Study team members will be provided with tracking sheets to collect data on the potential participants and enrollees. For example, they will track information on who was contacted via Twitter, when a Twitter user was contacted, their Web-based engagement with the outreach message, if targeted Twitter users used the contact form on the clinical trial webpage to contact the study team, and the results of the prescreening phone call. The USC Norris team members will use tracking sheets to track information about the potential participants who were screened, their eligibility, and enrollment.Study data will be collected using the system REDCap (Research Electronic Data Capture) at USC. REDCap is a secure Web-based application designed to support data capture for research studies , providiVerbatim transcription of audio-recorded interviews with the study team members of the enrolled clinical trials will be reviewed for completeness. Transcripts of interviews will be entered, managed, and coded using Atlas.ti (ATLAS.ti Scientific Software Development GmbH), a qualitative data management computer program.The data we collect will only be viewed by the study team for this project. Identifiers such as name, Twitter username, age, and gender data are collected and stored in a secure, Health Insurance Portability and Accountability Act–compliant database REDCap at USC for no longer than 1 year and will be deleted after that time. Additionally, the prescreened participants that we triage to the USC Norris team for further screening will be documented in the secure USC CTMS, which is based on the Web-based software system OnCore and designed to streamline the process of managing clinical trials. We will not store the internet protocol addresses of respondents. Names of noneligible individuals will not be maintained. The data for analysis will be deidentified.To facilitate the qualitative data analysis of the interviews with the study team members of the onboarded clinical trials, we will develop an initial code list based on the interview guide. The code list will be modified throughout the coding process. Each coded transcript will be discussed line by line until the coding team (including the co-PI) comes to an agreement about code definitions and how they should be applied. Important themes will be summarized and used to understand acceptance with the social media–based intervention for cancer clinical trials. Count outcomes will be presented as median and interquartile range; nominal outcomes will be presented as N (%). We will explore participant and study team characteristics between the 2 cohorts to examine where differences might lie by including them as potential covariates in the models. Comparisons of the before- and after-time periods will be made using generalized estimating equations for appropriate outcome type  accounting for the type of cancer.P values are of limited use to determine group differences, so we will focus on observed effect sizes . Additional quantitative data will be calculated using proportions of targeted Twitter users who engaged with outreach messages . To aid in the design of larger and more definitive studies, we also intend to estimate the effect size of the number of people enrolled associated with the use of targeted social media monitoring on Twitter as a tool for enhancing cancer trial recruitment.The impact of the social media intervention will be determined comparing monthly enrollment rates during the active intervention period  versus the prior 10 months as historical control for each disease trial group  using generalized estimating equations, accounting for intradisease random effects and trends. Analyses will be performed in SPSS v24 . As thisThis research project presents minimal-risk research. We will use public user data from the social network Twitter. Identifiable information such as human subjects’ names and Twitter handles will not be included in the analysis dataset. Patient identifiers do not apply. We have implemented the following measures to ensure data and information confidentiality and to minimize risk . We will further abide by USC IRB regulations and the USC Privacy of Personal Information policy. In general, all data will be entered into a computer and database that is password protected. The data will be stored using appropriate, secure computer software and encrypted computers. The IRB-approved study protocol details further information on procedures for monitoring and assessing study-related concerns , page 16We identified a number of scientific, ethical, and regulatory challenges to this study. Refer to the IRB-approved study protocol for further information on perceived ethical and regulatory issues and how we will manage these challenges and related risk . All listed authors and/or contributors are compliant with guidelines outlined by the International Committee of Medical Journal Editors for author inclusion in a published work. Public access to the study protocol and other necessary aspects will be made available through our ClinicalTrials.gov identifier (NCT03408561). Furthermore, to support research transparency and reproducibility, we will share the deidentified research data after publication of the study results. We will share the deidentified data on Figshare, a repository where users can make all of their research outputs available in a citable, shareable, and discoverable manner. We will use a data-sharing agreement that provides for (1) a commitment to using the data only for research purposes and not to identify any individual participant and (2) a commitment to securing the data using appropriate computer software.This study has been funded by the NCATS through a Clinical and Translational Science Award (CTSA) award . Study aWe recognize that this pilot study will not have sufficient resources to recruit a truly representative sample. Thus, the generalizability of the study results is somewhat limited. Twitter messages from locations outside of Los Angeles County, as well as messages in other non-English languages such as Spanish, and therefore nonspeakers of English, will not be included. Moreover, the social media intervention favors those with internet access and could therefore introduce potential bias into the clinical trials. Regardless of the fact that social media users “have grown more representative of the broader population” , TwitterThis pilot project will provide preliminary data and practical insight into the application of publicly available Twitter data to identify and recruit clinical trial participants at a comprehensive cancer center across 6 cancer disease types. If successful, the findings of this study will inform a multisite RCT to determine the efficacy of the social media intervention described here across different locations and populations. In addition, data from Twitter users and study team members of the onboarded clinical trials will be translated into a preliminary set of testable questions to further examine challenges around the use of social media monitoring in clinical trial recruitment in general and at comprehensive cancer centers.


Epithelial-to-mesenchymal transition (EMT) is one mechanism of carcinoma migration, while complex tumour migration or bulk migration is another - best demontrated by tumour cells invading blood vessels.Thirty cases of non-small cell lung carcinomas were used for identifying genes responsible for bulk cell migration, 232 squamous cell and adenocarcinomas to identify bulk migration rates. Genes expressed differently in the primary tumour and in the invasion front were regarded as relevant in migration and further validated in 528 NSCLC cases represented on tissue microarrays (TMAs) and metastasis TMAs.Markers relevant for bulk cancer cell migration were regulated differently when compared with EMT: Twist expressed in primary tumour, invasion front, and metastasis was not associated with TGFβ1 and canonical Wnt, as Slug, Snail, and Smads were negative and β-Catenin expressed membraneously. In the majority of tumours, E-Cadherin was downregulated at the invasive front, but not absent, but, coexpressed with N-Cadherin. Vimentin was coexpressed with cytokeratins at the invasion site in few cases, whereas fascin expression was seen in a majority. Expression of ERK1/2 was downregulated, PLCγ was only expressed at the invasive front and in metastasis. Brk and Mad, genes identified in Drosophila border cell migration, might be important for bulk migration and metastasis, together with invadipodia proteins Tks5 and Rab40B, which were only upregulated at the invasive front and in metastasis. CXCR1 was expressed equally in all carcinomas, as opposed to CXCR2 and 4, which were only expressed in few tumours.Bulk cancer cell migration seems predominant in AC and SCC. Twist, vimentin, fascin, Mad, Brk, Tsk5, Rab40B, ERK1/2 and PLCγ are associated with bulk cancer cell migration. This type of migration requires an orchestrated activation of proteins to keep the cells bound to each other and to coordinate movement. This hypothesis needs to be proven experimentally.The online version of this article (10.1186/s12885-018-4640-y) contains supplementary material, which is available to authorized users. Migration and invasion into the stroma is a requisite for cancer metastasis. Two mechanisms have been described in the last decade, namely epithelial to mesenchymal transition (EMT) and complex tumour cell migration. In EMT, as primarily described in experimental studies, the tumour cells downregulate adherence proteins, lose contact to cells, and change to a mesenchymal phenotype, often by exchanging keratin for vimentin or α-actin , 2. ThesDue to the fact that vascular invasion is a poor prognostic factor , we usuaAs a first step, we aimed to explore the frequency of bulk migration in AC and SCC, to evaluate molecules which might be associated with migration, invasion, and metastasis using immunohistochemistry. We are aware of limitations of this study: morphology provides a snapshot at a certain stage of development of a carcinoma, but cannot provide insight into dynamics of migration. However, we can compare expression patterns in the primary tumour with migrating and metastatic cells, which is not possible in a cell culture system. A morphological analysis can provide necessary information on which to base an experimental design.Thirty cases of AC and SCC were retrieved from the Lung Archive, all characterized by vascular invasion into pulmonary arteries or veins. The selected cases required having the central tumour, the invasion front to lung or pleura, and the vascular invasion on the same tissue slide to compare staining patterns side by side Fig. . As the Each case in the TMA was represented by at least 3 cores of tumour tissue and 1 core of normal lung tissue or metastatic site. Immunohistochemistry in TMAval/TMAmets was performed for those markers, which showed a different staining pattern in the study set between the central tumour and the two invasion sites (front and/or vascular invasion). Three pathologists  evaluated the slides independently. All cases were discussed and re-evaluated together in cases of discordant scores.Twenty-eight out of 30 carcinomas showed bulk cancer migration, with tumour bulks also seen in the blood vessel walls Fig. -c. One cAll carcinomas demonstrated nuclear positivity for Twist (30/30), there was no difference between invasion and vascular invasion, and the main tumour. Staining intensities ranged from moderate to strong Fig. , and in All carcinomas stained for E-Cadherin (E-Cad) in a membranous pattern Fig. . The intFascin antibodies stained 19 cases of central tumour and invasive front Fig. , in 7 adMother against dpp (Mad) and Brinker (Brk) were more intensely positive at the invasion site in 23/30 carcinomas Fig. . In 4 adStaining for the two invadipodia-inducing proteins Rab40B (member of RAS oncogene family) and Tks5 (scaffold protein TKS5) showed positivity exclusively at invasion sites Fig.  in 21/30Extracellular regulated MAP kinase (pERK1/2) was positive in the central areas of 10 carcinomas, and in 6 of them the invasion front was positive as well, however with lower intensity compared to the main tumour Fig. . In 16 cConnexin43, an adhesion molecule for homing to lung endothelia, was positive in 20/30 carcinoma Fig. . These w(ZFPL1) was positive in 8/30 tumours; however, in 7 of them, only few predominantly keratinized SCC cells stained; only one carcinoma was diffusely positive.Antibodies for Slug, TGFβ1, and basic leucine zipper ATF-like transcription factor 2 (SARI) reacted negatively in all carcinomas. 13/30 cases were minimally positive for Snail, but only in a small number of cells. SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 4 (SMARCA4) was positive in all cases. There was no difference when the tumour centre, the invasion front and vascular invasion were compared. Yes-associated protein 1 (YAP1) was positive in 29/30 cases, the invasion site always being less intensely stained Fig. . In 21 cFrom the validation set, 82 cases of SCC and 83 AC showed bulk migration, 2 SCC and 1 AC cases showed EMT migration pattern, and 31 SCC and 33 AC cases showed a mixed bulk and EMT migration pattern , and AC on the TMAval, the following was observed Table : E-Cad sThe TMA with combined primary tumour and metastasis tissue was tested for Connexin, Tks5, MAD, TWIST, vimentin, PLCγ, and CXCR4. In the TMAmets brain and extrathoracic lymph nodes metastasis were predominantly present, while adrenal glands, liver, skin, and bone appeared less frequently. There was a good concordance of positively stained carcinoma cells in the primary tumour and the metastasis for MAD, connexin43, CXCR4, and PLCγ, whereas TWIST, vimentin, and Tks5, were significantly more highly expressed in metastasis , and creating energy for movement – all depending on the initiation of a multi-layered genetic program.Here, we describe bulk cancer cell migration, also called complex movement or hybrid EMT , 18, as In bulk migration, there is no mesenchymal transition, as the tumour cells retained cytokeratin and E-Cad, although the latter downregulated at the invasion front. N-Cad was induced in all carcinomas more intensely stained at the invasion front, but always coexpressed with E-Cad. Although Twist was active in all tumours, its regulation was not controlled by TGFβ1 . In contIn EMT, the exchange of cytokeratin for vimentin is explained by the fact that vimentin gives carcinoma cells more flexibility to change cell shape and thus adapt for migration better. E-Cad contributes to tight binding of carcinoma cells. Our finding of tumour cells coexpressing vimentin and cytokeratin at the bulk border and of reduced expression of E-Cad in these bulks  probably results in more freedom of movement while still keeping the cell together . It mighFascin an actin-binding protein, was upregulated in tumour centres, but more intense at the invasion front. It might be another factor providing cross-talk within bulk cells. Fascin is crucial for filopodia formation . InteresInvadipodia formation is induced by either Rab40B or Tks5 or both , 30. CoePhosphorylated ERK1/2 was expressed in the carcinoma centre as well as at the invasion front, but less intense. As this expression was, in some cases, only present at the invasion front, ERK seems to play a role in migration, probably by interacting with other not yet identified proteins. There might exist underlying functional changes of ERK: Migrating cells usually do not proliferate, but when forming a metastatic focus, an increase in energy is required, probably reflected by ERK activation. PLCγ, another enzyme under the control of the RAS pathway, was exclusively upregulated at invasion sites and negative in tumour centres. In addition, it was highly expressed in metastasis compared with the primary tumour (invasion front), although this did not reach statistical significance. This confirms reports which already suggested that this enzyme is directly involved in migration and metastasis, probably by anaerobic decomposition of lipids –36. InteUpregulation of Mad, Twist, and PLCγ in metastasis points to important functions within the metastatic site, as well. Bulk movement is most likely the preferred mode of migration at metastatic sites, too. Mad, however, might be an early mediator of migration and is thus expressed equally in primary and metastatic tumours. Tks5 was unexpectedly lost in one third of metastases. It might be that the formation of invadipodia can be regulated by other proteins. The function of PLCγ requires further investigation, as this enzyme is important for migration as well as metastasis.Connexin43 (Con43), reported to be associated with intrapulmonary metastasis , was expChemokine receptors have been reported as being specifically associated with homing. CXCR4 has been reported as being responsible for metastasis to the brain , 42. In Focal adhesion kinase (FAK) was expressed in only few cases and in few tumour cells. The expression of SMARCA4  was positive in all carcinomas, at equal amounts within the tumour centre and the invasion front. SMARCA4 loss has been reported in dedifferentiated carcinomas and has been associated with rapid progression. This could not be proven in our series. Despite the fact that many of the carcinomas were well to moderate differentiated, they did not lose SMARCA4 .All markers identified in the study set were also found in the large cohort of carcinomas . There were some differences in the frequency of expression, especially for Mad, Tks5, PLCγ, and pERK, which might be due to the fact that, in tissue microarrays, punches are most often taken from central tumours, and therefore invasion front is not always represented and vascular invasion appears rarely, as well. The findings of frequent expression of Mad, Twist, vim, and PLCγ in metastasis confirms this interpretation. With respect to the different types, E-Cad, N-Cad, Mad, PLCγ, and Tsk5 were expressed in similar percentages in all carcinomas, while Connexin43 and Twist were more frequently expressed in SCC and LC compared to AC.By establishing a model to study bulk migration, several aspects will be taken into account: Cell cultures cannot be used, as a low density of seeded cells will automatically induce EMT, including expression of vimentin or smooth muscle actin to reach their neighbouring cell. The cells would also lose cytokeratin and E-Cad and express N-Cad , 47.In conclusion, we identified bulk cancer cell migration as the predominant form in AC and SCC, whereas pure EMT is rare. In addition, we identified a mixed form composed of EMT and bulk migrations which accounts for approximately 25% of AC and SCC. Several proteins known from EMT studies have been identified, but are differently regulated in bulk migration. Twist, vimentin, fascin, Mad, Brk, Tsk5, Rab40B, ERK1/2 and PLCγ are involved in bulk cancer cell migration, but they are not under the rule of TGFβ1 Additional file 2:Table S2. Raw data of the evaluation of the metastasis TMA. Prim = data of the primary tumor and META = data from the corresponding metastasis; in case of several metastasis the data were pooled and a mean was calculated. In addition statistical calculations for each marker were added (student t-test for paired data). (XLSX 27 kb)Additional file 3:Figure S1. Schematic cascade of migration factors. In EMT  TGFβ activates Twist, which activates ZEB1, SNAIL and Slug; ZEB1 suppresses cytokeratin, E-Cad, and RhoA, but upregulates Vim and N-Cad. In bulk migration (green) Twist is upregulated by an unknown factor, it does neither induce ZEB1, nor SNAIL or SLUG, but likely induce YAP1; E-Cad is down- and N-Cad upregulated. YAP1 might be upregulated by N-WASP, and itself upregulates probably ERK1/2; RhoA does not block YAP1 (via LATS), and Mad/Brk probably induce Tks5 and RAB40B; vascular growth factors of the chemokine family might induce RhoA and PCLγ. (JPEG 2662 kb)


Varicella zoster virus (VZV) is a highly contagious herpesvirus with potential for nosocomial transmission. However, the importance of nosocomial chickenpox outbreak in China has often been ignored. With the increasing immunocompromised population in China, a thorough review of issues related to nosocomial transmission and the seroprevalence rate of VZV among healthcare workers is necessary.Retrospective case finding for nosocomial transmission of chickenpox was conducted between January 1, 2013 and December 31, 2017. Cases were identified based on clinical features compatible with chickenpox. A cross-sectional study on the seroprevalence rate of VZV among healthcare workers (HCWs) was conducted between January 1, 2014 and December 31, 2017. The serum VZV antibodies of 1804 HCWs were measured by enzyme-linked immunosorbent assay (ELISA). The seroprevalence rate of VZV antibodies, the positive predictive value and negative predictive value of self-reported history of varicella were analyzed. The economic impact associated with nosocomial transmission of VZV was also assessed.P < 0.01). The seroprevalence rates of HCWs with different genders and occupations showed no statistically significant differences. The positive and negative predictive values of a self-reported history of varicella were 80.8 and 10.6% respectively. An estimation of 163.3 person-days of work were lost in each nosocomial transmission and 86.7 infection control unit person-hours were required for each outbreak investigation. The cost of VZV IgG ELISA screening was estimated to be 83 USD per nosocomial transmission.A total of 8 cases of chickenpox were identified in three nosocomial transmissions, including 4 HCWs who were infected nosocomially. The overall seroprevalence rate of VZV was 88.4%, which significantly increased with age (Nosocomial transmission of VZV occurred repeatedly in the hospital setting. An alarming 11.6% of HCWs were seronegative for VZV, which might increase the risk of nosocomial infection and outbreak for other susceptible co-workers and patients. This is especially important in the setting of a teaching hospital where many immunocompromised patients were managed. Furthermore, the positive predictive value of self-reported varicella on seroprevalence rate in our study was lower than those reported in other countries, therefore serological testing of VZV antibodies with subsequent vaccination for all non-immune HCWs should be considered. Varicella zoster virus (VZV) causes two clinically distinct forms of disease: varicella (chickenpox), as a primary infection, and zoster (shingles) due to reactivation of latent VZV –3. The tPrimary VZV infection is usually self-limiting in immunocompetent hosts, however, the severity increases with age. Furthermore, as HCWs care for patients in different wards and units, there is a high potential of nosocomial transmission among HCWs and patients if the HCW is infected, especially when the infectious period of VZV starts 2 days before the onset of rash. The situation would be worse if the infected HCW is involved in taking care of pregnant women and immunocompromised individuals (such as patients receiving high dose immunosuppressant or history of transplantation), as primary VZV infection in immunocompromised patients can be fulminant, and it can present as encephalitis, pneumonitis and hepatitis. The mortality rate of primary VZV infection could be up to 7% if left untreated. As a result, nosocomial transmission of VZV should never be overlooked.As nosocomial transmission of varicella is seldom reported in China, this study aims to report three nosocomial transmissions of chickenpox, and investigate the seroprevalence rate of VZV among healthcare workers in a tertiary teaching hospital in China.Retrospective case finding for nosocomial transmission of chickenpox was conducted, with specific attention being paid to infection control measures and outbreak investigations between January 1, 2013 and December 31, 2017. The clinical definition of varicella is “an illness with acute onset of diffuse  papulovesicular rash without other apparent cause”. All contacts in the hospital were followed up by the infection control unit staff to determine the number of susceptible contacts (non-immune to varicella) by contact tracing. Susceptible contacts were put under self-medical surveillance and requested to refrain from patient contact from Day 8 after 1st exposure to Day 21 after last exposure. Individuals were considered susceptible if they did not fulfill any one of the following criteria: 1) History of varicella; 2) Completion of varicella vaccine according to recommended schedule; 3) Positive serum VZV IgG. Post-exposure prophylaxis (PEP) for susceptible contacts were administered according to CDC recommendations [A cross-sectional study on the seroprevalence rate of VZV among healthcare workers (HCWs) was conducted between January 1, 2014 and December 31, 2017 in a tertiary teaching hospital in China, with 1804 hospital staff including doctors, nurses, technicians and non-clinical workers participated in this study. The study was approved by the Ethics Committee of the hospital. Through questionnaire, information including demographic data, history of varicella and vaccination were collected from the participants. Blood samples were collected from each individual and the serum VZV antibodies were measured by enzyme-linked immunosorbent assay (ELISA) using commercial kit .P-value of less than 0.05 was considered statistically significant. Positive predictive value (PPV) and negative predictive value (NPV) of a self-reported history of varicella were calculated, with a positive serum VZV IgG considered as the gold standard of immunity against VZV.Statistical analysis was performed using SPSS 20 software. Categorical variables were compared using Chi-square test. A In order to assess the economic impact of nosocomial transmission of VZV, two important parameters, person-days of work lost and infection control unit person-hours, were assessed. Person-days of work lost is defined as the total number of days of work loss in clinical staff for both susceptible contacts and infected cases. Infection control unit person-hours is defined as the number of hours required for infection control unit for outbreak investigation and control. The cost of performing VZV IgG ELISA was also calculated based on the price of equipment together with the manpower required to perform the test.There were three episodes of nosocomial transmission of varicella involving 8 individuals, in which 4 HCWs  were infected nosocomially. Table On May 5, 2013, a paediatrician (Case 2) presented with fever followed by generalized vesicular rash. She recalled contacting a patient (Case 1) with chickenpox on April 18, 2013 in the hospital, and she was susceptible to VZV, i.e., she was neither vaccinated nor previously infected with VZV. She was diagnosed to have varicella and requested self-isolation to prevent ongoing transmission. Contact tracing was initiated by the hospital infection control unit and a total of 23 healthcare worker contacts were identified with advice on prodromal symptoms to ensure they were not exposed to other susceptible HCWs or patients. No further new cases were identified after the above infection control measures. After this incident, serum VZV IgG testing for HCW was introduced by hospital health direction.On August 22, 2013, a non-immune operator from the hospital reception center (Case 4) presented with fever, generalized vesicular rash and sore throat. She recalled one of her colleagues (Case 3), who worked with her in the same office, developed chickenpox during August 10–17, 2013. She was subsequently diagnosed to have varicella. The infection control unit was informed of the incident, with 17 healthcare worker contacts identified through contact tracing. Further history taking and laboratory testing suggested 4 of the 17 contacts were susceptible, therefore they all received post-exposure prophylaxis with oral acyclovir. No further new cases were identified after the above infection control measures.On March 20, 2017, two nursing students (Case 5 and Case 6) from hospital outsourcing company reported to have fever and generalized vesicular rash. They worked as nursing assistants in the hospital and lived in a 8-person dormitory room. They were diagnosed to have varicella and requested self-isolation. The infection control unit was informed, with 42 healthcare worker contacts identified through contact tracing. Thorough investigation suggested 5 out of 42 contacts were susceptible. All susceptible contacts received VZV vaccination as Post-exposure Prophylaxis (PEP), with recommendation on self-isolation during incubation period provided.On April 5, 2017 and April 7, 2017, two nursing students (Case 7 and Case 8), who lived with Case 5 in the same dormitory, presented with similar clinical manifestations as Case 5, with VZV DNA detected from the vesicular fluid of skin lesions in both nursing students by PCR (Polymerase Chain Reaction). These two infected individuals were not classified as susceptible contacts during the first round of contact tracing, because both of them volunteered previous history of VZV vaccination. A further 45 healthcare worker contacts were identified through second round of contact tracing, and 4 out of 45 contacts were classified as susceptible individuals, with subsequent post-exposure prophylaxis provided. No further new cases were identified after the above infection control measures.Of the 1804 HCWs participated, there were 215 (11.9%) male and 1589 (88.1%) female HCWs, with their ages ranging from 17 to 60 years (median age: 27 years). Table Table A total of 20 person-days of work were lost by infected clinical staff during the above episodes, based on the fact that immunocompetent persons are unlikely to be infectious after day 5 of rash. Isolating susceptible healthcare worker contacts from duty from days 8 to 21 after last exposure would result in further working hours lost, with 470 person-hours loss for the above three episodes. Therefore, a total of 490 person-hours were lost during the above described three transmissions, with an estimate of 163.3 person-hours being lost per nosocomial transmission.During the first two transmissions, infection control unit required 40 h per transmission to contact trace, follow up and provide counseling to all involved healthcare workers. As the third transmission involved more clinical staff, infection control unit required 180 h for infection control measures. Therefore, a total of 260 infection control unit person-hours were required for the three transmissions, with 86.7 infection control unit person-hours required per transmission.The cost of each VZV IgG ELISA commercial kit is 11 RMB , and manpower for performing each ELISA kit is 2.5RMB  in our laboratory. With a total of 127 tests performed as a result of nosocomial transmissions, the total cost of VZV IgG ELISA screening is 1714.5 RMB (around 248 USD), with around 83 USD per nosocomial transmission.Varicella zoster virus (VZV) is a highly contagious herpesvirus with potential for nosocomial transmission, with the infectious period starting 2 days before the onset of rash, and lasting for 5–7 days after the appearance of rash –11. SeveNosocomial outbreaks of varicella involving both healthcare workers and patients are reported in other countries , 19–22. The seroprevalence rate of VZV in healthcare workers varies according to different geographical areas, with the prevalence of seronegativity ranging from < 5% in the USA, 8.9% in Taiwan, 19% in Saudi Arabia, 26% in India to approximately 50% in Sri Lanka , 24. In In our study, the seroprevalence rate of VZV in HCWs significantly increased with age, and this could be explained by accumulated time of exposure to VZV during lifetime in elderly. The seroprevalence rates of HCWs with different genders and occupations showed no statistically significant differences.In order to investigate whether self-reported history could replace serum VZV IgG screening in HCWs, self-reported history of varicella in HCWs was compared with the serum VZV IgG result to assess the reliability of self-reported history. Our study suggested that a positive history of varicella provided by HCWs is not a good predictor of positive serum VZV IgG, as our study demonstrated a false positive rate of 19.2%. On the other hand, 89.4% of HCWs with a negative history of varicella turned out to be seropositive, suggesting negative self-reported history could not rule out existing immunity towards VZV. Review of literature from different countries suggested a self-reported history of varicella has a positive predictive value of 92.5–99.5% and a negative predictive value of 2.5–14.4% –27. CompThe limitation of our study is that not all HCWs participated in our study, as 244 HCWs refused screening of serum VZV IgG, which may in turn lead to selection bias. However, further statistical analysis did not reveal any statistical differences by chi-square test between these HCWs and those who participated. Furthermore, this present study included a large number of HCWs with a wide age range and diverse occupations, therefore it still could provide a convincing approximation of the situation of VZV immunity in HCWs in China.With the advancement in the field of transplantation and medicine, we are encountering more immunocompromised patients in our daily practice. Our study clearly demonstrated that assuming seropositivity based on self-reported history of varicella is unreliable, and significant proportion (10.6%) of existing HCWs are still susceptible to VZV. Further intervention should be implemented in the health care system to prevent hospital transmission of varicella. It is time for us to implement routine serum VZV IgG testing and immunization of HCWs if serum VZV IgG result turns out to be negative, not only to reduce the cost of outbreak investigation, but more importantly to ensure safety of both HCWs and our patients. Such intervention should not only be performed on newly recruited members, but also to existing healthcare workers in the hospital.Nosocomial transmission of VZV occurred repeatedly in the hospital setting. An alarming 11.6% of HCWs were seronegative for VZV, which might increase the risk of nosocomial infection and outbreak for other susceptible co-workers and patients. This is especially important in the setting of a teaching hospital where many immunocompromised patients were managed. Furthermore, the positive predictive value of self-reported varicella on seroprevalence rate in our study was lower than those reported in other countries, therefore serological testing of VZV antibodies with subsequent vaccination for all non-immune HCWs should be considered.


The authors regret that the Acknowledgments section should have been as follows: “The present study was funded by the Grand Challenges Canada Stars in Global Health program, a Bill & Melinda Gates Foundation grant as part of the Pre-eclampsia Eclampsia Monitoring Prevention and Treatment (PreEMPT) initiative, and the Canadian Institute of Health Research grant KPE-124730.”


To facilitate assessment of the variability in sO2 and haemoglobin in tumours, for example in response to therapies, the baseline variability of these parameters was evaluated in subcutaneous head and neck tumours in mice, using a PAI system (MSOTinVision-256TF). Tumours of anaesthetized animals (midazolam-fentanyl-medetomidine) were imaged for 75 min, in varying positions, and repeatedly over 6 days. An increasing linear trend for average tumoural haemoglobin and blood sO2 was observed, when imaging over 75 min. There were no significant differences in these temporal trends, when repositioning tumours. A negative correlation was found between the percent decrease in blood sO2 over 6 days and tumour growth rate. This paper shows the potential of PAI to provide baseline data for assessing the significance of intra- and inter-tumoural variations that may eventually have value for predicting and/or monitoring cancer treatment response.Photoacoustic imaging (PAI) provides information on haemoglobin levels and blood oxygenation (sO Tempora2)  between days 1 and 6 of imaging, probably because they outgrew their blood supply before new vasculature could be formed. Slower growing tumours (n = 3) exhibited an increase or no change in sO2 [+12% and -1%], possibly because angiogenesis was occurring at a rate similar to the tumour growth rate. Interestingly, Tomaszewski et al. [2 (while O2 breathing) and ΔsO2 also decreased with tumour growth, from a 30 mm3 volume to time of culling. The CALR model used for this project has also been shown to develop rapidly and become hypoxic [In order to assess whether there was any dependence of tumour blood sOsurement  and the i et al.  used oxy hypoxic . This obA limitation of this study is that it evaluated the baseline variations in the photoacoustic signal for only one tumour type, a subcutaneous head and neck tumour model. Other tumour types might show different variation. For example, Tomaszewski et al.  showed t52 and HbT) and oxygen saturation over short (up to 75 min) and long (6 days) periods, and the variation with repositioning of the animal, in untreated subcutaneous head and neck tumours in nude mice. Time under anaesthesia is the largest source of temporal variability in the photoacoustic signal. To perform inter- and intra-animal comparison in longitudinal studies, for example when assessing the effects of treatment, ideally the imaging should be started at a fixed time point after anaesthesia and, if possible, sham-treated animals should be imaged at the same time points as the treated animals.This paper documents the variation in haemoglobin (Hb, HbOPositioning of the animal was found to influence image quality in a limited number of animals; when reconstruction artefacts or an unexpected absence in PAI signal are observed during the preview of the scan, the animal can be removed and re-positioned to obtain improved PAI signal. With a carefully planned experimental protocol, photoacoustic imaging could be used for assessing changes in tumour haemoglobin and oxygenation in longitudinal studies, including those designed to assess cancer therapies that may affect vasculature and, consequently, tumour oxygenation.All data and/or analysis performed in this study are available from the corresponding author on request.The authors declare that there are no conflict of interest.


In the Plastic, the 15N-urea was applied to the ridge only (Plastic-Ridge), or to the furrow only (Plastic-Furrow). Maize grain yield and net economic benefit for Plastic were significantly higher  than those for Open. Total plant 15N uptake was 72.5% greater in Plastic compared with Open, and 15N was allocated mostly to the grain. Losses of the applied urea-N were 54.5% lower in Plastic and much more residual 15N was recovered in 0–120 cm soil compared with Open . Lateral N movements from furrow to ridge and from ridge to furrow were observed and attributed to lateral movement of soil water due to microtopography of ridges and furrows and uneven soil water and heat conditions under mulching and plant water uptake. The ridges were the main N fertilizer source for plant uptake  and the furrow was the main source of N losses . Gas emissions, especially ammonia volatilization was probably the main N loss in furrow. Thus, appropriately localized N application – into the ridges, and management strategies should be designed for Plastic to maximize N use efficiency by crops, decrease N gas losses and maintain sustainable agricultural systems in drylands.A better understanding of the fate and transport of fertilizer nitrogen (N) is critical to maximize crop yields and minimize negative environmental impacts. Plastic film mulching is widely used in drylands to increase soil water use efficiency and crop yields, but the effects on fertilizer N use efficiency need to be evaluated. A field experiment with  ExcessiPlastic film mulching was introduced in China in 1978, originally only for vegetables but now is widely used for maize, wheat, potato and other staple crops . Plastic2O emission  has been one of the most effective cultivations to increase water use efficiency, soil temperature and yields for rain-fed croplands in Northwestern China, especially in the region with 400–600 mm of precipitation . This Pl15N tracer technique in the Plastic system, compared with a flat system without mulching (Open), to examine (1) plant N uptake and N losses; (2) vertical and lateral redistribution of residual N in soil; (3) differences in the fate of applied N for ridges and furrows.We hypothesize that plant N uptake and N losses will differ between the furrows and ridges in the Plastic system, and will have various advantages compared to the soil without plastic mulching. There are very few published data focused on this comparison and, in particular, no studies have simultaneously measured both lateral and vertical N transport. Therefore, we investigated the fate of fertilizer N for maize production in a semi-arid area using a 22.1The study site was located on the Loess Plateau at the Changwu Agricultural and Experimental Station of the Chinese Academy of Sciences  and has a semi-arid climate. Mean annual air temperature is 9.1 ℃; the average annual precipitation is 580 mm and more than 60% of total precipitation falls in July, August, and September . The ave2.22 (4.5 × 10 m). The urea application rates were the same in all plots: 260 kg N ha−1 , 40 kg P ha−1, and 75 kg K ha−1. All fertilizers were spread over the plots and mixed with the 0–15 cm surface soil by rake as a basal dressing, as is the current farming practice. Ridges and furrows were made after fertilizer application in Plastic. The Plastic comprised alternative furrows (30 cm wide) and ridges (15 cm high × 70 cm wide), and the ridges were covered with plastic film (0.008 mm thick and 90 cm wide). Maize was planted on each shoulder of the ridge with a spacing of 30 cm along the ridge, 40 cm across the ridge and 60 cm across the furrow (−1 on 30 April 2015 and harvested on 27 September 2015. The precipitation in this period was 346 mm (Two cropping systems were included : (1) a fe furrow . The Opee furrow . A high-s 346 mm , and no 2, covering a complete ridge and furrow, including 6 plants, 15N-labeled urea  and the P and K fertilizers, then the soil and fertilizers were returned to the micro-plot. In Plastic, the 15N-labeled fertilizer was applied in two positions in the separated micro-plots: 1) applied to the ridge only (Plastic-Ridge) and 2) applied to the furrow only (Plastic-Furrow). The fertilizer application rate for ridges and furrows in the micro-plots was the same as for the large plots, with the ridge: furrow ratio of 7:3 . In Open, the 15N-labeled fertilizer was applied evenly across the whole surface layer of the micro-plot. All micro-plots were enclosed by an aluminum sheet inserted into the soil to a depth of 2 cm and exposed 3 cm above the surface to prevent surface runoff.To study the fate of the applied fertilizer N, micro-plots . Soil samples were taken at the same depths in the micro-plots  were combined as a single sample for each depth . Mineral N was calculated as the sum of NO3− and NH4+. The content of microbial biomass N in soil was measured by the chloroform fumigation-extraction method The content of NO15N abundance in soil and plant samples was determined by isotope ratio mass spectrometer (IRMS) at UC Davis Stable Isotope Facility, University of California. The ammonium diffusion method . The natural abundance of 15N in soil and plant samples was also measured.The n method  followed2.4pu, kg ha−1), plant N derived from 15N-labeled fertilizer , plant N derived from soil , fertilizer N recovery , soil residual N  and N fertilizer lost to the environment  were calculated according to Eqs. p is plant dry matter yield (kg ha−1) and Np is plant N content (kg kg−1) dry matter;15N%p and 15N%f are the atom% 15N excess in the plant and soil, respectively;rate is the fertilizer N application rate (kg ha−1);15Nsoil is the soil 15N content (g kg−1 dry soil), soilbd is the soil bulk density (g cm-3) and d the depth of soil sampled (m);Plant total N uptake (Nhttp://www.npcs.gov.cn/ and http://china.guidechem.com/). Income refers to income from grain yield. The NEB was calculated by subtracting the input cost from the yield income . The total costs included the cost of field operations , fertilizer/pesticide/seed, and plastic film (d income .t-test was used and least significant differences (p < 0.05) were calculated to test for significance of differences in grain yield, dry matter biomass, Npu, Ndff, and Ndfs between Open and Plastic. The differences between treatments  were tested with one-way analysis of variance (ANOVA) and following Tukey-test (p < 0.05). The differences between depths we tested with one sample t-test. Homogeneity of variances was tested by Levene’s test, normal distribution of residues was tested by Shapiro test. All the data analyses were performed using SPSS 22. Graphs were produced with Origin 9.1.The paired sample 33.1p < 0.05), but stem, cob cores, bract and total aboveground biomass were similar (p < 0.05), compared with Open, especially in the grain and stems. The plant N uptake derived from the soil (Ndfs) was similar for the two systems (p > 0.05), accounting for 71.3% and 80.6% of plant N uptake for Plastic and Open, respectively. In contrast, the plant N uptake derived from urea-fertilizer (Ndff) was 71.1% higher for Plastic than for Open  accounted for 19.0–87.3% of total residual 15N in the soil layers for Plastic-Ridge at harvest in 2015, with an average of 48.2%. Respective values were 7.5–26.1% (average 15.1%) and 8.3–52.6% (average 29.6%) for Plastic-Furrow and Open, respectively. The NH4+-15N content was lower than NO3−-15N in all soil layers. The soil mineral 15N was higher in Plastic than that in Open (p < 0.05). Microbial biomass 15N was low, accounting for 0.3–12.4% of the total residual 15N in Plastic-Ridge, and 0.3–19.6% and 1.2–11.1% in Plastic-Furrow and Open, respectively. The larger 15N incorporation in microbial biomass occurred at soil depths of +15-0 cm, 0–20 cm, and 0–40 cm in Plastic-Ridge, Plastic-Furrow and Open, respectively (15N was larger in Open than in Plastic (p < 0.05).The mineral-ectively a–c. The 4+-15N and NO3−-15N in the soil were similar in Plastic-Furrow, Plastic-Ridge and Open at harvest  in Plastic-Ridge was 58.9% higher than that at harvest in 2015  for Plastic, while for Open it was mostly in the 0–60 cm soil layers  in the 0–20 cm soil layer (15N that moved from furrow to ridge (Furrow → Ridge) and from ridge to furrow (Ridge → Furrow) were 12.2 and 6.0 kg ha−1, respectively  for Plastic and Open, but the surplus N was different due to higher crop uptake and lower potential N loss in Plastic  was the same  the 15N fertilizer was applied in the top soil layers and the plastic film prevented N leaching by rainfall (15N by microorganisms at early stage of maize growing season) due to the higher temperature and moisture in mulched soil at late stage of maize growing season  the g season . Althoug Plastic  implied  harvest .Three processes: ammonia volatilization, denitrification and N leaching are associated with the potential N losses in the urea-fertilized field. The N leaching was low both in Plastic and Open . Nitrate2O) emission . Ammonia volatilization may be reduced with appropriate agricultural management, e.g. N fertilizer other than urea (e.g. KNO3)  . Mulching. KNO3) . Compare4.3−1, compared with 0.03 kg ha−1 for the ridge), this value accounted for only a small part of N loss. More than 96% of the plant 15N uptake was derived from the Plastic-Ridge, with less than 4% from the Plastic-Furrow, implying that the ridge was the main source of fertilizer N for plant uptake and the furrow was the main source for N losses. A much higher residual soil 15N was found in Plastic-ridge, compared to Plastic-Furrow , compared to the data at harvest. However, the mineral N content increased in Plastic-Ridge during this period. This difference can be attributed to the practice of keeping the mulch after harvest. Mulching can reduce N leaching in the fallow period. Additionally, the decomposition of crop roots and rhizosphere microorganisms during the fallow period  resulted4.4−1 for Open and Plastic, showing the high productivity in this area . This would improve N uptake and reduce gaseous N losses in the Plastic system to maintain the sustainability of agriculture in drylands.Compared with Open, Plastic significantly increased maize grain yields by 9.7% in a semi-arid area. Total N uptake and uptake of applied urea-N were both increased under Plastic mulch. Estimated losses of the applied urea N via ammonium volatilization were very high, but lower under Plastic, and residual soil N up to 1.2 m depth was greater. Lateral movement of N from furrow to ridge and from ridge to furrow occurred in Plastic, facilitated by lateral movement of soil water. In Plastic, the ridges were the main source of fertilizer N uptake by the plants (> 96%), and the furrows were the main source of N losses (c. 79%). Briefly, Plastic increased yields and net economic benefit but changed the fate and transport of applied urea-N in maize production in semi-arid rain-fed croplands. Therefore, appropriate N management strategies should be designed for plastic mulching systems, such as minimal or zero fertilizer application to furrows or the use of N fertilizer forms other than urea (e.g. KNO


As the human microbiota has been confirmed to be of great significance in maintaining health, the dominant bacteria in them have been applied as probiotics to treat various diseases. After the detection of bacteria in tumours, which had previously been considered a sterile region, these bacteria have been isolated and genetically modified for use in tumour therapy. In this review, we sum up the main types of bacteria used in tumour therapy and reveal the mechanisms of both wild type and engineered bacteria in eliminating tumour cells, providing potential possibilities for newly detected, genetically modified, tumour-associated bacteria in anti-tumour therapy. Bacteria have also been detected in some regions that had previously been considered sterile, including the placenta, breast milk, tumours and blood. This suggests the potential existence of probiotics or pathogens in those areas and may provide clues for the diagnosis and treatment of tumours th century, wild-type Clostridium perfringens was first detected in patients with cancer, and live anaerobic bacteria were injected into animal models to examine their oncolytic effects Salmonella typhimurium, Listeria monocytogenes) were designed to kill tumour cells with the development of advanced gene engineered techniques via the expression of tumour-related antigens, pro-drug-converting enzymes or agents toxic to tumours In the early 19Cancer is a devastating disease defined by abnormal cell growth with the potential to invade and spread to other parts of body, which is called metastasis All tumours contain two basic components: the parenchyma and stroma C. perfringensStreptococci, finally reaching the achievements of Coley's toxin (heat-killed Streptococcus and Serratia marcescens) The existence of bacteria in tumours provides a potential new direction for cancer therapy using wild type or engineered bacteria. The first breakthrough in cancer therapy dates back to 1813, when Vautier observed tumour regression in patients with gas gangrene after infection of Clostridium spp., Bifidobacterium spp., Salmonella typhimurium, Vibrio cholera, Listeria monocytogenes, and Bacillus spp. are bacteria that have been most widely identified inside tumours and have the ability to kill tumour cells in a natural or genetically modified form, indicating their potential for therapeutic effects in anti-cancer therapy  Later, in 1935, Connell found that the proteolytic enzymes secreted by Clostridium oncolytic therapy, J. R. Möse and G. Möse injected spores of non-pathogenic proteolytic strain C. butyricum intravenously into mice transplanted with solid Ehrlich carcinomas Clostridium spores with local irradiation and high-frequency hyperthermia increased the level of hypoxia in the tumour and led to an evident increase in the survival rate of mice bearing melanomas Aiming at finding an unharmful C. sporogenes ATCC 13732 and verified its non-pathogenicity in humans Clostridium spores in five patients with neoplastic disease. Oncolysis only occurred in large tumours, not in the surrounding normal tissues or smaller tumours and metastases Clostridium spore injection only affects large tumours rather than small tumours and metastases, and it cannot clear all tumour cells, which may result in the regrowth of the tumour or the discontinuation of clinical trials.In 1967, J. R. Möse and G. Möse injected themselves with spore suspensions of Clostridium became available, much effort was put into the development of Clostridium strains producing anti-tumour proteins. Because of their exquisite specificity to germinate in the hypoxic/necrotic regions of solid tumours, these strains could be used as vector systems for the specific delivery of therapeutic proteins in the tumour microenvironment C. sporogenes ATCC 13732 with the Escherichia coli-derived gene coding colicin E3, a bacteriocin with cancerostatic properties C. beijerinckii was capable of expressing E. coli enzymes nitroreductase and cytosine deaminase, which converts the non-toxic pro-drugs CB1954 and 5-fluorocytosine into toxic anti-cancer compounds, which diffuse into the tumour and trigger cell death by interfering in DNA replication/transcription Clostridium with the promotor recA driving the expression of the gene encoding for the cytokine TNF-α after 2 Gy of irradiation, indicating a promising prospect for gene targeting and ionising radiation C. novyi was the most promising C. novyi without its lethal toxin (C. novyi-NT) were intravenously injected with chemotherapeutic drugs, resulting in extensive necrosis of transplanted tumours within 24 hours C. novyi-NT and led to the dramatic regression of transplanted tumours in mice C. novyi-NT recombinated with specific anti-tumour genes.As soon as recombinant DNA techniques and specific transformation protocols for Bifidobacterium is a genus of Gram-positive, obligate anaerobic bacteria. They are naturally present in the dominant colonic microbiota, and represent up to 25% of the cultivable faecal bacteria in adults and 80% in infants Bifidobacterium is present in fermented foodstuffs  and its safety is supported by those foodstuffs and growing knowledge about Bifidobacteria taxonomy and physiology Bifidobacterium is considered as a safe candidate carrier due to the bacterial medicine Bifidobacterium bifidum , which is prescribed for patients in Japan. Experiments showed that after the intravenous administration of a Lac B suspension in tumour-bearing mice, they did not show any adverse symptoms E. coli (e-CD) B. longum carrying the e-CD expression vector or non-pathogenic E. coli as a control. The results showed that no inflammatory cytokines were induced by the injection of B. longum while E. coli induced the cytokines such as the interleukin-6 (IL-6), IL-1β and IL-18. This experiment demonstrated that the genetically modified B. longum did not trigger intense immunological response, thus verifying its safety. Similar results have also been reported showing that Bifidobacteria induces low levels of IL-12 and TNF-α in the blood regardless of the strain used. In contrast, strains Lactobacillus possess different abilities to induce IL-12 and TNF-α In order to examine the immunological reactions, further tests concentrated on inflammatory cytokine levels in the blood after the i.v. injection of Salmonella typhimurium is a pathogenic, Gram-negative, facultative anaerobic bacterial species that can be found predominantly in the intestinal lumen. Its toxicity is due to an outer membrane consisting of abundant lipopolysaccharides (LPS) which protect the bacteria from the environment. The LPS is made up of an O-antigen, a polysaccharide core and lipid A, which connects it to the outer membrane S. typhimurium has the ability to undergo acetylation of this O-antigen, which changes its conformation and makes it difficult for antibodies to recognise S. typhimurium, have been detected as the pathogen that causes self-limited enteritis in most healthy adults, infects many mammalian species and can easily be manipulated to carry therapeutic transgenes S. typhimurium exists as a facultative anaerobe, allowing it to survive in both oxygenated and hypoxic conditions; thus, it may colonise both small metastatic lesions and larger tumours. In 1997, Pawelek et al. found that Salmonella has the ability to infect and accumulate within implanted tumours in mice, achieving the ratios of concentrations in tumour/normal tissue up to 1,000:1, which indicates that this may be a clinically useful anti-cancer agent S. typhimurium limits its potential in preclinical studies of tumour therapy. For the earliest detection of attenuated Salmonella, Bacons and his co-workers reported the Salmonella with auxotrophic mutants had a great reduction in virulence S. typhimurium has been attenuated by partial deletion of the msbB gene, which is responsible for addition of a terminal myristyl group to lipid A S. typhimurium to induce the secretion of TNF-α in vitro in human monocytes and in vivo after the administration to mice and pigs purI gene, creating a growth requirement for external sources of purines, whose concentrations are high in the interstitial tissues in the tumour environment with the attenuated toxicities identified in mutant strains S. typhimurium (VNP20009) has a safety profile of possessing partial deletion of both msbB and purI genes, which limits the toxicity of S. typhimurium in normal tissues while retaining the tumour-targeting and tumour-inhibiting properties Wild-type Salmonella, in particular Salmonella spp. to retard tumour growth was assessed in various animal models. When single-cell suspensions of Salmonella were intravenously injected into animals with 4-5 mm tumour or in the case of metastatic models, the growth of the tumour and dissemination of metastases were strongly inhibited for a prolonged period In preclinical experiments, the ability of attenuated The mechanisms that lead to the accumulation of bacteria in tumours compared to relative lower levels in other tissues may include the different vascular structures and blood flow patterns, which may lead to the entrapment of bacteria in tumours. Additionally, bacteria may also attach or be phagocytosed by immune cells transferred to tumour sites Salmonella, recent studies show better therapeutic effects in combination with other anti-tumour therapies. As the bacteria alone could only delay the tumour growth rather than inhibit the recurrence of tumours, more rigorous genetic modulations could be applied to S. typhimurium to enhance the tumour inflammatory response. These strategies include the delivery of genes coding for cytokines, pro-drug-converting enzymes and agents toxic to tumour. For instance, engineered attenuated S. typhimurium is able to express cytosine deaminase, which could possibly catalyse the non-toxic pro-drug 5-fluorocytosine to anti-tumour drug fluorouracil, which mimics uracil in tumour cell replication and inhibit this process S. typhimurium detected in mice models. In this situation, an attenuated S. typhimurium was engineered to express a Toll-like receptor (TLR) 5 agonist Vibrio vulnificus flagellin B protein (FlaB), leading to the recognition of lipopolysaccharide on the surface of the S. typhimurium and triggering an immune response inside the tumour; the efficacy was stronger than with a single TLR4 or TLR5 agonist S. typhimurium expressing the cytokine LIGHT was injected intravenously to mice models of both primary and established pulmonary metastases, carrying out anti-tumour effects by binding to LIGHT receptors, transducing signals to induce the expression of chemokines Concerning the clinical trials about attenuated The genes that can be chosen should maintain a high tumour to normal tissue ratio, resulting in the largest beneficial effect to patients. Meanwhile, genes delivered to the bacteria should have very low toxicity or even no influence on normal tissues. Ideally, the secreted substance or the activated drug should have a short circulating half-life and exert anti-tumour effects. It should be also shown that the synergetic toxic substances produced by bacteria do not influence the replication and persistence of the bacteria in tumours. It may happen that toxicities of expressing substances evolve from the modification of enzymes inside our bodies. If it comes to the situation that toxicities are too strong to sustain, it may be possible for patients to kill the bacteria and eliminate the toxicity using antibiotics. Even under the worst circumstances, the tumour would be exposed to far greater concentrations of the agent than normal tissue and presumably even limited exposure might have a substantial therapeutic benefit.With the numerous effector genes that could be engineered into bacterial hosts, therapies could be extended to sequential or concurrent administration of similar or different bacteria that contain separate gene products. Demonstration of the central concept of selective intratumoural accumulation of bacteria in cancer patients can be expected to lead to a vast and novel repertoire of therapeutic options for the treatment of metastatic diseases.Listeria monocytogenes is a Gram-positive, facultative anaerobic, rod-shaped bacterium that has tumbling motility and is unable to form spores L. monocytogenes lives and replicates within the cytosol of the host cell. It is one of the most virulent foodborne pathogens and is usually phagocytosed by antigen-presenting cells (APC). Once phagocytosed, the vast majority of the bacteria will be degraded inside the phagosome; however, about 10% of the bacteria are able to take advantage of virulent factors to break down the phagosome, which enables them to live freely in the host cytosol. These virulence factors mainly include liseriolysin O (LLO), which is a pore-forming haemolysin and phospholipase L. monocytogenes starts to undergo rapid replication L. monocytogenes that extend towards nearby cells, forming a double-membrane protrusions and infecting them L. monocytogenes is not exposed to the extracellular defences of the host, primarily accounting for why host cells do not develop a protective humoral response to it. The genes related to this cycle  are regulated by the transcriptional activator listeriolysin A (PrfA) As a facultative anaerobic intracellular bacterium, L. monocytogenes, antibodies have been regarded for a long time to play no role in Listeria resistance, as there have been studies showing no protective effects after transferring the serum of Listeria-immune mice to naïve mice ListeriaL. monocytogenes escape from the phagosome is considerably reduced due to the opsonisation of LLO, thereby limiting intracellular growth. However, the amount of anti-LLO antibody used by Unanue and colleagues in these studies far exceeded physiological levels, putting into question to what extent the antibody can work well in neutralising the bacteria and demonstrating the humoral response may be too weak to provide effective protection to Listeria infection L. monocytogenes can spread to nearby hosts, the rest of the bacteria are killed through a series of mechanisms mediated by our immune systems. Early resistance to L. monocytogenes is controlled mainly by macrophages, neutrophils and NK cells through various cytokines and chemokines L. monocytogenes phagocytised by macrophages are degraded after fusion of the phagosome and lysosome and protein fragments are presented on major histocompatibility complex (MHC) II molecules induced by IFN-γ, which in turn present antigen to CD4+ T cells. The remaining 10% of L. monocytogenes escape from the phagolysosome and replicate inside the cytosol. The proteins produced here are degraded inside the proteasome and transported into the lumen of the endoplasmic reticulum (ER) through the transporter associated with antigen processing (TAP) and loaded onto MHC I molecules, thereby triggering the CD8+ T cell response L. monocytogenes is an ideal vector for delivering antigens to be processed and presented through both the MHC class I and II antigen processing pathways, which is a hallmark of host immunity to L. monocytogenes.Considering the host immune response to L. monocytogenes has been used as a vector to generate cell-mediated immunity against a wide range of pathogen antigens: influenza nucleoprotein (NP), HPV E7, HIV gag, simian immunodeficiency virus (SIV) env, tumour antigens tyrosinase-related protein (trp)-2 and human epidermal growth factor receptor (HER)-2/neu in preclinical experiments by normally i.v. injection. As a vaccine vector, the recombinant L. monocytogenes strains that have been developed for clinical uses are highly attenuated compared to wild type Listeria. With attenuation, the existing anti-vector immunity does not affect the therapeutic efficacy, which was verified by using a mutant Listeria missing the actA gene as the antigen vector Owing to this property, unusual among intracellular pathogens, + T cell response that is strong enough to kill tumour cells, the antigen that is responsible for triggering the immune response needs to be secreted outside the cell wall of the bacteria. In order to achieve this goal, L. monocytogenes is always engineered in a way by fusing target antigens with a secreted bacterial protein such as LLO to form a fusion protein. The promoter and signal sequence of LLO have been used to increase the expression of the target antigen For the purposes of generating a CD8L. monocytogenes as a vector for cancer immunotherapy was carried out in a recombinant influenza NP (Lm-NP) against several tumour cell lines  + and CD8+ T cells, demonstrating a potent anti-tumour immunity triggered by recombinant L. monocytogenesThe first test of L. monocytogenes was utilised to carry tumour-associated antigens HPV-16 E7, which can be either part of the Listeria genome following the haemolysin signal sequence (Lm-E7) or recombined into the plasmid with hly promotor followed by a non-haemolytic fusion of LLO-E7 (Lm-LLO-E7). Both of the strains were tested in established TC-1 tumours, derived from murine lung epithelial cells immortalised by HPV-16 E6 and E7 as well as transformed with activated oncogene rasBased on this successful approach, L. monocytogenes are designed to express tumour-related antigens to trigger both innate and adaptive immunity clinically L. monocytogenes vaccines expressing human papilloma virus (HPV) serotype 16 E7 (ADXS11-001), which is known to be overexpressed in HPV-related cervical cancer, have reached phase III clinical trials. Additionally, live-attenuated strains (ADXS31-142) that express prostatic specific antigen (PSA) and HER-2 expressing vaccines are in phase I/II clinical trials. Attenuated L. monocytogenes vaccines expressing mesothelin (ANZ-100/CRS-207) is being tested in mesothelioma, pancreatic, ovarian and Fallopian cancers Despite the preclinical studies carried out in animal models, engineered Bacillus spp. is a genus of Gram-positive, rod-shaped, obligate aerobic or facultative anaerobic bacteria that can form spores Bacillus spp. are relatively abundant, exhibiting antifungal, anti-bacterial and even anti-tumour activities Bacillus thuringiensis, isolated firstly as a pathogen in the sotto disease of the silkworm, Bombyx mori, has the ability to produce crystalline parasporal inclusions when sporulating th century to possess strong insecticidal effects to several insects B. thuringiensis has garnered the interest of scientists as an insecticide for decades, while recent studies reveal that the most common B. thuringiensis strains distributed in nature are non-insecticidal, raising the question as to whether the parasporal inclusions of non-insecticidal B. thuringiensis have specific bioactivities Although B. thuringiensis, containing 1700 Japanese isolates from Kyushu University and 44 reference types from the Institut Pasteur, Paris B. thuringiensis, named parasporins (PS) http://parasporin.fitc.pref.fukuoka.jp/), the 81 kDa parasporin characterised by Mizuki et al. in 2000 constitutes the PS1Aa1 family. Currently, this protein group is subdivided into six major families (PS1-PS6) 2+, with no change in plasma membrane permeability, leading to the apoptosis of cancer cells B. thuringiensis have only been tested in vitro, they pave the ways for further investigations in treating cancer in vivo.In 1999, Mizuki et al. first screened the proteins inside the parasporal inclusions of 1744 strains of B. thuringiensis, another closely related strain is B. cereus. The only discrimination between them is the crystalline proteinaceous inclusions produced by B. thuringiensisB. cereus have been identified as probiotics when administered in certain amounts orally B. cereus; fraction BC1 showed cytotoxicity against HepG2 cells by damaging DNA and triggering apoptosis in liver cancer cells B. thuringiensis, this fraction did not contain proteins Beyond Bacillus, including B. subtilis, B. licheniformis and so on have also been discovered to induce cytotoxicity in cancer cells via various compounds Bacillus possesses strong bioactivity by producing various compounds that are toxic to cancer cells in vitro, providing direction for further investigations into the metabolites of Bacillus in the treatment of cancer.Other species of Clostridium spp., Bifidobacterium spp., S. typhimurium, Vibrio cholera, E. coli and L. monocytogenes have been observed to live in tumours; some of them have been applied to treat tumours using wild type or gene-modified strains S. pyogenes OK-432, a poorly virulent strain of type III group A S. pyogenes that can be treated with penicillin G, has been used as agent to treat unresectable lymphangiomas. Mycobacterium bovis (BCG), acquired from a virulent strain of M. bovis by incubating it on a special medium, can be used to treat bladder cancer Although existing oncolytic bacteria are under perpetual development, various challenges remain before they can be used successfully in the clinic. Problems include the concentration of toxic agents, targeting efficacy, bacterial toxicity, genetic instability and the combination with other anti-cancer therapies. Considering the concentration of toxic agents produced by bacteria in tumours, it should be high enough to exert therapeutic effects but not induce systemic toxicity. Targeting efficacy is influenced by tumour size, location and blood supply, which is distinct in different patients F. nucleatum, which is indigenous to the oral cavity, has also been found in the gut microbiota and shows associations with colorectal carcinomas. Comparing the microbiome of colorectal carcinoma with that of a healthy colon, high enrichment of F. nucleatum in colonic tissues as well as stools of patients with colorectal adenomas and adenocarcinomas has been identified by metagenomic analyses F. nucleatum-associated colorectal cancers have revealed that the influence of tumourigenesis is not exerted by inducing inflammation or exacerbating colitis-associated colorectal cancer; rather, it is conducted by increasing the infiltration of CD11b+ myeloid cells, a phenotype of myeloid-derived suppressor cells (MDSCs) in mice, which facilitate tumour growth and angiogenesis + T cells through the expression of arginase 1 and inducible nitric oxide synthase (iNOS) F. nucleatum-fed mice by a mean of 13.4 times and 7.8 times, respectively, compared with controls, which indicates the modulatory effects of F. nucleatum to tumour-immune microenvironments, thereby promoting tumour progression and metastasis Upon discovering oncolytic bacteria living in tumours, scientists found that some bacteria show specificities for certain types of tumours. F. nucleatum-associated colorectal cancers in human were identified. They include TAM-associated genes , MDSC-associated genes , PTGS2 (COX2), IL-1β and TNF-α. Their expression levels correlate with the abundance of F. nucleatum in Fusobacterium-associated tumours and some of these genes suggest an NFκB-driven proinflammatory response . This signalling pathway was later found to be more highly activated with a higher abundance of F. nucleatum in tumours After collecting supportive data from the mouse models, the immune cell biomarker genes related to Enterobacteriaceae and Pseudomonadaceae are dominant In another similar situation, Straussman and colleagues detected the existence of bacteria in samples of human pancreatic ductal adenocarcinomas (PDACs) and showed that the most common species, accounting for more than half of all reads, belong to the Gammaproteobacterium, in which members of F. nucleatum and Gammaproteobacteria have excellent potential to be remoulded directionally for anti-tumour therapy. With the mechanisms of these bacteria in promoting tumour progression being revealed, the genes responsible for these processes can be selectively knocked out and recombined with genes expressing tumour-associated antigens, enzymes converting pro-drugs or agents toxic to tumours. By taking advantage of their characteristics of targeting and colonising certain types of tumours, incorporated with genetic modifications, bacteria living inside specific tumours have excellent potential for treating cancer and represent an innovative direction in complementary and direct anti-tumour therapy.Based on the extensive tumour-specific colonisation of these bacteria, together with the rapid development of gene-modification techniques, bacteria living in certain types of tumour such as


Polycystic ovary syndrome (PCOS) is a common reproductive disease with high heterogeneity. The role of excess androgen in PCOS etiology remains disputed, since around 20%–50% of PCOS women do not display hyperandrogenemia. The microenvironment of the ovary critically influences follicular development. In the present study, we assessed the role of androgen in PCOS by investigating whether excessive follicular fluid androgen was present in PCOS patients with normal serum androgen levels and influenced by follicular fluid insulin resistance (IR). Follicular fluid samples of 105 women with PCOS and 105 controls were collected. Levels of steroid hormones, glucose and insulin in the follicular fluid were examined and compared with data from serum biochemistry tests. We found that 64.9% (63/97) of PCOS patients with normal serum androgen levels displayed abnormally high follicular fluid androgen level. The follicular fluid androgen level was positively correlated with follicular fluid IR within a certain range and follicular fluid estrogen-to-testosterone (E2/T) ratio was significantly reduced in these patients. These results indicated that there existed a subgroup of PCOS patients who displayed excessive follicular fluid androgen and IR despite their normal circulating testosterone (T) levels. Our study highlights the importance of ovary hyperandrogenism and IR in the etiology of PCOS. It affects 6%–18% of women of reproductive age. We determined whether follicular fluid IR contributed to excessive follicular fluid androgen by testing follicular fluid insulin and glucose levels with RIA. Our data showed that the glucose level in the follicular fluid was significantly higher in PCOS women when compared with the control group (P = 0.02), but no significant difference of the insulin level was detected (Table 2). The follicular fluid HOMA-IR value was 10.05±6.17 in the PCOS group, and was significantly higher than 7.66±4.33 in the control group (P<0.01). In Group 1 follicular fluid T was found to be positively correlated with follicular fluid glucose level  and HOMA-IR , although it was not significantly correlated with follicular fluid insulin . Paradoxically, in Group 2 follicular fluid T showed no significant correlation with insulin , glucose  and HOMA-IR  in the follicular fluid. These data suggested that ovary IR might be a promoting factor of high ovary T level in a subgroup of PCOS patients.IR has been found to have promoting effects on HAIn this study, we found that follicular fluid androgen level could be significantly increased in women with PCOS even though these patients displayed normal circulating androgen level. Such ovary HA was partially positively correlated with the local IR level in the ovary. These observations have important implications for improvement of PCOS diagnosis and treatment as well as for understanding of PCOS etiology.–16.Although HA is thought to be responsible for oligo-ovulation and follicular atresia, varied prevalence of PCOS women with HA has been reported and the role of androgens in PCOS etiology is still an open question. Furthermore, little is known about the relationship between circulating and ovary HA in PCOS patients, though they could occur together in patients with PCOSIn the present study, the frequency of PCOS patients with biochemical androgen excess was only 7.62% (8/105), lower than expected. In contrast, the significant higher frequency  of ovary androgen excess was detected in the PCOS patients. Ovary androgen excess was not always in parallel with that in circulating serum. PCOS women who displayed normal circulating androgen levels had great possibility to have excessive ovary androgen. These observations, on one hand, supported the critical role of abnormal androgen metabolism in the etiology of PCOS. On the other hand, our study implied a non-negligible limitation of using excessive circulating androgen as diagnostic criteria for PCOS. Ovary HA could be a hidden and more direct factor of ovarian malfunctions. These findings had potential clinical and therapeutic implications and suggested that abnormal ovary androgen metabolism should be carefully considered in diagnosis and treatment of PCOS.BMI has been confirmed to be positively related to circulating androgen excess. The low percentage of PCOS patients with circulating biochemical HA observed in this study might be partly explained by the low rate of overweight PCOS patients we collected, since losing weight, rather than IVF, was a preferred choice for overweight PCOS patients at our Center. The independent increase of follicular fluid T in PCOS patients and its diagnostic value deserved further explorations in large samples.[17–. The HOMA-IR (plasma glucose × plasma insulin/22.5) is a well-defined criterion for evaluation of IR, which is correlated with the gold standard clamp test for IR. Since there is still no standard to measure IR level in the follicular fluid, we used HOMA-IR to estimate IR in the ovary in this study. We found that the level of follicular fluid HOMA-IR was significantly higher in women with PCOS than in the control group. Chi-square analysis showed that follicular fluid IR was positively correlated with follicular fluid HA either in the PCOS group or the control group. These data strongly suggest that ovary IR and HA may intrinsically interact with each other. Little is known about the intra-ovary interaction between T and IR. Few reports available are conflicting. For example, T treatment was reported to down-regulate Glut4 expression and downstream insulin signaling, leading to IR in endometrial cells, while other reports showed HA had no significant effect on insulin signaling in granulosa cells. On the other hand, insulin was reported to stimulate androgen production by enhancing the expression and activation of P450c17 in theca cells in vitro[23–. The follicular fluid IR might prevent follicular fluid T from converting to E2, since the E2/T level was significantly lower in the follicular fluid of PCOS patients. Determination of intro-ovary CYP19a1 activity and expression of PCOS patients with follicular fluid HA and follicular fluid IR will provide useful clues. At present, we do not know how excessive follicular fluid androgen could exist in PCOS patients with normal androgen level in serum. We proposed that follicular fluid IR may partly contribute to the local high androgen, since follicular fluid T level was positively correlated with follicular fluid HOMA-IR values. Follicular fluid androgen excess and IR might disturb local hormone metabolism, thus contributing to the pathogenesis of PCOS.HA and IR are considered to be strongly associated with PCOSAnother interesting question raised by this study is whether ovary IR could occur independent of circulating IR level as in the case of HA. Unfortunately, the data of circulating insulin level of the patients examined in this study were not available. Further investigation is required.One limitation of the present study is the small sample size of the PCOS patients with high circulating androgen levels. Further study with a larger patient population is required to determine whether follicular fluid T levels are even higher in PCOS patients with high circulating androgen levels than those without.In conclusion, our present study revealed that a large proportion of PCOS patients with normal circulating androgen displayed follicular fluid HA. Follicular fluid T level was positively correlated with follicular fluid IR in a certain range. Our study highlights the potential roles of ovary androgen excess and IR in PCOS etiology and has significant implications for PCOS diagnosis and treatment.