Several cost-effective programs are being implemented around the world that use mobile technology to improve Sexual and Reproductive Health (SRH) uptake and awareness among youth. Mobile phone applications are a viable and effective means of increasing access to SRH services and tools in low and middle-income countries. This paper presents a protocol for a pilot study of a novel program, a mobile phone-based sexual and reproductive health services awareness and delivery application with the objective of increasing the demand for SRH services amongst the youth in Uganda.The study employs rigorous evaluation methods to ascertain the impact of the mobile application. We propose a randomized control trial study to determine the causal effect of the mobile phone app in creating awareness and increasing uptake of sexual and reproductive health services in Uganda. The main outcome of the impact evaluation is the percentage change in the SRH services and tools uptake, SRH knowledge and sexual behavior. We will also conduct a model-based incremental cost-effectiveness analysis (CEA) and budget impact analysis (BIA). The main outcomes of the economic evaluation will be the average cost per app user, cost per app service and tool provided. We will also test the in-app advertising model as a way to generate revenue to sustain the program subsidies and related costs.The study seeks to establish the proof of concept of using a mobile application to increase create awareness and increase uptake of SRH tools and services among youth in Uganda. The study results will lead to the development of a demand-driven, culturally-relevant, and easy-to-use mobile app to enhance the uptake of SRH services among the youth in Uganda and globally.MUREC1/7 No. 07/05–18. Registered 29th June 2018. We purpose to develop a mobile phone application (app) to increase awareness and uptake of sexual and reproductive health (SRH) services among youth aged 18 to 30 years in Uganda. This app is intended to link users to health facilities where they can access SRH goods and services. It will also reduce on the challenge of poor access to SRH services due to lack of privacy and confidentiality: the app allows users to make orders online and goods are delivered to pick up points of individuals’ choice. We will partner with an app developer to develop the app. We will then pilot this study among students of Kyambogo university, and participants who will download the app will be followed up at 1, 3 and 6 months. We will collect data on app usage from the server, and perform analyses to determine technical performance, impact (increase in SRH demand and usage), acceptability, usability, and cost effectiveness. The prototype app includes an advertising interface to enable corporations and other business entities to collaborate as potential advertisers and to generate revenue for sustainability. If successful, we propose to extend the program to all the youth in the country who own a smart phone but, also, include other services.Across the globe, youth account for 17.6% of the population size, and in low and middle-income countries (LMICs), while 90% are aged between 10 and 24 years  and overIn Uganda, youth face many SRH risks such as the unmet need for contraception (30%), which leads to unplanned pregnancies (43%) that result in unsafe abortions (30%) and sexually transmitted infections (17%) , 9]. Th. Th9]. TAwareness and uptake of SRH services remains sub-optimal in LMICs , 12, 14,The question researchers and policymakers should be asking is, what works for the youth? Earlier reviews suggest that SRH interventions targeting youth should be accessible, equitable, appropriate and effective . In our What strategies are currently in place to improve access to SRH services and tools? A largely successful strategy involved SRH public media campaigns , while sensitizing the youth –22. Levetext messaging interventions may induce both short and long-term behavioral change, and improve outcomes related to smoking cessation [Is there a role for mobile phone applications to increase SRH access and uptake? It certainly seems smart and possible to leverage the high mobile phone coverage rates to increase SRH services uptake. The literature is replete with evidence that essation , 26, phyessation –19, diabessation –34. ThisSeveral programs are being implemented around the world that integrate mobile technology to improve SRH uptake among youth, and deliver SRH information, and have been proven to be cost-effective in Kenya and Tanzania , 35. ConIn a review of the literature, we found some mobile phone apps for SRH , a compeIn this study, we propose to develop a mobile phone-based sexual and reproductive health services awareness and delivery application with the objective of increasing the demand for SRH services amongst the youth in Uganda. Our proposed innovation bridges the privacy and confidentiality gap by allowing users to make orders online using the application and have them completed and delivered to their preferred pick-up points. Using the application, users will also be able to schedule a visit to any of the partner clinics. The proposed innovation will reduce both the knowledge and financial barriers to access of SRH information and services by providing comprehensive SRH information and subsidizing SRH services. In the long run, the SRH App will be self-sustaining through revenues from in-app advertisements.The concept of using mobile apps to link users to services, make orders online, and have orders delivered is itself not novel. Mobile apps like Uber  for car mobile phone-based app to increase awareness and uptake of SRH services among the youth aged 18 to 30 years in Uganda, 2) assess the effectiveness of the app on SRH uptake and awareness among the youth aged 18 to 30 years in Uganda, 3) determine the acceptability and usability of the app among the youth aged 18 to 30 years in Uganda, 4) assess the cost-effectiveness and budget impact of the app services delivery model from the perspective of the Ugandan government and taxpayer, and 5) pilot test in-app advertising as a way of generating revenues to sustain the SRH service subsidies associated with the app.In this protocol, we describe a pilot study to develop and evaluate a mobile phone application (app) aimed at increasing uptake of SRH services among the youth in Uganda. We anticipate that this study will lead to the development of a demand-driven, culturally-relevant, and easy-to-use mobile app to enhance the uptake of SRH services among the youth in Uganda and beyond. In addition to reducing the financial and knowledge gap in SRH services, the app that is planned for development and pilot testing in this study is unique in three ways: 1) it will be developed in a collective process that involves local stakeholders and the target population in the design process to ensure high acceptability and user satisfaction, 2) it bridges the gap of privacy and confidentiality by allowing users to anonymously make orders online, and have them delivered at their preferred pick up points and also allows users to choose a visit to a preferred clinic, and 3) it will have an advertisers interface to generate revenue to sustain SRH service subsidies. The objectives of this study are to 1) develop a This is a pilot project that will be implemented in two phases. In the first phase, we will use a stakeholder-driven approach to develop the SRH mobile app. This will involve Focus Group Discussions (FGDs) to understand app preferences, followed by iterative app design by the app developer, in-house testing through subsequent FGDs and a one-day field test of how the app runs in its current status. The second phase is a prospective impact evaluation that will involve three complementary sub-studies: 1) a randomized controlled trial to evaluate the effect of the app on SRH services awareness and uptake, 2) a cross-sectional study to assess the acceptability, user satisfaction, and the use of in-app advertising for revenue generation, and 3) a cost-effectiveness and budget impact analysis. Details on the study design are presented in Fig. The primary setting of this pilot project is Kyambogo University (KYU) in Kampala, the second largest university in Uganda. The campus is located 8 KM from Kampala city center along the Kampala-Jinja highway on Kyambogo hill. Kyambogo University has a population of over 25,000 students with a near-100% cell phone penetration and it is projected that more than 50% of students own internet-enabled phones, giving an estimated 12,500 eligible participants. The app will be tested among students aged 18 to 30 years residing in the university residency halls and hostels.We propose to design a user-friendly SRH services awareness and delivery mobile application. The app will create an SRH tool delivery platform where different SRH tools such as condoms, contraceptive and emergency pills, home testing kits, etc. can be ordered and delivered to the user at their preferred pick-up point at a subsidized cost. The app will also link users to their preferred clinics, chosen from a pool of nearby clinics providing SRH services like HIV/STI/pregnancy testing and counseling, and contraception placement. During the study, we will cover their cost of transport to and from the selected clinic. The app will also include: 1) a menstrual cycle tracker for women to track ovulation and menstruation with an aim of understanding when they are at highest risk of becoming pregnant, 2) an interface with SRH tips and tools, and (3) a chat box where users can ask any questions anonymously and receive responses. We also propose to build an advertisement interface and test its use as a way of generating revenues for sustainability. Details on the app modules are presented in Table  a menstr an interThe app will be integrated with three existing service providers: (1) partner health facilities for SRH service provision, (2) SafeBoda for transportation, and (3) Beyonic Uganda Ltd. for managing the payments system.We propose to provide a subsidy for every tool or service ordered or accessed using our SRH app. We will pay 50% discount of every SRH service cost at the clinic and the client will pay 50%. We propose to provide a 50% discount on the delivery cost incurred by transporting the tool from the clinic to the preferred pick-up point or picking the user from their preferred location to the clinic. We anticipate that revenues from the in-app advertisements will sustain the subsidies after the pilot phase.The study control group will receive no intervention but will however be followed at one and six months respectively.Inclusion criteriaKyambogo University students between the ages of 18–30 yearsSelf-reported sexually activity in the past six monthsLeft with more than one academic year to completionWilling and able to provide written informed consent to participate in the projectAccess to an internet-enabled android smart-phone deviceb)Exclusion criteriaNot sexually active i.e. never had sex and no self-reported sexual activity in the past six monthsRefusal to provide informed consentFinal year at the universityNot in possession of an internet enabled android smart-phone deviceThis study will be conducted among the male and female students of Kyambogo University in Uganda. The participants will be included or excluded from the study based on the following criteria;Study designb.Focus Group DiscussionsWe will employ qualitative methods involving Focus Group Discussions (FGDs) to guide app development. The key stakeholders in this phase of the project include the students (intended target of the application) and the Behavioral Change Communication (BCC) group at the Community Health Department within the Division of Reproductive Health in the Ministry of Health. We have enlisted the BCC group to support our team in development of the SRH tips in the application. They will be involved in design and conduct of the FGDs and in-house testing of the app. The app will be developed in collaboration with an experienced local app developer, and students at Kyambogo University.c.App designWe propose to conduct two sets of Focus Group Discussions (FGDs) each lasting 45 to 60 min with students from Kyambogo University. In the first set, we propose a maximum of 6 FGDs of 8–10 students each—3 male only and 3 female only groups. The second set consists of 2 FGDs, of 8–10 students each stratified by sex  and will be used for in-house testing of the app. We have proposed to use FGDs separated by sex to encourage freedom of expression. However, to encourage diversity of opinion we will attempt to capture a broad age range  and whether or not they are residents and non-residents of on-campus student halls. The first set of FGDs will be used to: 1) understand students’ perspectives and experiences of what drives risky behavior, and 2) understand the SRH app preferences that students are likely to respond to. The interviews will be semi-structured, comprising of open-ended questions guided by an interviewer. We shall make every attempt to encourage the flow of conversation among the students, only using probing questions where we feel we need more information. We anticipate saturation to occur after 6 FGDs. The second set of FGDs will be used to preview the app and provide feedback to the development team for improvement. We anticipate that after the 2 discussions, the final iteration of the app will be ready for deployment. All FGDs will be recorded with two digital voice recorders and supplemented by notes taken by two interviewers. Focus group data will be transcribed by a professional transcriber. Interviews will be anonymized and transcribed and then entered into Atlas Ti scientific software as primary documents from which codes and patterns will be generated. New themes will also be generated from open coding, and key themes will be identified to beef up the quantitative analysis. The research team will synthesize the data to generate content and design for the app.Data from the FGDs will be used to guide the design of the SRH mobile app on an Android platform. We propose to contract with a locally established app developer to design the app. The app developer will participate in both the first and in-house app testing FGDs and in the one-day field event. After the final version of the app has been developed, it will be deployed on the Google Play store where study participants will be able to download and use it. Although we propose to design an app that is driven by the target audience’s preferences, we have enlisted the participation of a Behavioral Change Communications (BCC) expert from the Ministry of Health and an IT expert from the National Information Technology Authority (NITA) who will ensure that the app is consistent with recommended standards for Uganda and conforms to all local regulatory requirements. All versions of the app will be submitted to the Mbarara University of Science and Technology (MUST) Institutional Review Board (IRB) before in-house testing and prior to the pilot testing in phase 2 of the study and, later, to the Uganda National Council for Science and Technology (UNCST) for clearance. After the iterations of app design are complete, we will conduct a one-day field test of the app to test the app’s integration with the partner health facilities, SafeBoda, and the Beyonic payments system. We will also test the responsiveness of the clinics in preparing the orders and the average time it takes for the SafeBoda to connect to the clinic and deliver the package to the client. Based on the outcomes from this activity, recommendations will be made to improve the process.App effectiveness, acceptability, user-satisfactionWe will employ a randomized controlled trial design. Consenting participants will be randomized to download the app or to a control group. The app will not be transferable from one device to another, except through download from Google play store.The study treatment is randomization to receive the app. We anticipate that the app will be available for download from Google play store and will be password protected. To ensure that the treatment is not contaminated, we propose that the app developer use a unique one-time password, only useable once by a person (telephone number/ username) that have been randomized to receive the app.We will seek administrative clearance and operational guidance from the Reproductive Health Division within the Ministry of Health and the Kampala Capital City Authority to implement the project at Kyambogo University, Kampala. We shall work together with the University administration and student leadership to sensitize participants and encourage participation at recruitment.the SRH app and will be able to receive and process orders from the clients. We will employ trained personnel at every clinic to manage the orders, pack, and deliver products ordered. The team will also manage the appointments for clients requesting for services at the clinics via the SRH app. We will work with SafeBoda to deliver the products to the preferred pick up points as shall be suggested by the client. We will also integrate with Beyonic Uganda Ltd. to manage the clinic payments and receiving of payments for services from the clients via mobile money.This project will be implemented by GHE Consulting researchers, who will undergo rigorous training in research methods, research ethics, and Sexual and Reproductive Health (SRH). We will enroll four clinics and pharmacies that provide high-quality SRH services and are located in the neighborhood of KYU. The enrolled clinics and pharmacies will be integrated with The primary outcomes of the app effectiveness study are the changes in the prevalence of modern contraceptive use, STI testing, STI treatment, and SRH awareness. We propose to estimate the study’s sample size based on the prevalence of modern contraceptive use among the youth aged 18 to 30 years. According to the 2016 Uganda Demographic and Health Survey, the prevalence of modern contraception among the youth aged 18 to 30 years in Uganda is 30.2%. The study utilizes the sample size formula for Z test for two sample proportions used for behavioral studies to compute sample size estimates.Sample size formula;Where:n = sample size.d = design effect.p1 = the prevalence of modern contraception.p2 = projected prevalence at the end of the study, so that (p2 − p1) is the magnitude of change you want to be able to detect.p = (p1 +p2)/2.Zα1 − = the z-score corresponding to desired level of significance.Zβ1 −  = the z-score corresponding to the desired level of power.The table below shows the required sample size for each group with varying magnitude of change in proportions to be detected, different power sizes, and two-sided level of significance of 0.05.The study will require a sample size of 435 participants per randomized group in order to obtain 90% power to detect a 15% change in the prevalence of modern contraception at a two-sided of 5% level of significance. The sample size estimations assumed no design effect i.e. d = 1. After adjusting for the 10% non-response rate, a sample size of 479 was computed leading to a total sample size of 958.We propose to randomly recruit the study participants from the university halls and private affiliated hostels at KYU. A listing of university halls and affiliated hostels and their estimated population will be prepared with the help of student leaders at the university. Permission to access the halls and hostels will be sought from the respective wardens. We will further conduct a listing of room numbers across the university halls and hostels identified. Rooms will be uniquely identified by the name of hall/ hostel and the room number. A random sample of 958 rooms will be taken and, in every room, only one participant who meets the criteria will be selected. In the event that there is more than one eligible participant in the room, a simple draw lot will be used to select one participant. Several iterations of this sample plan will be made till when the required sample size is achieved. We aim at recruiting 70% females and 30% males on the program. Trained research assistants will move to the selected rooms and recruit the study participants. Recruited participants will first be screened for eligibility before enrollment in the study.Enrolled participants will be randomized to two arms of the study in a 1:1 ratio, participants that will receive the App and control participants that will not receive the App. At the time of consent and enrollment, we shall explain to the participants that there is a 50% chance that they receive the app, that the research team will have no control over this, and will not know until the end of the study whether or not they were randomized to receive the app. We will only record the telephone number of the consenting participant, link this to a unique study ID and pass this on to the app developer. The app developer will use a computer generated random sequence of numbers, matched to telephone numbers and study ID to allocate participants to the trial. The app will be available for download from the developer’s repository and will be password protected. To ensure that the treatment is not contaminated, the developer will use a unique one-time password, only useable once by a person (telephone number/ username) that have been randomized to receive the app. Participants randomized to receive the app will receive text messages with the app link and guidelines on how to install the app on their phones. They will further receive more guidelines on how the app will be used and its benefits.We shall make every attempt to blind the research team at GHE Consulting to the allocation sequence. Only the app developer will know or have access to this sequence. We propose to use an electronic system of data collection that is managed by the app developer. The statistician at GHE Consulting will have access only to data from the standard questionnaire to track response rates. The app developer will retain all data on app usage and downloads until the end of the study.the SRH App in increasing SRH awareness and uptake. A standardized questionnaire has been developed for this study. At enrollment, the research assistant will obtain background information from eligible and consenting participants. We will collect data on the demographic and socioeconomic characteristics, SRH knowledge, sexual behavior and attitude, and SRH uptake . Information on SRH uptake will be self-reported at both baseline and end line. Questions on SRH knowledge and sexual behaviors will be modified from the World Health Organization (WHO) SRH guiding questionnaire [Enrolled study participants will be followed up at one and six months to determine the effectiveness of ionnaire  and the ionnaire . QuestioWe plan to use the smart-phone based Open Data Kit (ODK) app to collect data both at baseline and end line. Research assistants will be trained on the use of smart-phone and ODK for data collection. This will enable the team to collect high-quality data in real time, thereby saving time.The app developer will also track app download and usage statistics so that at the close of the study we can determine who of those randomized to the study actually downloaded and used the app to access SRH tools and services. We believe this is not only a cost-efficient way to collect the data but will contribute to blind assessments as the GHE Consulting team will not have direct access to mobile contacts. While app use will be managed by the developer, the PI will have access to user statistics through the administrator module of the app.We will separate the responsibility of GHE Consulting and the app developer for data management to ensure the integrity of the research and confidentiality. All data collected at all times will not be identifiable. The app developer will maintain telephone contacts and the random allocation sequence, while the statistician at GHE will maintain app usage statistics. GHE Consulting will maintain records of the standard questionnaire responses and will administer the end-line acceptability and user satisfaction questionnaire. These two data systems will be maintained separately and only linked through unique study IDs.The app developer will hand over all data to GHE (U) Ltd. at the close of the study. Direct access to analyze, verify and reproduce study data or reports will be granted to the PI, research coordinator, and study statistician. Regulatory entities like Grand Challenges Canada (GCC) and the MUST IRB that may want to monitor the study will be granted access to study procedures and data where requested. Pursuant to our contract with Grand Challenges Canada, the original source documents, study records, and reports will be stored intact for a period of ten years after the study ends.The primary outcome of the study is the magnitude of change in the SRH services uptake measured by the proportion change of modern contraceptives use and testing and treatment for sexually transmitted infections at month six. The secondary outcomes are changes in participant’s knowledge of SRH and sexual behaviors at month six.We will assess the differences at baseline between the intervention and control arms. Observed group differences for each variable will be summarized using means and proportions. The primary analysis will be an intention to treat analysis (ITA). The magnitude of change in proportion between the two groups provides a potentially unbiased estimate of the average treatment effect (ATE) of the SRH App. We will use a two-sample proportion Z-test to test the null hypothesis that the two sample changes in proportions are significantly different at month six at 5% level of significance. We will also carry out a difference in difference (DID) analysis to identify the magnitude of change as a result of the SRH App. We will also compare the changes in the different contraceptive methods and STI tests self-reported at six months by study arm.We will conduct binary regression analysis with the primary outcome as the response variable and the participant characteristics as the explanatory variables. We shall adjust for any confounding influence that may arise from the participant belonging in any study arm or time point of data collection by adding the study arm and time point as explanatory variables in the regression model.We are also interested in the effect of the SRH App among those who download and use the App. Using the data from the app developer, we shall construct a binary index of app use. We shall then conduct an instrumental variables analysis to obtain an estimate of the effect of app use on SRH uptake and awareness. We shall conduct the standardized test to verify whether the instrument has good predictive power in the first stage of the regression and whether it is valid in a multiple regression analysis with the observed characteristics included in the model.p-values will be reported. All results will be considered statistically significant at a 5% level of significance.We also further assess if SRH awareness is associated with changes in SRH uptake among the study participants in both arms of the study. We will construct an index for SRH awareness and the conduct a binary regression analysis with SRH uptake as the response variable and SRH awareness as the explanatory variable. We check the validity of the association in a multiple regression analysis with observed characteristics included in the model. Both adjusted and crude odds ratios with their corresponding confidence intervals and To assess acceptability and user-satisfaction, we will analyze the download and usability statistics and reviewer comments on the app from those randomized to receive the app. Further, we have included questions on acceptability and user satisfaction (user rating of the app) and will report simple proportions for each question.b)Cost-effectiveness and budget impact analysisWe have identified three quality assurance areas for services provided using the app: 1) quality of SRH services and products provided by the clinics and pharmacies, 2) preventing contamination of the tools on delivery, and 3) ensuring that the product is received by the user that ordered for it. We propose to recruit registered and certified clinics and pharmacies to provide the SRH App services. Partner clinics and pharmacies will be recruited by GHE Consulting medical and pharmacy specialists. Product will be delivered in sealed packages. The user will receive the notification that the order has been sent and after receiving it they will confirm and the notification will be sent to the sender that the order has been received. We will also partner with SafeBoda that are identifiable, trackable and promote safety in the bodaboda business in the country. The app will include a live chat feature, allowing users to ask questions and make complaints about services delivery.We propose to conduct a model-based incremental cost-effectiveness analysis (CEA) and budget impact analysis (BIA). The main outcomes of the economic evaluation will be the average cost per app user, cost per app service provided, and cost per disability-adjusted life year (DALY) averted. The cost per app user will be stratified distance measured in kilometers. The crude estimate of distance will be estimated by the distance between the health facility and the pickup point. We also propose to perform a static aggregate budget impact analysis (BIA) to examine the potential economic value of scaling up the App services delivery to the government and the individual taxpayer.We will perform a primary micro-costing of the SRH App services delivery model from both the payer’s perspective, i.e. include all direct medical and all direct non-medical costs, and the modified societal perspective, i.e. add indirect costs of lost productivity as a result of app use. The direct medical costs will include all resources used in providing SRH services in terms of the medical devices and supplies. The direct non-medical costs will include transport costs incurred in delivering the services, communication costs involved, app administration costs, other program costs and the opportunity costs. Data on costs will be collected through the app use statistics and health facility personnel costs.c)In-app advertisingWe will also estimate the aggregate national budget impact as well as the cost per Ugandan taxpayer per year of implementing the SRH app services delivery model. The BIA will involve the following steps: 1) characterization of the population of Uganda to estimate the potential number of people to be impacted by the app, 2) selection of a time horizon between 1 and 3 years, 3) documenting the current state of SRH services demand creation models nationally, 4) estimating the cost of SRH App services delivery model at scale, and 5) presenting budget impacts tailored to different audiences. The main analysis will be the generation of an estimate of the potential annual fiscal implication for the taxpayer and the government.We propose to develop the advertisement interface in the app to test a way of generating revenues to the sustain the subsidies that will be extended to the users of the SRH App. We will develop high-impact pitches and approach the different companies like telecommunication companies, hospitals, and pharmacies, beverage and breweries companies, among others and invite them to buy advertising space in the app. We will develop metrics for cost estimation and content for the different adverts based on the Uganda Communications Commission guidelines and market prices and sell advertising space to corporations.We will assess companies’ willingness to advertise with the app at scale-up through the key informant interviews. A key informant interview is attached. We will also assess the satisfaction level of app users on the advertisement interface. Satisfaction questions are included in the acceptability and user satisfaction questionnaire.We do not anticipate any local ICT regulatory requirements either from the Uganda Communications Commission or the Ministry of Information and Communications Technology. However, should these arise we are prepared to present all necessary documentation. We will involve the BCC experts in the Ministry of Health and the IT experts at the National Information Technology Authority (NITA) and we shall share all iterations of the app with both the Ministry, NITA and MUST IRB.This study will be submitted for approval by the Mbarara University of Science and Technology (MUST) Institutional Review Board (IRB) and the Uganda National Council of Science and Technology (UNCST). All investigators have or will receive ethics training prior to the start of the study. All participants will provide informed consent and will be allowed withdraw consent at any time. Study documents will be kept under highest standards of confidentiality and records will be kept under lock and key.This is a data-sensitive project, especially with regard to confidentiality of participants’ mobile phone contact numbers, and SRH service and tools clinical records. We will make every effort to limit access to the client records. We shall discuss possible encryption technologies with the app developer. In any case, it is only the app developer who will have access to telephone numbers, and all records of numbers will be destroyed at the completion of final data collection phase.The SRH App poses minimal risks to the user. In the event that the user registers any adverse event or side effect as a result of the use of SRH app tools and services, they can make an appointment with our clinic or go to the nearby partner clinic for help. We will report any such cases to MUST review board within 7 days of the occurrence.The study has already hired an experienced app developer, conducted the first set of Focus Group Discussions, and the app prototype is currently under development.In this pilot study, we will develop a sexual and reproductive health services delivery and awareness phone application and assess its effectiveness in increasing awareness and uptake of sexual and reproductive health services among the youth aged 18 to 30 years using a randomized controlled design. The study will also assess the acceptability, usability, cost-effectiveness of this services delivery model. Further to this, the study will also assess effectiveness of in-app advertising as a way of generating revenues possible transition to scale. Existing evidence reveals that sexual and reproductive health programs tailored to the youth should be accessible, equitable, appropriate and effective . The chaThis paper has described the steps that will be taken to develop and evaluate the impact of mobile phone application in increasing awareness and uptake of SRH services among the youth in Uganda. Unlike existing non-self-sufficient programs, this study will provide evidence on the use of in-app purchases as a means of funding the transition to scale. The pilot study will culminate into a demand-driven, culturally-relevant, and easy-to-use mobile app that will be popularly used and effective among the youth in Uganda.


The HERV-K (HML-2) viruses are the youngest of the human endogenous retroviruses. They are present as several almost complete proviral copies and numerous fragments in the human genome. Many HERV-K proviruses express a regulatory protein Rec, which binds to an element present in HERV-K mRNAs called the RcRE. This interaction is necessary for the nucleo-cytoplasmic export and expression of HERV-K mRNAs that retain introns and plays a role analogous to that of Rev and the RRE in HIV replication. There are over 900 HERV-K RcREs distributed throughout the human genome. Thus, it was of interest to determine if Rev could functionally interact with selected RcRE elements that map either to HERV-K proviruses or human gene regions. This interaction would have the potential to alter the expression of both HERV-K mRNAs and cellular mRNAs during HIV-1 infection.In this study we employed a combination of RNAseq, bioinformatics and cell-based functional assays. Potential RcREs were identified through a number of bioinformatic approaches. They were then tested for their ability to promote export and translation of a reporter mRNA with a retained intron in conjunction with Rev or Rec. Some of the selected elements functioned well with either Rev, Rec or both, whereas some showed little or no function. Rev function on individual RcREs varied and was also dependent on the Rev sequence. We also performed RNAseq on total and cytoplasmic RNA isolated from SupT1 cells expressing HIV Rev, with or without Tat, or HERV-K Rec. Proviral mRNA from three HERV-K loci  accumulated in the cytoplasm in the presence of Rev or Tat and Rev, but not Rec. Consistent with this, the 3′ RcRE from 3q12.3 functioned well with HIV-Rev in our reporter assay. In contrast, this RcRE showed little or no function with Rec.The HIV Rev protein can functionally interact with many RcREs present in the human genome, depending on the RcRE sequence, as well as the Rev sequence. This leads to export of some of the HERV-K proviral mRNAs and also has the potential to change the expression of non-viral genes. The lysates were then incubated at 37 °C for 30 min.Total and cytoplasmic RNA from three biological replicates of SupT1 cells transduced with the empty MSCV vector (EV) or MSCV vectors expressing Rev, Tat, Tat and Rev and Rec together with the fluorescent proteins, were isolated from cells sorted by flow cytometry. The methods used for total and cytoplasmic RNA extraction were previously described , with thTotal and cytoplasmic RNA lysates were then extracted once with an equal volume (1:1) of phenol and chloroform-isoamyl alcohol (24:1), followed by two extractions with only chloroform-isoamyl alcohol (24:1). Sodium acetate  was then added to the final extractions for a final concentration of 0.3 M prior to precipitation with 2.5 volumes of 200 proof ethanol (− 20 °C). The RNA was pelleted for 1 h at 4 °C 3000 RPM (3014 g) in a Baxter Cryofuge 6000 centrifuge, then washed twice with 70% ethanol and air dried prior to the addition of RNase/DNase free water. Total RNA was DNase digested  then extracted and precipitated as before. Final RNA concentrations were determined with a Qubit RNA HS assay kit .Stranded libraries were prepared by Novogene  from total and cytoplasmic RNA using reagents from New England Biolabs (NEB). First, polyA+ RNA was isolated from total or cytoplasmic RNA using poly-T magnetic beads. The polyA containing RNA was then fragmented with fragmentation buffer (NEB) and first strand cDNA was synthesized using random hexamers and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was performed using DNA Polymerase I and RNase H while substituting dTTP with dUTP. The double stranded cDNA was purified using AMPure XP beads . To ensure that the library was stranded, the second strand cDNA was digested by USER  prior to selecting for fragments approximately 150–200 bp in length with AMPure XP beads. The final library was amplified by PCR, then re-purified with AMPure XP beads prior to sequencing on the Illumina platform at Novogene.FASTQ files were trimmed with Trimmomatic-0.38 using the standard settings (ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36), then assessed for quality using FASTQC software. Trimmed reads were aligned to hg19 (GRCh37) using HISAT2  . After 72 h, cell supernatants were harvested and supernatant p24 was measured by ELISA. (A) Vectors expressing Rev from HIV 1A, 6AG and 8G, prRev or Rec were transfected together with reporter plasmids that contained the prRcRE or the RcREs from the seven indicated gene regions. Supernatant p24 was measured at 72 h post transfection. (B) For each of the Rev (or Rec) sequences, the activity of the Rev (or Rec)/prRcRE pair from part A was normalized to 1, and the activity of that specific Rev with each of the other RcREs was plotted relative to this value. This allows a direct comparison of the activity of each Rev (or Rec) protein with each of the different RcREs relative to its activity on the prRcRE.Additional file 2: Figure S2. Quantitative analysis of HERV-K proviral transcripts from total and cytoplasmic RNAseq data. After normalization of the data, the fold difference in the number of unique reads mapping to the 22q11.23 (left panel) or 4p16.1b (right panel) loci were quantified using DESeq2 for total or cytoplasmic RNA from the Rev, Tat, Tat and Rev or Rec transduced samples, compared to the samples transduced with the empty vector.Additional file 3: Figure S3. Visualization of NEAT1 reads from total and cytoplasmic RNAseq data. Total and cytoplasmic DESeq2 normalized read counts for NEAT1 were visualized with IGV. Note that DNA reads from total RNA map across the entire NEAT1 gene region and include both the NEAT 1_1 and NEAT 1_2 RNA isoforms. In contrast, most of the reads from cytoplasmic RNA map only to the region corresponding to the NEAT1_1 RNA isoform.


Breviolum minutum with those of the homologous symbionts freshly isolated from the sea anemone Exaiptasia diaphana, a widely used model for coral hosts. Additionally, we assessed expression levels of a list of candidate host transporters of interest in anemones with and without symbionts. Our transcriptome analyses highlight the distinctive nature of the two algal life stages, with many gene expression level changes correlating to the different morphologies, cell cycles, and metabolisms adopted in hospite versus free‐living. Morphogenesis‐related genes that likely underpin the metamorphosis process observed when symbionts enter a host cell were up‐regulated. Conversely, many down‐regulated genes appear to be indicative of the protective and confined nature of the symbiosome. Our results emphasize the significance of transmembrane transport to the symbiosis, and in particular of ammonium and sugar transport. Further, we pinpoint and characterize candidate transporters—predicted to be localized variously to the algal plasma membrane, the host plasma membrane, and the symbiosome membrane—that likely serve pivotal roles in the interchange of material during symbiosis. Our study provides new insights that expand our understanding of the molecular exchanges that underpin the cnidarian–algal symbiotic relationship.Coral–dinoflagellate symbiosis underpins the evolutionary success of corals reefs. Successful exchange of molecules between the cnidarian host and the Symbiodiniaceae algae enables the mutualistic partnership. The algae translocate photosynthate to their host in exchange for nutrients and shelter. The photosynthate must traverse multiple membranes, most likely facilitated by transporters. Here, we compared gene expression profiles of cultured, free‐living  The symbiosis between corals and dinoflagellate algae underpins the evolutionary success of corals reefs. Our study focuses on algal symbiont gene expression and targets differentially expressed putative transporters that might serve pivotal roles in the interchange of material during symbiosis. Our study emphasizes the great significance of transmembrane transport to the success of the symbiotic relationship. We examined this data set for genes that are differentially expressed between the two life stages. In addition to transporters, we aimed to generate a gene expression compendium providing a molecular window into the dramatic morphological, physiological, and life‐history differences for Symbiodiniaceae in hospite or free‐living states.Here, we identify a set of transporter genes that are likely involved in the nutrient exchange between Symbiodiniaceae endosymbionts and their cnidarian host. To identify host transporters involved in host–symbiont nutrient exchange, we searched the literature for 22.1Exaiptasia diaphana sea anemones were collected from the National Sea Simulator at the Australian Institute of Marine Science . These animals are of Great Barrier Reef (GBR) origin , and all were found to host the same Symbiodiniaceae strain , and once a week, filamentous algae were removed immediately prior to a full water change.Three genotypes . Three replicate subcultures were generated in cell culture flasks (0.2‐μm membrane vented cap) with 0.2 μm filtered seawater (FSW) supplemented with 1× Diago's IMK medium (Novachem). Cultures were kept in a growth chamber  under a constant temperature of 26°C, 12:12 light:dark photoperiod cycle, and 60 μmol m−2S−1 photons. Cultures were routinely monitored by light microscopy to assess their health . The replicate subcultures were kept and maintained for at least a month prior to RNA extractions. Prior to RNA extraction, cells were counted using an automated cell counter (countess™ II FL automated cell counter with EVOS™ GFP light cube for detection of fluorescence).2.4B. minutum, cultured, and freshly isolated, and from symbiotic and aposymbiotic E. diaphana anemones.RNA was extracted from the two life stages of 7 cells from each of the three replicate flasks were processed for RNA extraction. RNA was extracted according to Rosic and Hoegh‐Guldberg  symbiotic Symbiotic and aposymbiotic anemones: Three replicates of each, representing three distinct genotypes (AIMS2–4), were taken for RNA extractions. RNA was extracted using TRIzol (Invitrogen Thermo Fisher Scientific) following the manufacturer's instructions with a few adjustments: Frozen anemones were homogenized in cold TRIzol (kept at 4°C) using glass homogenizer and, following centrifugation, were further homogenized by 425–600 mm acid‐washed glass beads (Sigma) using a TissueLyser II (Qiagen) for 30 s at 30 Hz. RNA was precipitated in 250 μl ice‐cold isopropanol and 250 μl 0.8 M sodium citrate in 1.2 M sodium chloride.B. minutum RNA quality and quantity were further assessed by the Australian Genome Facility (AGRF) using the Agilent 2100 Bioanalyzer system with a RNA 6000 Nano kit.The concentration of the RNA was measured using a DeNovis DS‐11 spectrophotometer, and the RNA integrity was assessed by agarose gel electrophoresis and evaluated based on clear 28S and 18S ribosomal RNA bands. 2.5CTs method was used, and fold changes were calculated using the 2−ΔΔCt formula to estimate the relative difference in the number of transcripts between the aposymbiotic and symbiotic samples. Four potential house‐keeping genes were tested: 28S ribosomal RNA gene  using the SPSS software .Real‐time polymerase chain reaction (qPCR) analysis was performed for 14 candidate transporters of interest that were found to be significantly down‐regulated in anemones without endosymbionts comparing to symbiotic anemones in previous transcriptome/proteomic papers  was used for Illumina TruSeq Stranded mRNA library prep kit, with polyA purification, and sequenced on the Illumina HiSeq platform  by AGRF. The Illumina bcl2fastq 2.20.0.422 pipeline was used to generate the sequence data. The data generated met the AGRF quality standards. The Fastq files were deposited at SRA database under the accession number PRJNA544863.Total RNA (400 ng) of cultured and freshly isolated https://www.bioinformatics.babraham.ac.uk/projects/fastqc/; Andrews, http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) was used for trimming sequencing adapters and low‐quality reads. In order to minimize host and bacterial contamination in the assembled transcriptome, the reads were searched against a custom‐made database comprised of publicly available Symbiodiniaceae and cnidarian transcriptomes in order to maximize the sequences coverage  mapped to an endosymbiotic alga were further used for the assembly.RNAseq analysis was conducted using the Galaxy interface, a web‐based platform for data‐intensive biological research  with EdgeR method was used to achieve statistics, to compute normalized log2(FC) (in hospite vs. cultured) to the transcripts and to draw a multidimensional scaling (MDS) plot. A hierarchical clustering heat map was generated using TPM values (transcript per million) and based on Euclidian distances in Excel XLSTAT.Transcript quantification was generated via Salmon  were defined as those with an adjusted p‐values of the GO categories within the group , and the best resulting homologous sequences were further used for localization predictions.In an attempt to further predict which of the host's candidate transporters of interest might be localized to the symbiosome membrane, we focused on those that were predicted to the lysosome/vacuole. We used host's transcripts that were predicted to the lysosome/vacuole, to search the genome of the nonsymbiotic anemone Sequences of differentially expressed transporter transcripts were in silico translated into protein sequences; those that were predicted to localize to the plasma membrane or to the host's lysosome/vacuole were further analyzed using InterPro as a diagnostic tool  groups in the case of down‐expressed and constitutively expressed genes but were also up‐regulated and high‐ranked for n Figure . Transmen Figure . Constitn Figure . A totaln Figure  and S6.3.2Eleven of the fourteen host candidate transporters of interest examined were found to be significantly down‐regulated in aposymbiotic anemones when compared to symbiotic anemones Figure : viz. am3.3Predicted locations for the up‐ and down‐regulated algal transporters place the transporters in six different subcellular compartments Figure . ApproxiB. minutum transcriptome revealed that out of 134 transcripts . In addition to inorganic carbon fixation, the chloroplast provides multiple functions such as assimilation of ammonium, assembly of iron–sulfur complexes, and stress signaling  proteins in Toll‐like receptors are associated with the first line of defense in the innate immune response of cnidarians Dunn, . LRR dom4.1.3in hospite. Thus, we speculate that free‐living algae utilize endocytosis for the uptake of molecules that are too big for membranal protein‐assisted transport  that are defined by a 35‐amino acid structural motif  was identified, and while not significantly enriched, it might be related to host–symbiont recognition. It has been suggested that recognition at the initial phase of the symbiosis involves glycans that are on the surface of the algal cells that are identified by the cnidarian host  in the up‐regulated group, compared with the down‐regulated group. Moreover, an examination of the list of potential sugar transporters , and three out of seven transcripts were up‐regulated when the medium was supplemented with glucose  transporters  underpin symbiont ammonium assimilation  differeNitrogen cycling has been shown to be drastically different between symbiotic and aposymbiotic cnidarians  domains and putative localization within symbiont cell membrane were both up‐ and down‐regulated. Recently, transcripts of an ABC transporter were found to be up‐regulated in another Symbiodiniaceae species, 5B. minutum depending on whether it is free‐living or in symbiosis with its anemone host. Gene expression changes correlate with both morphological and metabolic changes adopted during the two radically different life stages. Many substrate transporters, both in the symbiont and the host, are shown to undergo extensive gene expression changes as part of the symbiosis. Further, we pinpoint candidate transporters localized throughout the holobiont compartments that might serve pivotal roles in the interchange of materials during symbiosis. Transfer of sugar and ammonium between the symbiont and the host comprises the most significant exchange of compounds in the symbiosis, and we identify promising candidate transporters that may assist in these processes, which are putatively localized to the algal plasma membrane and the symbiosome membrane. Future studies should focus on clarifying the missing details of how the two organisms successfully interact as a holobiont, by confirming both the subcellular localization of these putative transporters and defining their actual substrate preferences. These insights will expand our understanding of the material exchanges that are the crux of the symbiotic relationship.This study reveals extensive changes in gene expression for the alga None declared.KM‐L, MJHvO and GIM designed research. KM‐L performed research and analyzed data. MJHvO and GIM analyzed data. KM‐L, MJHvO and GIM wrote the paper. Click here for additional data file. Click here for additional data file.


While most anatomic TSA stems allow some intra-operative adjustments, the default configuration assumes that head offset is directly proportional to stem diameter. Some authors reported that humeral head diameter is proportional to intra-medullary canal width and humeral head offset, but none investigated the direct relationship between head offset and endosteal measurements. The purpose of the study was to determine whether global humeral head offset is proportional to intramedullary canal width at the distal metaphysis and proximal diaphysis.We analyzed 100 Computed Tomography shoulder scans of patients aged 59.1 ± 20.5 with no signs of gleno-humeral arthritis nor humeral dysplasia. The width of the intramedullary diaphyseal canal was determined at four transverse sections 65, 70, 100 and 105 mm below the head center. The inter-observer agreement was excellent for intramedullary canal width (ICC = 0.96), head diameter (ICC = 0.97) and global head offset (ICC = 0.85). Correlations were analysed using Pearson’s coefficients and multivariable regressions were performed to determine associations between head offset and five independent variables .r = − 0.31, p = 0.002), but not correlated with intramedullary canal width . Multivariable regression confirmed that global head offset was independently associated with head diameter , but not with intramedullary canal width .Global head offset was negatively correlated with head diameter (The present study revealed that humeral offset is not correlated with intramedullary canal width. Implant manufacturers and shoulder surgeons should be aware of the subtle morphologic features, to enhance humeral stem design and restore native anatomy. Success of total shoulder arthroplasty (TSA) requires accurate restoration of anatomy  and length , excluding shoulders with signs of: (i) osteoarthritis or rheumatoid arthritis, (ii) Hill Sachs lesions, (iii) humeral head necrosis or deformities, and (iv) mal-unions secondary to displaced humeral neck fractures. The cohort comprised 62 men and 38 women aged 59.1 ± 20.5 years . The patients presented with fractures of the scapula or clavicle (45), rotator cuff tears (39), shoulder dislocation or instability with no signs of osseous damage at the proximal humerus or glenoid (9), tumor at the scapula or distal humerus (5), calcific tendinitis (1) and thoracic syndrome (1). All patients provided informed consent to use of their images and data for research and publishing purposes. As the study was performed using pre-existing CT scans, institutional review board (IRB) approval was not required.The Digital Imaging and Communication in Medicine (DICOM) files were processed using Osirix  in standard resolution. Four series of 8 points were digitalised on endosteal transverse sections 65, 70, 100 and 105 mm below the head center Fig. . The appr < 0.24) between native head offset and intramedullary canal width, with alpha = 0.05 and beta = 0.80, was calculated a priori to be a minimum of 71 patients.The sample size necessary to test the hypothesis, that there is no correlation  were excellent for intramedullary canal width , head diameter  and global head offset .P-values < 0.05 were considered statistically significant.Descriptive statistics were used to summarize the data. Shapiro–Wilk tests were used to assess the normality of distributions. For non-Gaussian quantitative data, differences between groups were evaluated using Wilcoxon rank-sum tests (Mann–Whitney U test). For continuous variables, correlations were analysed using Pearsons coefficients. Uni-variable and multivariable linear regressions were performed to determine associations between head offset and five independent variables . Both uni- and multi-varaible analyses were deemed necessary to identify potential confounding variables. Statistical analyses were performed using R version 3.3.3 . p = 0.094).The average intramedullary canal width was 14.5 ± 2.5 mm , and the average head diameter was 44.9 ± 4.4 mm , with a retroversion of 24.6° ± 19.2°  . Global head offset was negatively correlated with head diameter , but not with intramedullary canal width , thus confirming the study hypothesis , but not with intramedullary canal width . Posterior head offset was neither correlated with head diameter , nor with intramedullary canal width .Intramedullary canal width was positively correlated with head diameter (p = 0.001) and patient age  but not with intramedullary canal width  (Table p = 0.005), but not with intramedullary canal width , reaffirming the study hypothesis.Uni-variable regression revealed that global head offset was significantly associated with head diameter  in arthritic shoulders, subchondral damage to the humeral head often renders calculations of its diameter and centre difficult and inaccurate; and (ii) the goals of TSA are to replace damaged articular surfaces and restore adequate joint architecture, which is often deformed due to congenital or progressive pathologies in arthritic shoulders.While shoulder arthroplasty was initially intended for elderly patients with low functional expectations, TSA is now performed in younger patients with greater functional demands, which renders reconstruction of native anatomy all the more essential. Modern TSA implants allow for some adjustments of humeral head offset , we found it to be weak and negative (r = − 0.28). These contradictory results might be explained by differences in measurement techniques, as Pearl et al. used cadaver bone x-rays, and referred to the reamed canal for the CC axis.Our morphologic measurements are within the ranges reported in other published studies Table : 43–51 mThe limitations of this study include: (i) the inability to determine the transepicondylar axis to establish the frontal humeral plane and calculate true head retroversion, (ii) the digitization of endosteal transverse sections at fixed rather than proportional distances below the head center, which was not possible because the total heights of the humerus and of the patient were unknown, (iii) the population studied was Caucasian, and may not be representative of other ethnicities (Aroonjarattham et al., The present study revealed that humeral offset is not correlated with intramedullary canal width. These findings are relevant to implant manufacturers and shoulder surgeons, who should be aware of the subtle morphologic features, to enhance humeral stem design and restore native anatomy.


Micropterus salmoides) against Nocardia seriolae, gray mullet  against Lactococcus garvieae, orange-spotted grouper (Epinephelus coioides) against Vibrio harveyi, and koi carp (Cyprinus carpio) against Aeromonas sobria, using RNA-seq techniques. We demonstrated that only 39 differentially expressed genes (DEGs) were present in all species. However, the number of specific DEGs in each species was relatively higher than that of common DEGs; 493 DEGs in largemouth bass against N. seriolae, 819 DEGs in mullets against L. garvieae, 909 in groupers against V. harveyi, and 1471 in carps against A. sobria. The DEGs in different fish species were also representative of specific immune-related pathways. The results of this study will enhance our understanding of the immune responses of fish, and will aid in the development of effective vaccines, therapies, and disease-resistant strains.Transcriptome analysis is a powerful tool that enables a deep understanding of complicated physiological pathways, including immune responses. RNA sequencing (RNA-Seq)-based transcriptome analysis and various bioinformatics tools have also been used to study non-model animals, including aquaculture species for which reference genomes are not available. Rapid developments in these techniques have not only accelerated investigations into the process of pathogenic infection and defense strategies in fish, but also used to identify immunity-related genes in fish. These findings will contribute to fish immunotherapy for the prevention and treatment of bacterial infections through the design of more specific and effective immune stimulants, adjuvants, and vaccines. Until now, there has been little information regarding the universality and diversity of immune reactions against pathogenic infection in fish. Therefore, one of the aims of this paper is to introduce the RNA-Seq technique for examination of immune responses in pathogen-infected fish. This review also aims to highlight comparative studies of immune responses against bacteria, based on our previous findings in largemouth bass ( Transcriptome analysis is used to study principal pathways of development, cellular fate, physiology, activity, and disease progression. RNA sequencing (RNA-Seq) is a modern technology for transcriptome profiling that uses next-generation sequencing (NGS). Advancements in bioinformatics has significantly supported RNA-Seq technology to accelerate the knowledge on transcriptomes .In the aquaculture field, there is wide utility for RNA-Seq in various applications, such as understanding the development of embryo and larvae, toxicology, environmental stress, effect of dietary conditions, and discovery of novel transcripts –4. In adMicropterus salmoides) against Nocardia seriolae, gray mullet  against Lactococcus garvieae, orange-spotted grouper (Epinephelus coioides) against Vibrio harveyi, and koi carp (Cyprinus carpio) against Aeromonas sobria.In this paper, we firstly introduce the RNA-Seq technique and current knowledge for investigations of immune responses in pathogen-infected fish. This review also aims to highlight comparative studies of fish immune responses against bacteria based on our previous studies that we demonstrated the transcriptome of bacteria infected fish, largemouth bass  (Oncorhynchus mykiss)   , rainbow mykiss) , and Atlo salar) . Althougo salar) , 6. The equences . RNA-SeqEpinephelus coioides), a total of 79,128 unigenes were identified and aligned with each database; 58,926 (74.47%) in NT, 43,576 (55.07%) in NR, 14,750 (18.64%) in COG, 34711 (43.87%) in KEGG, and 4232 (5.35%) in GO (Cyprinus carpio), a wide range of data on tissue-specific gene expression and translation , NCBI non-redundant protein (NR), Clusters of Orthologous Groups (COGs) , Kyoto E%) in GO . The num%) in GO –19. Althnslation  has beenAlthough fish are constantly exposed to risk of microorganism pathogens, fish could keep in shape to act immune mechanisms against pathogens. In a first line of immune response, fish are protected by non-specific humoral factors including growth inhibiting substances (transferrin and antiproteases), lysins , and making a link with non-specific phagocyte responses. Second, fish produce antibody constitutes for a specific humoral defense inhibiting bacterial adherence and invasion of non-phagocytic host cells and counteracting toxins from bacterial . After dAeromonus hydrophila is a Gram-negative bacterium, and causes a wide spectrum of diseases in vertebrates , RNA-Seq analysis was conducted with RNA from several tissues, and 238 differentially expressed unigenes were identified in infected fish (Ctenopharygodon idella), 2121 DEGs were identified in spleens of A. hydrophila (6 hpi)-infected fish, some of which were involved in phagocytosis, the complement system, and cytokine production , DEGs in A. hydrophila-infected livers were mainly associated with Th1/2 cell differentiation pathways, as well as in pathogen recognition and complement system , the transcript profile of F. columnare-infected gills was examined using RNA-Seq to investigate differences in susceptibility to F. columnare (F. columnare-infected and non-infected mandarin fish (Siniperca chuatsi) have been reported using the head kidney F. columnare-infected and non-infected group  (ter fish . This diter fish . In chanolumnare . In resiolumnare  was upreed group . The resed group . A simillburnus)  was alsolburnus) .Streptococcus agalactiae, a Gram-positive round bacterium, is a harmful aquaculture pathogen that leads to enormous economic losses in various teleost (Oreochromis spp.) after S. agalactiae infection was conducted, and results indicated that DEGs are mainly involved in immune-related pathways, especially Toll-like receptor signaling and leukocyte transendothelial migration (Oreochromis niloticus) spleens following S. agalactiae infections  revealed that the expression of genes involved in the complement pathway and antimicrobial peptides is enhanced upon V. alginolyticus infection (Epinephelus lanceolatus) larvae infected with Vibrio alginolyticus suggested that TLR5 signaling induces secretion of several cytokines (IL-1β and IL-8)  .four research papers that conducted transcriptome analysis on infected fish, namely largemouth bass  against Nocardia seriolae  against Lactococcus garvieae (Epinephelus coioides) against Vibrio harveyi (Cyprinus carpio) against Aeromonas sobria  were found in mullets against L. garvieae, 909 DEGs (601 up-regulated and 308 down regulated) were found in groupers (Epinephelus coioides) against V. harveyi, and 1471 DEGs  were found in carps against A. sobria  , CCL19 (C-C motif chemokine 19), and SOCS1  . The como salar) . From oto salar) , complemNocardia seriolae is a filamentous Gram-positive bacterium that causes nocardiosis with high mortality in many fish species in Japan, Taiwan and Japan. The infected fish showed a lethal granulomatous disease of the skin, muscle, spleen, kidney, and liver tissues , administrated recombinant IL-12 and formalin-killed N. seriolae showed the higher survival rates after challenged with N. seriolae, compared to vehicle and FKC only groups  and CXCR4 (C-X-C motif chemokine receptor 4) in were also up-regulated in koi carps during A sobria infections  of koi carp against A. sobria and orange-spotted grouper against V. harveyi , cytokines and chemokines (CSF3 and CCL21), lysosome related genes (LYPLA3 and SLC11A1) and caspase recruitment domain-containing protein (Card) 9  of largemouth bass against N. seriolae and gray mullet against L. garvieae ( harveyi . However(Card) 9 . N. serigarvieae . Up-regugarvieae . Althougin vivo. We also summarized current knowledge on immune responses to pathogenic challenges via RNA-Seq in teleost. In this study, we could identify the specific pathway in each fish against bacteria species, Noth1 signaling and IL-12 signaling pathway in largemouth bass against N. seriolae, CXCL12 and CXCR4 signaling pathway in koi carp against A. sobria, TGFα signaling pathway in orange-spotted grouper against V. harveyi, and IL-17 signaling pathway in gray mullet against L. garvieae. These types of studies are increasing, and have enormously aided in our understanding of pathogenic strategies and immune defense systems in aquaculture fish. However, there remains certain limitations of RNA-Seq analysis in aquaculture species. Additionally, the RNA-seq technology could be used to expand existing datasets on splicing variants in mRNA and SNP. Currently, differences in immune response against different pathogens are not well-described. In this study, we attempted to investigate both species-specific and common immune related genes that are up-regulated during bacterial infections based on our previous RNA-seq data. Secondary use of RNA-seq datasets may be essential for preparation of future RNA-seq studies in aquaculture species, which can further deepen our understanding of specific immune functions against pathogens. In aquaculture field, these deep and particular understanding of immune response against each pathogens will provide us to more accurate diagnosis of disease and develop a more effective vaccine and adjuvant of each pathogens.In this review, we first introduced applications of the RNA-Seq technology in aquaculture studies. The RNA-Seq technology has allowed us to identify many novel genes, and to investigate the expression patterns at various conditions in non-model teleost. Therefore, findings based on this technology have accelerated research in the aquaculture field. Additionally, high throughput quantification by RNA-seq could be used to identify pathogen, and to evaluate the efficacy of vaccines and adjuvants against pathogen SM analyzed transcriptome data and wrote the paper. P-CW and S-CC reviewed the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNAs (circRNAs) are implicated in cancer development. This study aimed to evaluate whether circulating circRNAs may serve as novel biomarkers for non‐small cell lung cancer (NSCLC). We used RNA sequencing (RNA‐seq) and quantitative real‐time PCR to explore cancer‐related circRNAs. Bioinformatics and functional analyses were performed to reveal biological effects of circRNAs on lung cancer cells. A total of 5471 distinct circRNAs were identified by total RNA‐seq, in which 185 were differentially expressed between cancerous and adjacent normal tissues. A circRNA derived from exon 5–7 of the FARSA gene, termed circFARSA, was observed to increase in cancerous tissues (P = 0.016), and was more abundant in patients’ plasma than controls (P < 0.001). Overexpression of circFARSA in A549 cell line significantly promoted cell migration and invasion. In silico analysis suggested that circFARSA might sponge miR‐330‐5p and miR‐326, thereby relieving their inhibitory effects on oncogene fatty acid synthase. Summarily, this study revealed circRNA profile of NSCLC for the first time and provided the evidence of plasma circFARSA as a potential noninvasive biomarker for this malignancy.Emerging evidence indicates that circular  Lung cancer is the most common and deadly malignancy worldwide, with more than 1.8 million new cases and nearly 1.6 million deaths estimated in 2012 Circular RNAs (circRNAs) were regarded as the by‐products of splicing or splicing errors with low abundance until recently Compared to liner RNAs that terminate in 5′ caps and 3′ tails, circRNAs form covalently closed loops without 5′ or 3′ polarities and have been confirmed relatively stable and resistant to RNase R FARSA gene, termed circFARSA, was found to be significantly upregulated in cancerous tissues and abundant in corresponding plasma. We further determined circFARSA levels in plasma from 50 patients with NSCLC and 50 healthy controls using qRT‐PCR. Bioinformatics and functional analyses were also performed to show biological clues of cirFARSA in lung cancer.This study aimed to assess the potential value of plasma circRNAs as novel biomarkers for NSCLC. We first performed high‐throughput RNA sequencing (RNA‐seq) to identify differentially expressed circRNAs in 10 paired NSCLC tissues and adjacent normal counterparts. A circRNA derived from exon 5–7 of the This study was approved by the institutional review board of Nanjing Medical University and conformed to the Declaration of Helsinki. A two‐stage design was performed Fig. . In the Total tissue RNA was extracted using RNeasy Mini Kit  and was analyzed by an Agilent 2100 Bioanalyzer system  with the RNA 6000 Nano Labchip Kit. Only samples of high‐quality RNA (RNA Integrity Number ≥7.5) were used in the subsequent construction of RNA‐seq libraries. Ribosome‐minus RNA was fractionated from total RNA samples, and RNA‐seq libraries were constructed orderly by RNA fragmentation, random hexamer‐primed cDNA synthesis, linker ligation, and PCR amplification using a TruSeq RNA Sample Prep Kit . The purified cDNA libraries were sequenced on the Illumina HiSeq1500 platform .Illumina BCL files were converted to FASTQ format using CASAVA v1.8.2. Initial quality controls were conducted by Trimmomatic version 0.32.43 to trim off adapters and low quality bases (ILLUMINACLIP: adapter.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:20). Qualified reads were sent to CIRCexplorer v 1.1.10 pipeline to identify and quantify circRNAs with default parameters Total plasma RNA was extracted using TRIzol LS Reagent  and RNeasy Plus Mini Kit (Qiagen), according to the manufacturer's instructions. As an internal control, 100 ng total RNA of C. elegans was artificially added into each plasma specimen (200 μL) before RNA extraction. The purity and concentration of RNA samples were determined with the NanoDrop ND‐2000 . The integrity of RNA was assessed by agarose gel electrophoresis. Template cDNA was synthesized by reverse transcription using random primers .™ II  on ABI 7900 system. Cel‐circRNA 9 (cel_9), which is highly expressed in C. elegans and RNase R resistant −ΔCt represented a relative expression level of circRNAs. The assay was performed by triplicate in three independent experiments to guarantee reliability.The qRT‐PCR was performed by using the SYBR Premix Ex TaqGAATTCTGAAATATGCTATCTTACAGGACTCTGAAGACCTACTGGGTGAG‐3′and 5′‐CGGGATCCTCAAGAAAAAATATATTCACCTCGAAGGAAGAAGGTGTCGTGCT‐3′. The target fragment of 394‐bp orderly contained EcoR I site, splice acceptor AG, circFARSA sequence, splice donor GT, and Bam HI site. After digestion with EcoR I and Bam HI enzymes, the fragment was cloned into the pLCDH‐ciR vector  for the circulation of target transcripts The cDNA encoding circFARSA in SH‐SY5Y cells was PCR‐amplified by using primers 5′‐CGA human lung cancer cell line, A549, was purchased from ATCC (American Type Culture Collection) and confirmed by short tandem repeat (STR) profiling. Proliferation assays were performed by using CCK‐8 kit . At 0, 12, 24, 36, 48, and 72 h, the CCK‐8 reagent was added into each well and incubated at 37°C for 2 h. The optical density at 450 nm was measured by an automatic microplate reader . Cell migration and invasion were assessed by using Costar Transwell plates , as described previously http://www.cytoscape.org/).CircRNA sequence was treated as a seed to analyze the circRNA–miRNA interaction by using Circular RNA Interactome based on the TargetScan algorithm https://portal.gdc.cancer.gov/).RNA‐seq data (level 3) for 488 LUAD  and 489 LUSC patients  were downloaded from the TCGA data portal (http://www.kmplot.com/lung). Jetset scoring was used to identify the optimal microarray probe set for FASN The prognostic value of the FASN was analyzed by a web‐based Kaplan–Meier plotter with multiple microarray data  and R software version3.3.0 (Free Software Foundation's GNU project). DESeq2 package in Bioconductor was used to identify differentially expressed circRNAs in RNA‐seq data. Hierarchical clustering analysis was performed using the most significant differentially expressed circRNAs by Cluster and TreeView programs. We assessed the relationship of circRNAs between tissues and plasma by calculating Pearson correlation coefficient. Differences in plasma circRNAs between cases and controls were assessed by paired t‐tests after log‐normal transformation of raw expression data. P < 0.05), among which 120 circRNAs were upregulated while 65 circRNAs were downregulated. The top 10 upregulated and downregulated circRNAs ranked by P values are shown in Table A total of 5471 circRNAs were detected by RNA‐seq in 10 pairs of samples. The volcano plot filtering showed differentially expressed circRNAs between NSCLC and adjacent normal tissues .To prioritize candidate circRNAs for detection in circulation, we combined the published data of serum circRNA profile with our findings of upregulated circRNAs r = 0.64, P = 0.047)  Fig. B.http://www.circbase.org)  Fig. .P < 0.001). We also tested the level of FARSA mRNA in plasma and found that its abundance was too low to be detected.The level of plasma circFARSA was determined in 50 NSCLC cases and 50 healthy controls by qRT‐PCR Fig. C. We fouP = 0.048).We further analyzed the relationship between plasma circFARSA and clinicopathological features of patients Table . No statThe ROC curve was plotted to evaluate the diagnostic value of circFARSA in distinguishing patients with NSCLC from cancer‐free individuals Fig. D. The arOverexpression of circFARSA in transfected A549 cells was confirmed by qRT‐PCR Fig. A. ComparUsing Circular RNA Interactome, the top five miRNAs targeted by circFARSA were identified as follows: miR‐330‐5p, miR‐326, miR‐1178‐3p, miR‐620, and miR‐1270. Based on KEGG annotation, 12 pathways were identified and fatty acid biosynthesis was the most statistically significant pathway Fig. A. Cytoscn = 57, P < 0.001) and squamous carcinoma , compared with paired normal tissues  indicated the prognostic role of FASN expression in lung cancer patients   which emerges as an oncogene in various cancers. In TCGA datasets, FASN expression was increased in both lung adenocarcinoma  Fig. B.In the present study, we utilized RNA‐seq and revealed 120 upregulated circRNAs and 65 downregulated circRNAs in NSCLC tissues. To prioritize candidate circRNAs for detection in plasma, we integrated the published data of serum circRNA profile with our findings. Then, we demonstrated that circFARSA exhibited the highest abundance among nine serum‐detectable circRNAs. In addition, plasma circFARSA was higher in patients with NSCLC than in healthy controls. The area under the ROC curve was 0.71, suggesting that plasma circFARSA might represent a potential biomarker for NSCLC.Recent studies suggest that specific circRNAs are abundant and stable in saliva CircRNAs have been demonstrated to act as miRNA sponges and regulate the expression of miRNA‐targeted transcripts The present study has several strengths. First, using high‐throughput RNA‐seq, we provided a global insight of differently expressed circRNAs in NSCLC. Second, it is the first attempt to explore the potential of plasma circRNAs as novel biomarkers for patients with NSCLC. Third, we demonstrated that a C. elegan circRNA, cel_9, was stable in human plasma and suitable as internal reference. Finally, both in silico and in vitro evidences suggested the crucial role of circFARSA in lung cancer progression. However, there are also several limitations. First, although we conducted two‐stage design with the screening and validation stages, the sample size was relatively small. Other circRNAs in the profile might also have the potential as lung cancer biomarkers. Future studies with large sample size in the first tissue‐screening stage and second plasma‐validation stage are warranted. Second, additional in‐depth functional studies are essential to unravel molecular mechanism of circFARSA–miRNA–FASN interactions in NSCLC.In conclusion, the present study provides the precursory evidence to foster the potential of plasma circFARSA as a novel biomarker for NSCLC. Our findings would shed light on future follow‐up studies on circulating circRNAs as noninvasive biomarkers for cancer.The authors have no conflict of interest to disclose.Figure S1. Flow chart of circRNA selection.Figure S2. Specific melting peaks for qRT‐PCR products of circFARSA and cel_9 in plasma (A)circFARSA; (B)cel_9.Figure S3. Sequencing result of qRT‐PCR product and the schematic diagram of circFARSA.Figure S4. (A)The expression levels of circFARSA in A549 cells after transfection with or without circFARSA plasmid. Data are shown as mean ± SD, ***P < 0.01, n = 3; (B) The expression levels of FARSA mRNA in A549 cells after transfection with or without circFARSA plasmid. Data are shown as mean ± SD, ns: P value not significant, n = 3.Figure S5. CCK8 cell proliferation assay of A549 cells after transfection with or without circFARSA plasmid.Figure S6. FASN expression in lung cancer and its association with prognosis.Click here for additional data file.Table S1. Clinicopathological features of 10 NSCLC patients in RNA‐seq analysis.Table S2. Top 10 up‐regulated and down‐regulated circRNAs between NSCLC cancerous and adjacent normal tissues.Table S3. Top 10 candidate circRNAs in plasma from NSCLC patients.Table S4. The relationship between plasma circFARSA and clinicopathological features of NSCLC patients.Click here for additional data file.


Given the rapidly changing demographics, there will be an increasing number of individuals with dementia who will need significant support from informal caregivers. Providing care for an individual with dementia has been associated with negative outcomes in a number of domains including physical health, mental health, financial status and social functioning. There is a small but growing base of literature suggesting that fostering psychological flexibility, including acceptance, with dementia caregivers may be a helpful intervention. Experiential avoidance, which is the less adaptive alternative to acceptance, is the aversion from negative internal experiences including thoughts, feeling and physical sensations. Experiential avoidance has been found to be significantly related to depression and negative affect. The present study utilized online dementia caregiver support group samples (n = 158) to evaluate the relationship between experiential avoidance and general demographics, other aspects of psychological flexibility, and caregiver distress. Experiential avoidance was positively correlated with cognitive fusion (r(134) = .231, p < .01), caregiver burden (r(127) = .258, p < .01), and distress associated with dementia related behaviors (r(140) = .225, p < .01). Experiential avoidance was negatively correlated with engaged living (r(133) = -.244, p < .01), mindfulness (r(123) = -.187, p < .05), and self-rated health (r(138) = -.193, p < .05). Additionally, experiential avoidance was significantly higher for male caregivers (t(136)=2.462, p=.015) and those age 65 and over (t(134)=-2.421, p=.017). These findings support previous research that suggests experiential avoidance may be an important construct to target in future interventions with dementia caregivers.