Large numbers of women who participate in cervical screening require follow-up for minor cytological abnormalities. Little is known about the psychological consequences of alternative management policies for these women. We compared, over 30-months, psychosocial outcomes of two policies: cytological surveillance  and a hospital-based colposcopy examination.Women attending for a routine cytology test within the UK NHS Cervical Screening Programmes were eligible to participate. 3399 women, aged 20–59 years, with low-grade abnormal cytology, were randomised to cytological surveillance  or initial colposcopy with biopsies and/or subsequent treatment based on colposcopic and histological findings (n = 1696). At 12, 18, 24 and 30-months post-recruitment, women completed the Hospital Anxiety and Depression Scale (HADS). A subgroup (n = 2354) completed the Impact of Event Scale (IES) six weeks after the colposcopy episode or first surveillance cytology test. Primary outcomes were percentages over the entire follow-up period of significant depression (≥8) and significant anxiety . Secondary outcomes were point prevalences of significant depression, significant anxiety and procedure-related distress (≥9). Outcomes were compared between arms by calculating fully-adjusted odds ratios (ORs) for initial colposcopy versus cytological surveillance.There was no significant difference in 30-month percentages of significant depression  or anxiety  between arms. At the six-week assessment, anxiety and distress, but not depression, were significantly less common in the initial colposcopy arm . Neither anxiety nor depression differed between arms at subsequent time-points.There was no difference in the longer-term psychosocial impact of management policies based on cytological surveillance or initial colposcopy. Policy-makers, clinicians, and women themselves can be reassured that neither management policy has a significantly greater psychosocial cost.ISRCTN 34841617Controlled-Trials.com  All screening programmes involve a balance between benefit, harm and affordability Various follow-up strategies are available In this paper, we use data from the TOMBOLA trial to extend this evidence-base. Our primary aim was to compare – over a 30 month period - the psychosocial impact on women of management policies based on repeat cytology tests  versus a colposcopic examination and, where necessary, related procedures . The 30-month period was intended to broadly represent the interval between screening rounds in the NHS CSPs and facilitate comparison between psychosocial and clinical results The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. Full details of the design and recruitment processes are reported elsewhere The alternative management policies under evaluation were designed to mimic real-world practice in the NHS CSPs. Cytological surveillance involved repeat cytology tests every six months in primary care. Women returned to routine recall  if they had three consecutive normal results. Women who had a cytology test showing moderate dyskaryosis or worse, or three consecutive inadequate tests, were referred to an NHS colposcopy clinic and were managed according to local protocols. Otherwise, women remained on six-monthly tests for the duration of follow-up within the trial.Women randomised to immediate colposcopy received an appointment to attend a hospital outpatient clinic for a colposcopy examination. Those who attended were invited to take part in a second randomisation - to immediate treatment by large loop excision of the transformation zone (LLETZ) or 2–4 targeted punch biopsies and selective recall for treatment if these showed CIN2/3 or more severe disease Women in both arms were invited to attend an exit colposcopy examination approximately three years after trial recruitment.This analysis was based on all women recruited to TOMBOLA from February 2001 onwards, the time at which the psychosocial assessments were introduced: these women were invited to complete psychosocial assessments at five time-points - at recruitment, and at four points during follow-up: 12, 18, 24 and 30 months post-recruitment. Hence, the outcomes for the analysis were self-reported by women. A sub-group of these women  were invited to complete an additional assessment; this took place six weeks after either the first surveillance cytology test or completion of the colposcopy episode . The recruitment assessment was intended to provide information on potential explanatory variables and women completed the questionnaire at their recruitment clinic appointment. The six-week assessment was designed primarily to assess distress related to the procedure the women had recently undergone; subsequent assessments were designed to assess more general longer-term psychosocial effects . The six-week and subsequent questionnaires were administered by post.The primary outcomes were significant depression and significant anxiety over the entire follow-up period. These were the percentages of women who reported significant depression or anxiety at least once over the 30-month period : henceforth this is referred to as “30-month percentage”. Significant depression and significant anxiety were assessed by the Hospital Anxiety and Depression Scale (HADS) As a secondary outcome, we investigated short-term distress related to receipt of an abnormal cytology test result and its subsequent follow-up: this was the percentage of women who reported distress, as determined from the Impact of Event Scale (IES) The recruitment questionnaire included a section on socio-demographics and lifestyle . It also included the Multi-dimensional Health Locus of Control Scale (MHLCS) which measures three dimensions of health locus of control – chance, powerful others and internal Questionnaire response rates at each assessment time-point were calculated based on the total number of women who remained in the trial at that time. Comparisons of trial arms were by intention-to-treat. The numbers of women who only partially completed a HADS subscale or the IES were small (between 10 and 26 at each follow-up time-point) and no imputation of missing values, or pro-rating for missing responses, was done. These women were excluded from the secondary analyses of the individual time-points, but we made the conservative decision to include them in the primary analysis of the entire 30-month window . HADS and IES scores were strongly positively skewed and so, instead of treating these as continuous variables, for each subscale/instrument, a binary outcome variable was created based on whether or not each woman scored above a specific threshold value on that subscale/instrument. For the HADS, Zigmond and Snaith over 30-months, of a policy of cytological surveillance versus a policy of initial colposcopy, we compared the 30-month percentages of significant depression and significant anxiety between the trial arms. These 30-month percentages were calculated as follows: for anxiety in the cytological surveillance arm, we divided the number of women who scored ≥11 on the HADS anxiety subscale at one or more of the 12, 18, 24 or 30-month assessments (n = 394) by the total number of women who were recruited after the psychosocial assessments were introduced and who were randomised to cytological surveillance .To test the null hypothesis of no difference in the psychosocial impact on women (assessed in terms of significant depression and significant anxiety), within the 30-month window, we compared the point prevalence of significant depression, significant anxiety and procedure-related distress in each arm at all relevant individual time-points: these secondary analyses were intended mainly for descriptive purposes. Point prevalence was computed as follows: for anxiety in the cytological surveillance arm at 12-months, we divided the number of women who scored ≥11 on the HADS at the 12-month assessment (n = 218) by the number of women who completed the HADS anxiety subscale at 12-months .To explore our secondary aim relating to differences in patterns of psychosocial effects between the two management policies a priori to be potential confounders are shown in Odds ratios for initial colposcopy compared to cytological surveillance were derived from logistic regression models run in STATA®  and used to test whether management arm predicted the psychological outcomes. Separate models were built for each psychosocial outcome. Our interpretation of the results was based on “fully-adjusted” odds ratios and the process for developing the fully-adjusted models was as follows. We first computed unadjusted odds ratios. Then, as recommended for trials employing minimisation To assess whether randomisation resulted in balance between the trial arms in significant depression and significant anxiety at the start of follow-up, we compared the prevalence between arms at recruitment using z-tests.and 30 months. Additional sensitivity analyses investigated the effect of changing the cut-points for the definitions of significant depression (≥11), significant anxiety (≥8) and procedure-related distress (moderate or worse ≥26).We conducted a sensitivity analysis of 30-month percentages which included the six-week assessment . This analysis was limited to the subgroup of women who were eligible to complete the six-week assessment. To investigate whether there was any evidence of bias, the primary analyses were repeated, restricting the study population to women who had completed the psychosocial questionnaires at 12, 18, 24 The trial was approved by the joint research ethics committee of NHS Grampian and the University of Aberdeen, the Tayside committee on medical research ethics, and the Nottingham research ethics committee, and was registered (ISRCTN34841617). All participants provided written informed consent.In total, 3399 women were eligible to take part in the psychosocial study, of whom 1703 were randomised to cytological surveillance and 1696 to initial colposcopy . Forty-t2354 women were included in the sub-group who received the six-week assessment, 1285 in the cytological surveillance arm and 1069 in the colposcopy arm. The characteristics of these women were similar to the full study population .Ninety-eight percent of women in each arm completed the psychosocial questionnaire at recruitment . QuestioThe 30-month percentage of significant depression was 16.0% in the initial colposcopy arm compared to 16.7% in the cytological surveillance arm; this translated into a fully-adjusted odds ratio (OR) of 0.99 . The comparison of arms was unchanged when the analysis was repeated using a subscale score of ≥11 to define significant depression (not shown). There was no difference in the results when the analysis was repeated including only those women who had competed psychosocial questionnaires at all time-points .and 30 months, the results were also unchanged (not shown)The 30-month percentage of significant anxiety was 22.6% in the initial colposcopy arm compared to 23.1% in cytological surveillance arm. The fully-adjusted odds ratio indicated that there was no difference between the arms , and this difference was statistically significant in fully-adjusted analysis , this could have served to bias the odds ratios towards the null. Finally, we chose to administer the questionnaire at six-weeks because we anticipated that women would have received results of their cytology test or colposcopy and any related procedures before receiving the questionnaire. Colposcopy and histology results were reported to women by the TOMBOLA trial office and the turnaround time was generally two weeks or less. By contrast, cytology results were reported directly from laboratories. We know that at some times during the trial there were significant delays in cytology reporting, and it is therefore possible that some women may not have received their result before completing the questionnaire. This could have artificially inflated the prevalence of anxiety at six-weeks in the cytological surveillance arm.Various factors may complicate the interpretation of the six-week comparison between arms. The six-week assessment was conducted in a sub-group of the study population, and women who did not attend for colposcopy or a first surveillance smear were not included. Default from colposcopy was higher than for cytological surveillance (6.7% vs 2.4%) Although the statistical methodology was not optimal for the detection of temporal changes, one of the striking findings from the secondary analysis related to the pattern of anxiety during the follow-up period. The heightened level of anxiety at recruitment  has previously been described by ourselves and others The pattern of depression during follow-up was quite different to that for anxiety. Around 8.5% of women scored in the range for significant depression shortly after receipt of the abnormal cytology result; there was a modest dip, to 7%, at six-weeks followed by a modest rise to 10% at 12-months, remaining at that level thereafter. The prevalence of depression in the current study was lower than among women who were undergoing cytology surveillance (16%) or had recently had colposcopy (24%) in the study of Bell et al This large, population-based, trial was nested within the NHS CSPs. The overall participation rate in the trial was 52%. Two strands of evidence suggest that the generalisability of the results is likely to be high. Firstly, the ratio of BNA to mild cytology results among trial recruits was similar to that observed in the CSPs Trial participation was lower among younger women, and we have previously shown that the prevalence of anxiety is higher in this group a priori evidence on which to base selection of the time-points at which to conduct the psychosocial assessments; we therefore selected these to avoid being too close to expected follow-up visits, and thus avoid the spikes of anxiety which may be associated with these. Other than the short information leaflets which we provided as part of the trial, we had no control over the information women received (or sourced for themselves) about the management policies or their results. Whilst we would expect that there would be substantial differences between individual women in terms of information seeking and receipt, the randomisation should have provided balance between the arms in this regard.This is the first study to have prospectively assessed the psychosocial impact of low-grade cytology tests and their subsequent management beyond 12 months. The instruments that we used have been extensively validated. There was little In this large, population-based, trial we found no difference in the psychosocial impact of management policies based on cytological surveillance or initial colposcopy over 30-months. Policy-makers, clinicians, and women themselves can be reassured that neither management policy has a significantly greater psychosocial cost.Checklist S1CONSORT Checklist - The Trial Of Management of Borderline and Other Low-grade Abnormal smears (TOMBOLA).(DOC)Click here for additional data file.Protocol S1Protocol for the Trial Of Management of Borderline and Other Low-grade Abnormal smears (TOMBOLA).(PDF)Click here for additional data file.


PCBA-capped CdSe NPs), using carboxylate ligands with [60]fullerene capping groups that provides an effective synthetic methodology to attach fullerenes noncovalently to CdSe, is presented for usage in nanotechnology and photoelectric fields. Interestingly, either the internal charge transfer or the energy transfer in the hybrid material contributes to photoluminescence (PL) quenching of the CdSe moieties.A new form of fullerene-capped CdSe nanoparticles ( A parPCBA) (see below) for use as n-type materials in hybrid bulk-heterojunction solar cell -phenyl Cr (PCBA) , hexadecr (PCBA) ,33,34, a2 gas, PCBA  was added along with 10 mL anhydrous THF. The mixture was then sonicated at room temperature for an hour while the nanoparticles gradually became fully soluble in the THF to form an optically clear solution. The exchange procedure was monitored by FT-IR (diminution of the carboxylic acid band at 1705 cm-1), which showed nearly quantitative formation after 3 h. The solution was then dried by bubbling Ar and then stored in glove box.To the pyridine-capped CdSe nanocrystals (17 mg) dried with N61-butyric acid methyl ester (PCBM) is a soluble form of fullerenes as well as the most widely used organic n-type material. The PCBM can be hydrolyzed with a strong acid to the corresponding carboxylic acid PCMA which is a hardly soluble, not processable material. The introduction of a branched side chain as a solubilizer on the phenylene of PCBM (PCBA) [60.-Phenyl CM (PCBA)  allows tPCBA afforded the PCBA-capped CdSe NPs which is readily soluble in organic solvents  -capped CdSe nanoparticles (CdSe NPs) were prepared using the reported procedures ,33,34. T) Figure .-1), the FT-IR spectrum of the pristine PCBA, displays a strong absorption band at 1,705 cm-1 assigned to the carboxylic acid, which is not present in the pyridine-capped CdSe NPs. In contrast, the PCBA-capped CdSe NPs reveal somewhat similar to the PCBA; however, it appears that the carboxylic acid band is diminished and shifted to lower energy at 1,730 cm-1 with a relatively decrease in intensity. The inspection of FT-IR of the PCBA-capped CdSe NPs such as the disappearance of the carboxylic acid band and new broad bands around 1,570 cm-1 is clearly suggestive that carboxylate binding are formed between PCBA and CdSe through the ligand exchange reaction.To ensure that the ligand exchanges had successfully gone to completion, the NPs were characterized by FT-IR Figure . On a loPCBA-capped CdSe). The TEM image of the pyridine-capped CdSe is characteristic of uniformly shaped spheres, with a mean size of 5.0 ± 1 nm  were investigated in chloroform solution. As shown in Figure PCBA-capped CdSe, the distinguishable band at 330 nm corresponds to the characteristic of C60 not only exhibits but also the absorption feature is almost identical to that of the pristine PCBA. This implies that PCBA is completely binding along the CdSe unit.The UV–Vis and photoluminescence (PL) properties of all the NPs  [PCBA-capped CdSe of the intracomplex quenching is indeed charge injection of a conduction band electron of the photoexcited CdSe NPs to the surface-bound PCBA, we are complementing our studies by time-resolved transient absorption spectroscopic measurements with CdSe NPs and PCBA-capped CdSe.In PL spectrum of the pyridine-capped CdSe NPs, the bathochromic shift of the onset of the emission of ca. 60 nm is observed when compared to the HDA-capped CdSe NPs. This low-energy emission can be due to aggregated formation and increased intermolecular interaction in the NPs . In the (<10 nm) . To confPCBA may be a significant key in further improving the efficiency of an organic solar cell.Both PCBM and CdSe derivatives have been applied as the electron transport materials in the most widely used configuration of the photovoltaic cells ,21,37-39In addressing this fundamental question, we are currently studying the hybrid nanostructures fabricated with conjugated polymers, in that enables to create the positive aspect in the hybrid solar cells and other hybrid materials-based optical and electrooptical applications via the ligand exchange.PCBA-CdSe by directly exchanging the ligands onto the surface of pyridine-capped CdSe, which is fully characterized by FT-IR, UV–Vis absorption, PL spectra, and TEM. The resulting PCBA-capped CdSe in solution shows a dominant emission peak centered at 795 nm, which is originated from the PCBA units. It clearly indicates that either the internal charge transfer or the energy transfer from the CdSe to PCBA moieties in solution occurs, so that the emission band (λmax = 512 nm) from the CdSe units is nearly quenched. We envision that a hybrid-polymeric nanoarchitecture can be utilized for constructing one layer of solar cell to explore the possible optoelectronic device application, which will open up new opportunities for the development of solar cells based on organic–inorganic nanocomposites. This work is currently under investigation.In summary, we have demonstrated the preparation of soluble nanoparticle of


IP based Wireless Sensor Networks (IP-WSNs) are gaining importance for their broad range of applications in health-care, home automation, environmental monitoring, industrial control, vehicle telematics and agricultural monitoring. In all these applications, mobility in the sensor network with special attention to energy efficiency is a major issue to be addressed. Host-based mobility management protocols are not suitable for IP-WSNs because of their energy inefficiency, so network based mobility management protocols can be an alternative for the mobility supported IP-WSNs. In this paper we propose a network based mobility supported IP-WSN protocol called Sensor Proxy Mobile IPv6 (SPMIPv6). We present its architecture, message formats and also evaluate its performance considering signaling cost, mobility cost and energy consumption. Our analysis shows that with respect to the number of IP-WSN nodes, the proposed scheme reduces the signaling cost by 60% and 56%, as well as the mobility cost by 62% and 57%, compared to MIPv6 and PMIPv6, respectively. The simulation results also show that in terms of the number of hops, SPMIPv6 decreases the signaling cost by 56% and 53% as well as mobility cost by 60% and 67% as compared to MIPv6 and PMIPv6 respectively. It also indicates that proposed scheme reduces the level of energy consumption significantly. A senetc. But with the advent of the Internet of things and federated IP-WSNs, this demand is going to be blurred. The huge number of IPv6 addresses, the necessity for end to end communication and advancement of micro-electronics have changed the concept of the research community. Now a tiny sensor node can hold a compatible TCP/IP protocol stack , the mean number of times the process is in state jS starting from state iS, is given by:Let k steps, starting from state iS, can be computed from the total number of times that the process is in the boundary states starting from state Sj, the initial state. The average number of location updates (Ubu) in the analytical model, is given by:The total number of boundary updates in Generalizing:We can use the above equation to determine the number of binding update messages. Because we need to send a binding update message whenever the sensor node moves between IP-WSNs, each time a node enters a boundary state, a binding update message is generated. Therefore, we need to determine the expected number of times that the process enters into a boundary state within k steps.buU binding update messages, given that the sensor node experiences a total of k transitions between SMAGs. Therefore, the ratio of intra-IP-WSN mobility is denoted as Mra_IP_WSNint and is expressed by:Thus, sensor nodes need to send Mer_IP_WSNint and is expressed by:The ratio of inter-IP-WSN mobility is denoted as 4.3.We have evaluated our proposed model based on signaling cost, mobility cost and energy consumption. In the subsequent section we discuss the signaling cost analysis and energy consumption analysis with the help of different parameters mentioned in sn-smag and the distance between SMAG and SLMA is denoted by Dsmag-slma. The corresponding node (CN) considered as the specialist doctor is communicating with the visiting doctor. There are some visiting doctors who take the patient in continuous basis. In this analytical model, different distances are used for calculating signaling cost. The cost is incurred due to transmission of data and control signal. The cost will vary dew to different types of signal transmission. There may be five types of mobility scenario which has already been discussed in the earlier section. If patients move to a remote site then the signal will be transmitted through PMIPv6 domain. Other wise signal transmission will be restricted with in the SPMIPv6 domain.mipv6) of the proposed scheme based on MIPv6 is calculated by summing up the individual cost of intra-IP-WSN mobility (Mintra-ip-wsn. Csd) and inter-IP-WSN mobility (Minter-ip-wsn.(Csd + Cbu)). Where, Csd and Cbu are the sensor mobility cost and binding update cost respectively:sd and Cbu are calculated in term of MIPv6 as follows:From pmipv6) of the proposed scheme based on PMIPv6 is calculated by summing up the individual cost of intra-IP-WSN mobility (Mintra-ip-wsn. Csd) and inter-IP-WSN mobility (Minter-ip-wsn.(Csd + Cbu)). Where, Csd and Cbu are the sensor mobility cost and binding update cost respectively:sd and Cbu are calculated in term of PMIPv6 as follows:From spmipv6) of the proposed scheme based on SPMIPv6 is calculated by summing up the individual cost of intra-IP-WSN mobility (Mintra-ip-wsn. Csd) and inter-IP-WSN mobility (Minter-ip-wsn.(Csd + Cbu)). Where, Csd and Cbu are the sensor mobility cost and binding update cost respectively:sd and Cbu are calculated in term of SPMIPv6 as follows:From 4.4.tx and Erx is the distance-independent amount of energy consumed by the transmitter and receiver electronics and the digital processing of each. Here α and β are path loss exponent (2 < α < 5) and a constant [J/bit m2], r is a transmission range. Lctrl is the length of control packets in bits, LE is the energy needed by the transmitter device to transmit or receive a packet, and T is the time period between two consecutive topological changes of the IP-WSN. nffdp(id) indicates the number of fully functional neighboring node for a path p and range d:For energy consumption analysis, we consider an IP-WSN with densely deployed IP sensing devices. The network consists of two types of IP sensing device: fully functional IP sensing devices (IP-FFD) and reduced functional IP sensing devices (IP-RFD). IP-FFD holds the complete 6LoWPAN protocol stack and performs the routing functions. IP-RFD performs the sensing functions and forwards the data to the IP-FFD. We have followed the energy consumption model used in ,24–27. TSince same type of transmitting and receiving device is concerned in IP-WSN:So energy consumed by MIPv6 scheme can be calculated by the following mathematical derivation. Here Energy consumed by PMIPv6 scheme can be calculated by the following mathematical derivation. Here Finally energy consumed by SPMIPv6 scheme can be calculated in the same way. Here 5.In this section, we present the results of experiments evaluating the performance of our scheme, and compare the performance of our proposed scheme to MIPv6 and PMIPv6. First, we evaluate the performance of our proposed approach by mathematical analysis. Then, we set signaling cost and mobility related cost for the number of IP based sensor nodes and number of hops traverse during the mobility phase in order to evaluate the consequences of our proposed scheme with PMIPv6 and MIPv6. Finally, we summarize the key characteristics of our proposed approach as compared to PMIPv6 & MIPv6 approach.We have implemented the model and evaluate the parameter such as the signaling cost and mobility related cost as presented in this paper. The experiments were conducted on a computer with AMD Athlon™ 2.5 GHz CPU and 2 GB primary memory. We use network simulator tool NS2 on VMware based Linux Fedora 4.The Difference of mobility cost becomes more reasonable when the number of mobile sensor node increases. The SPMIPv6 scheme reduces signaling cost by 62% and 57% with respect to MIPv6 and PMIPv6 in terms of the number of IP-WSN nodes.The The 6.In this paper we present a sensor proxy mobile IPv6 protocol that enhances the mobility issue in IP-WSNs. Mobility in sensor networks is the most challenging issue that must be addressed with special attention to energy efficiency. Since SPMIPv6 is a sensor network-based localized mobility management protocol, it meets the demands for energy efficiency in terms of reducing signaling costs and mobility costs. Here we also present its architecture and messages formats and evaluate its performance by analyzing signaling costs and mobility costs. Our experiments show that SPMIPv6 reduces both signaling cost and mobility cost in comparison to MIPv6 and PMIPv6. In this paper, we focus on a 6LoWPAN based IP-WSN of the same vendor and protocol stack. In future we will focus on sensor networks consisting of multi vendors and heterogeneous protocol stacks.


Infection with herpes simplex virus 1 (HSV-1) typically results in the formation of cold sores; however, in individuals with a compromised immune system the infection can be severe. The virus life cycle is characterized by two distinct phases: a lytic replication phase occurring in mucous membranes, and a latent state that is established in neurons of the trigeminal ganglia.in vitro results in reduced viral yields and the formation of syncytial plaques, where the plasma membranes of infected cells fuse together. In vivo, the absence of UL24 results in reduced viral titers and a drastic reduction in viral reactivation ex vivo. Previously, we showed that UL24 is both necessary and sufficient to induce the redistribution of the nucleolar protein nucleolin. The UL24 protein contains 269 amino acids and is expressed late in the virus life cycle. A bioinformatics study identified an endonuclease motif within the conserved N-terminal portion of the protein; however, its function remains to be demonstrated. Although several roles have been attributed to UL24, the mechanisms involved are unknown.The UL24 protein of HSV-1 is implicated in both the lytic and latent phases of infection. Infection with a UL24-deficient virus We hypothesized that UL24 functions, at least in part, through protein-protein interactions. To investigate this possibility, we used a virus that expresses UL24 with a hemagglutinin (HA) tag fused to its N-terminus, vHA-UL24. Lysates from infected cells were harvested, and fractionated on glycerol gradients. We found that UL24 was present in fractions corresponding to a much higher molecular mass than would be expected for UL24 alone, suggesting an association of UL24 with cellular or viral proteins during infection. To isolate potential UL24-binding proteins, we performed immunoprecipitations on fractions containing HA-UL24, using antibodies directed against the HA epitope. Immunoprecipitated proteins were resolved by denaturing polyacrylamide gel electrophoresis. Silver staining of the gel enabled the detection of proteins that appeared to specifically co-precipitate with HA-UL24. Candidate binding proteins will be identified by mass spectrometry. The identification of UL24-interacting partners will contribute to a better understanding of the roles of UL24 in different steps of the virus life cycle.


To date, insertable cardiac monitors (ICM) have been implanted in the hospital without critical evaluation of other potential settings. Providing alternatives to in-hospital insertion may increase access to ICM, decrease waiting times for patients awaiting diagnosis, and reduce hospital resources.This was a prospective, non-randomized, clinical trial involving nine clinical sites throughout the United States designed to assess the feasibility of ICM implants in a non-hospital setting. Other than the Reveal® ICM, implant supplies and techniques were left to physician discretion in patients who met indications. Patients were followed up to 90 days post-implant. The primary objective was to characterize the number of procedure-related adverse events that required surgical intervention within 90 days.Sixty-five patients were implanted at nine out-of-hospital sites. The insertion procedure was well tolerated by all patients. There were no deaths, systemic infections or endocarditis. There were two (3%) procedure-related adverse events requiring device explant and four (6%) adverse events not requiring explant. ICM use led to 16 diagnoses (24.6%) with 9 patients proceeding to alternate cardiac device implants during the course of the 90-day follow up.Out-of-hospital ICM insertion can be accomplished with comparable procedural safety and represents a reasonable alternative to the in-hospital setting.Clinicaltrials.gov registration number:NCT01168427 Subcutaneous insertable cardiac monitors (ICM) are indicated for use in patients with clinical syndromes or symptoms suggestive of cardiac arrhythmias Movement of minor surgical procedures to a less resource-intensive, in-office setting has been adopted by multiple clinical specialties including vascular and plastic surgery, gynecology, and urology The purpose of this study was to document the feasibility of out-of-hospital insertion of the Medtronic Reveal®  ICM outside of the hospital environment. The primary objective was to document all procedure-related adverse events that required surgical intervention within 90 days of device placement. Secondary objectives included documenting all other procedure-related adverse events, time and resource utilization, insertion technique, and device functionality.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see The study protocol and informed consent was approved by the Western Institutional Review Board, Olympia WA for 8 of the 9 participating institutions and by the local Scripps IRB, La Jolla, CA for the Scripps Clinic at Torrey Pines. All patients were provided with and signed the IRB approved written informed consent.This was a prospective, non-randomized, clinical trial  involving experienced implanters at nine clinic sites in the United States. The Medtronic Reveal® DX or Reveal® XT ICM was implanted in adult, non-pregnant subjects with established clinical indications using techniques and procedures similar to those for in-hospital insertion. Patients were screened and eligible patients enrolled as they presented to the practices of the study physicians. Patients with limited life expectancy (<12 months), active infection (within previous 30 days) or otherwise at high risk for infection, and those with unusual thoracic anatomy or scarring at the implant site were excluded from the study.The insertion procedure was required to take place in a setting other than an operating room or electrophysiology laboratory. The room utilized for the procedure was left to physician discretion. The Reveal® DX or the Reveal® XT device and the 2090 CareLink® Programmer were the only required components for this study. Physicians had access to disposable items similar to those used for in-hospital insertion  with specific supplies utilized at the discretion of the study investigator. The type and route of administration of perioperative prophylactic antibiotics were also left to individual physician practices. All centers were instructed to adhere to infection control procedures and room preparation guidelines. Patient sedation was limited to oral anxiolytics and analgesics at the physician’s discretion. In-office follow-up visits occurred at 30 and 90 days post-implant to identify any adverse events that could be attributable to the implant procedure. If necessary, the 90-day visit could be conducted by telephone.All complications were documented throughout the study. An Adverse Event Adjudication Committee (AEAC) comprised of four independent, non-participating physicians adjudicated each event and classified it as procedure-related, infection-related, serious or not serious, and whether the event was a complication. Procedure-related adverse events were defined as events due to procedures related to the implantation or surgical modification of the device. Complications were defined as adverse events that resulted in death, involved any termination of significant device function, or required invasive intervention. Serious complications were defined as events leading to death or serious deterioration of the health of the subject. The primary analysis used the Kaplan-Meier method to estimate the event rate and associated upper bound of 95% two-sided confidence interval at 90 days post-implant. For this analysis, days of follow-up were computed as the days from implant to the onset date of the first event for subjects with events. For subjects without an event, days of follow-up were calculated as days since implant to last follow-up date. The last follow-up date was defined as the date of the 90-day visit, death, or exit, whichever came first. A simulation study using Kaplan-Meier and Exact binomial method was conducted to determine the sample size of 65 patients, assuming that 30 day and 90 day event rate were 4% and 4.5% Baseline characteristics of the study patients are listed in Fifteen physicians with extensive prior ICM experience performed the implants and included five cardiologists and ten electrophysiologists. Of the 15 implanting physicians, 9 placed 2–5 devices, representing 34 insertions; 3 implanters placed 9–10 devices for 28 procedures; and 3 implanters placed one ICM each.Fifty-nine patients (89%) completed the 30-day follow up  and 52  procedure-related adverse events which required resolution by surgical intervention within 90 days of the implant procedure. There were four (6%) additional adverse events that were characterized as not serious by the AEAC and did not require explant. No patient required parenteral antibiotics for infection-related adverse events. There were no deaths, systemic infections or cases of endocarditis.The two procedure-related adverse events leading to surgical intervention and subsequent explantation of the device were adjudicated by the AEAC as implant site infections. One of these events occurred on day 25 in a 37 year old with no co-morbidities and was preceded by a vesicular erythematous rash immediately under the occlusive dressing tape. This patient was found to have a family history of tape allergy. The second infection-related event requiring explant occurred on day 53 in a 73 year old with diabetes and hypertension. It was preceded by pain and erythema and treated with oral antibiotics prior to explant. Both patients received prophylactic oral antibiotics at appropriate intervals prior to implant. The pocket was not flushed with antibiotic-containing solutions in either procedure. Physicians in both cases wore sterile gowns, gloves and masks during the implant and did not report problems with the procedure. Based on these events, the Kaplan-Meier estimate of the primary event rate requiring surgical intervention at 90 days post implant was 3.4% with a 95% confidence interval upper bound at 12.9%. Both events resolved upon device explantation.Of the four minor events, one was a scalp rash attributed to prophylactic vancomycin infusion at the time of implant (treated with diphenhydramine); one minor dressing allergy manifested as a self-limiting rash, and two were superficial infection events that resolved with oral antibiotic therapy. Both patients with superficial infections received prophylactic antibiotics; one intravenously (IV) and one orally. Both ICM pockets were flushed with antibiotic-containing solutions during the procedure. One patient had a history of Type-2 diabetes. The four, non-primary events all resolved without surgical intervention and all occurred within 12 days of the procedure.All 65 (100%) of patients received preoperative antibiotics generally targeting gram-positive cocci with 35 patients (54%) receiving oral antibiotics and 30 patients (46%) receiving IV antibiotics. The most common oral and IV antibiotics were cephalexin and cefazolin, respectively. Forty patients (65.5%) had the pocket flushed with an antibiotic-containing solution. A majority (61.5%) of patients received only preoperative antibiotics with the remaining 25 patients receiving some form of oral, postoperative antibiotics for varying durations.The patient and physician preparation as well as the type of room used for implant procedure was left up to the discretion of the investigator . The majThe overall procedure time was 118±51 (range: 52–238) minutes from the start of the procedure preparation until the end of subject recovery, with a mean “skin to skin” time of 26±10 (range: 8–56) minutes. The implant procedure typically required one physician and one registered nurse. Most patients (72%) were prepped in the procedure room. Physicians prepped in the implant room in 43 cases (66%). The device was inserted above the fourth rib in 60 (92%) cases and ICM orientation was vertical in 28 (43%) and horizontal in 13 (20%) with the remainder at various angles.This study allowed physicians to insert an ICM in indicated subjects utilizing their own techniques outside of the hospital operating room or cardiac catheterization lab. The major finding of this trial of 65 patients who had ICM implants outside of the traditional hospital setting was that this approach is indeed feasible with 91% of patients having no adverse events. Only 2 patients exhibited complications within 90 days requiring surgical intervention and device explantation. Four patients had AEAC-adjudicated minor adverse events. Despite the variation in techniques employed, this study demonstrated that with careful selection of an out-of-hospital environment, ICM insertion can be performed with comparable complication rates to the in-hospital setting.There has been gradual evolution in the procedure-related aspects of cardiac implantable electronic devices such as pacemakers and defibrillators. These devices were initially implanted in surgical suites by surgeons to minimize bacterial contamination and infection. More recently, complication rates for in-hospital cardiac catheterization or electrophysiology laboratory implantation were found to be comparable The concept of moving a surgical procedure to a less resource-intensive setting is not new The most costly adverse event that can occur as a result of an ICM implant, in both resources and patient health, is surgical intervention. Because of this, the study focused on these events as a primary objective. The Kaplan-Meier estimate of rate of adverse events requiring surgical intervention within 90 days was 3.4% and was comparable to previously reported in-hospital ICM insertion complication rates In this study, all patients received preoperative antibiotic prophylaxis in light of previous data showing beneficial results from randomized, double-blind, placebo-controlled trials for other implantable cardiac devices Two separate adverse events were recorded as allergic reactions to the occlusive dressing. Allergic reactions due to dressings have been previously reported This study was designed to assess the feasibility of out-of-hospital ICM implants and therefore patient preparation and physician practices were not strictly controlled. In 15% of the cases, the procedure room was similar to the hospital setting (physician owned outpatient diagnostic laboratory). This was a laboratory distinctly separate from the hospital and had not previously been used to implant cardiac devices. Nonetheless, it reflects a different environment than the other out-of-hospital locations. Only 52 (80%) of the 65 patients were followed out to the planned 90 day observation period. As identified in The results of this trial in a non-structured, out-of-hospital setting demonstrate that the ICM can be inserted with comparable patient outcomes to in-hospital insertion. More study is needed to clarify potential gains in associated cost savings, workflow, and the utility of a structured implant protocol.Checklist S1CONSORT checklist.(DOC)Click here for additional data file.Protocol S1Trial protocol.(PDF)Click here for additional data file.


Indoleamine 2,3 dioxygenase (IDO) is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold) in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation. Indoleamine 2,3-dioxygenase (IDO) is a monomeric, heme-containing enzyme that catalyzes the rate limiting step of conversion of tryptophan to kynurenine . RecentlA critical aspect of an immunological intervention using IDO is the requirement to achieve effective expression of this gene. However, genetic manipulation by non-viral transfection approaches has been challenging due to the issues of low transfection efficiency, loss of cell viability, and difficulty in obtaining stable transfection ,6. PreviNevertheless, the immunogenicity and transient gene expression of adenoviral vectors may hinder its clinical use in transplantation. Thus, in order to sustain the efficacy of IDO activity in the local environment of transplanted organ, it is essential to prolong the expression of functional IDO protein. In fact, it was shown that lentiviral vectors can maintain efficient long-term target gene expression in vivo for more than 4 years . It is hA lentiviral construct for expressing the IDO gene was generated using the pLC-E vector  modified2 cell culture flasks , and incubated in a humidified incubator at 37°C supplied with 5% CO2. Fibroblasts at passages three to five were used in all experiments.Skin samples were collected from 6- to 8-week-old male C57BL/6 (B6) mice according to the guidelines of the Animal Policy and Welfare Committee of the University of British Columbia. The samples were then washed in sterile dulbecco's modified eagle medium (DMEM)  supplemented with antibiotic–antimycotic preparation . Cultures of fibroblasts were established as previously described  and grow2 round plate, 10 μg IDO/pLC-E vector, 7.5 μg R8.9 plasmid, and 2.5 μg VSV-G plasmid were used. The culture medium was replaced with a fresh medium containing DMEM and 5% FBS 12–16 h after transfection. Supernatant was harvested 30 h after the medium change. The vector stocks were concentrated by centrifugation at 126,000g for 90 min using Beckman Ultracentrifuge and stored at -80°C until use.Replication-defective lentiviral vectors were generated as previously described ,16 by trFor transduction, 293T cells or mouse fibroblasts were seeded on flat-bottom 6-well cell culture plates  and incubated with the same titer of IDO-lentiviral vector for 24 h in the absence or presence of 10 ug/ml polybrene. Cell supernatant containing lentiviral vectors were removed from the culture 30 h later.For detection of the IDO protein expression, transduced cells were harvested 24 h post-transduction and washed twice with PBS. Cells were then lysed in lysis buffer . Equal amounts of total protein from each individual 293T cell culture were separated by 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to a PVDF membrane  and immunoblotted with a polyclonal anti-human IDO antibody  at final dilution of 1:5,000. Horseradish peroxidase-conjugated goat anti-rabbit IgG was used as the secondary antibody for the enhanced chemiluminescence detection system . Blots were then stripped and reprobed for β-actin as a control for protein loading.The lentiviral-mediated IDO expression in transduced 293T cells, and mouse fibroblasts were examined by fluorescence microscopy with a Zeiss Axiovert 200 M microscope. Images from identical areas of cultured cells were recorded using both fluorescence and bright-field microscopy. Images were captured using Northern Eclipse image analysis software.L-kynurenine, present in the conditioned medium of transduced cells. The amount of L-kynurenine was measured by a previously established method [The biological activity of IDO was evaluated by measuring the level of tryptophan degradation product, d method . Protein6 cells/mL for flow cytometry and at 107 cells/mL for sorting in PBS±2% FBS. For flow cytometry, BD LSRII with 630 LP and 670/30 detectors were used. For sorting, BD FACSAria with a blue laser detector was used. In each set, live cells were gated using forward scatter channel vs side scatter channel followed by gating on the IDO-expressing mCherry-positive cells. The number of mCherry-positive cells for fibroblasts transduced with IDO-lentiviral vector in the presence of polybrene after sorting was also determined by flow cytometry analysis. The average purity was greater than 95%.To determine the transduction efficiency of the IDO-lentiviral vector, and in order to sort mCherry-positive cells, non-transduced cultured fibroblasts, fibroblasts transduced with IDO-lentiviral vector in the absence or presence of polybrene were trypsinized, collected, and centrifuged. The cell pellet was washed twice with PBS and resuspended at 10d, majority of transduced cells express red fluorescent mCherry protein as a reporter gene for IDO expression compared with that of control cells . Figure c and a show the images of the same cells in bright-field. Furthermore, to examine the protein expression of the exogenously introduced IDO gene, lysates of the transduced cells were subjected to SDS-PAGE and immunoblotted with an anti-IDO polyclonal antibody  in the conditioned media. In comparison with non-transduced cells, the concentration of L-kynurenine in the conditioned media of transduced cells increased nearly threefold . The results show that the IDO-expressing lentiviral vector generated by transient transfection of 293T cells was functional and suitable for transduction of primary fibroblasts.Schematic diagram of the IDO–mCherry lentiviral vector is shown in Figure 1by Figure . As seend Figure  and the a and e as well as b and f represent non-transduced fibroblasts and IDO-lentiviral vector-transduced fibroblasts, respectively. To facilitate delivery of the IDO-lentiviral vector, polybrene was added to cells at the time of transduction. The cells were similarly examined by fluorescence microscopy 48 h post-transduction . Transduced fibroblasts in the presence of polybrene were gated and sorted by FACS and examined by fluorescence microscopy . As the fluorescent images show, the level of mCherry-positive cells increased in the presence of polybrene and after sorting. The expression of IDO protein in these cells was determined by Western blot analysis . IDO protein level increased in the presence of polybrene and after sorting. The functionality of the overexpressed IDO protein was assessed by kynurenine assay . The kynurenine content in the conditioned media of mouse fibroblasts transduced in the presence of polybrene increased approximately twofold when compared with that of control with no polybrene treatment . This finding revealed that polybrene significantly enhanced the transduction efficiency in primary mouse fibroblasts as well as the IDO protein and its enzymatic activity.Dermal fibroblasts were transduced with the IDO-expressing lentiviral vector. To analyze lentiviral-mediated IDO expression, transduced cells were visualized by fluorescence microscopy 24 h post-transduction Figure . The Figs Figure , panel ay Figure , panel ba of Figure b and c of Figure d, shows that more than 95% of the cell population after sorting were expressing IDO, and this was confirmed by flow cytometry, fluorescence microscopy, and kynurenine assay. The transduction efficiency was shown as percentage of mCherry-positive fluorescent cells in the total cell population studied . Transduction of mouse fibroblasts in the presence of polybrene showed a significant increase in the number of IDO-expressing cells . As a result, we successfully obtained a cell population that contains more than 95% IDO-expressing primary fibroblasts .To maximize the chances for successful transplantation and reduce the risk of immunorejection, it would be advantageous to have a highly pure population of IDO-expressing cells. By FACS, we sorted the IDO–mCherry fluorescent protein-expressing cells Figure . Panel ad Figure , panel eAs efficient gene delivery is a prerequisite for successful gene therapy, we believe that polybrene significantly increases lentiviral-mediated delivery of the IDO gene into target cells and that should make this enzyme a promising and highly attractive strategy for improving the outcome of cell and possibly organ transplantation.The authors declare that they have no competing interests.


Effective eHealth interventions can benefit a large number of patients with content intended to support self-care and management of both chronic and acute conditions. Even though usage statistics are easily logged in most eHealth interventions, usage or exposure has rarely been reported in trials, let alone studied in relationship to effectiveness.The intent of the study was to evaluate use of a fully automated, Web-based program, the Electronic Self Report Assessment-Cancer (ESRA-C), and how delivery and total use of the intervention may have affected cancer symptom distress.Patients at two cancer centers used ESRA-C to self-report symptom and quality of life (SxQOL) issues during therapy. Participants were randomized to ESRA-C assessment only (control) or the ESRA-C intervention delivered via the Internet to patients’ homes or to a tablet at the clinic. The intervention enabled participants to self-monitor SxQOL and receive self-care education and customized coaching on how to report concerns to clinicians. Overall and voluntary intervention use were defined as having ≥2 exposures, and one non-prompted exposure to the intervention, respectively. Factors associated with intervention use were explored with Fisher’s exact test. Propensity score matching was used to select a sample of control participants similar to intervention participants who used the intervention. Analysis of covariance (ANCOVA) was used to compare change in Symptom Distress Scale (SDS-15) scores from pre-treatment to end-of-study by groups in the matched sample.P=.01) in the intervention group users compared to the matched control group.Radiation oncology participants used the intervention, overall and voluntarily, more than medical oncology and transplant participants. Participants who were working and had more than a high school education voluntarily used the intervention more. The SDS-15 score was reduced by an estimated 1.53 points . Clinicians and researchers have developed eHealth solutions that supplement the limited time for patient report and communication within the confines of the ambulatory care, face-to-face visit . ReporteThe Electronic Self Report Assessment for Cancer (ESRA-C) is a patient-centered technology developed with rigorous participatory design methods  and evalThe purpose of this analysis was to determine the impact of the ESRA-C intervention exposure on cancer symptom distress and describe frequency of intervention use by participants in the ESRA-C II trial.This analysis addresses one component of our program of research founded on the Quality Health Outcomes Model, a framework proposed by Mitchell and colleagues  to illusTeaching Tips and View My Reports components.The ESRA-C II was a randomized trial conducted at two comprehensive cancer centers. Full details of the trial  and systTeaching Tips tab or the View My Reports tab within the intervention home page  “Why does this happen?”, (2) “What can I do about this?”, and (3) “What do I tell my clinical team?” a. After ome page b-c by clTeaching Tips, but did have the option to report SxQOL, and click the (non-pushed) Teaching Tips tab and the View My Reports tab.Intervention group participants were invited to access ESRA-C at any time between prompted sessions and clinic visits. These sessions were defined as any intervention use that was not prompted. During voluntary, non-prompted sessions, the participant did not receive pushed Teaching Tips and/or View My Reports. As a conservative measure of exposure to Teaching Tips, if at least one pushed tip was delivered during a prompted session, this was considered comparable to a single click on the non-pushed Teaching Tips. For example, a participant with at least one pushed teaching tip at each of three prompted sessions would have a total of three pushed teaching tips. Total exposure to the intervention consisted of three components: (1) the number of pushed teaching tips during prompted sessions, (2) the number of clicks on the non-pushed Teaching Tips tab during prompted and non-prompted sessions, and (3) the number of clicks on the View My Reports tab during prompted and non-prompted sessions. Voluntary exposure only occurred during non-prompted sessions and consisted of (1) the number of clicks on the (non-pushed) Teaching Tips tab, and (2) the number of clicks on the View My Reports tab.The ESRA-C intervention was considered delivered if the participant accessed the At prompted study time points, all participants were presented a set of SxQOL self-report assessments that included the Symptom Distress Scale-15 (SDS-15)  and EORTP=.02) in the intervention group compared to the control group [A total of 752 participants were randomized in the parent trial: 374 intervention and 378 control. Of those, 523  had complete SDS-15 baseline and end-of-study scores. In the primary analysis of covariance (ANCOVA), the average SDS-15 score was reduced by an estimated 1.21 points , work status, frequent computer user, gender, married/partnered, education , and service . Stage of disease was not considered for two reasons: (1) the highly associated relationship of stage and working status, a phenomenon documented in our prior work , and (2)Propensity score matching was performed with the complete data method in which no missing information could exist in the covariates used to compute the propensity score. A sensitivity analysis was conducted with the goal to balance the missingness within the two most common missing factors, work status and minority status. The propensity score matched sample was obtained using the R package “MatchIt”  and nearTotal and voluntary exposure to the intervention components can only be calculated within the intervention group. The median total exposure was 2 (range 0-29). Intervention use was defined as at least two exposures. Of the 374 intervention participants, 233 (62.3%) used the intervention. A software error precluded exposure to pushed teaching tips for any intervention participants with an appointment date from June 30, 2010 to May 12, 2011. Of the 141 not receiving pushed teaching tips in the intervention, this error precluded three possible exposures in 48 (34.0%), two exposures in 21 (14.9%), and 1 exposure in 15 (10.6%) intervention group participants. There were 55 participants on the intervention arm that required a clinic/assisted point of access; their median total exposure was 2 (range 0-13) and 16 (29%) viewed the intervention voluntarily in the clinic. There were 319 participants on the intervention arm that indicated home/independent access; the median total exposure for these remote users was 3 (range 0-29) and 111 (34.8%) viewed the intervention voluntarily.P=.02, P=.06) and education (P=.05). Participants that used the intervention were working and had more than a high school education. Additionally, there was a significant difference in the proportion of participants voluntarily using the intervention by service (P=.001). More radiation oncology participants 58/125 (46.4%) voluntarily used the intervention compared to medical oncology 61/211 (28.9%) and transplant 8/38 (21%) participants  in the intervention group compared to the matched control group. The sensitivity analysis that balanced the missingness within the work status and minority status factors produced similar results as the complete data analysis (data not shown).Teaching Tips, accessed non-pushed tips, and viewed reports of SxQOL outcomes graphed over time. About a third voluntarily accessed the intervention in between clinic visits. We discovered that use of the intervention significantly reduced the estimated symptom distress score when compared to participants who did not use the intervention. The magnitude of the estimate (1.53) was larger than in the primary outcome analysis when we compared study groups (1.21) [The results of this propensity analysis suggested that reduction of cancer symptom distress, the primary outcome of our randomized trial, was associated with use of the ESRA-C intervention components. More than half of participants in the intervention group were exposed to pushed timate 1.3 was lars (1.21) , illustrUse of, and adherence to, Web-based health care interventions have primarily been evaluated in health promotion and chronic disease self-management settings . Few stuNot surprisingly, ESRA-C was accessed remotely and voluntarily more frequently by those with higher education, who may have been likely to use personal computers or tablets on a regular basis. This is consistent with the finding, while of borderline statistical significance, that working individuals also used ESRA-C more often in between clinic visits. Working when beginning cancer therapy has been shown as a significant variable in two of our earlier analyses, predicting a lower rate of emergency department and unplanned admissions , yet preParticipants who enrolled in the trial as they were about to undergo radiation treatments also accessed ESRA-C voluntarily significantly more often than those enrolled when beginning medical cancer therapies or stem cell transplant. We are not aware of differences in usual care symptom support between modality services at the cancer centers; yet, if differences existed, patients may have turned to ESRA-C more often in radiation. Alternatively, these participants were reminded of ESRA-C almost every day of the week as they entered the radiation setting where each had consented to the trial.Our findings are limited by the fact that about a third of the intervention group participants never received pushed teaching tips in the assessments prior to on-study clinic visits. Thus, the effect of the intervention may have been different if all had the opportunity to see the tips. Our participant sample was less diverse with regard to race and ethnicity than the rates of cancer diagnoses in minority groups in the United States  and all First, propensity analyses could be replicated in other eHealth trials as a method to investigate the relationship of usage to health outcomes. Although our participants’ raw exposure to the intervention was not high in an absolute sense, we were able to study the association of symptom distress with usage rather than report raw usage. Second, investigators could evaluate whether usage was related to various characteristics, and whether they are characteristics of the intervention, of the user, or of the condition addressed by the intervention. We provided some rationale for the mechanism that triggered usage by certain demographic groups, but this could have been a combination of aspects of the intervention itself in addition to participant demographics. Although some investigators found that educated, older, employed women were the most active users of Web-based, chronic disease  and healOur findings have implications for the many patients treated at institutions that have deployed a patient portal as a component of an electronic medical record system. There may be patients at risk for failed symptom and distress screening and/or failed symptom support delivery if such systems are available only to those Web-savvy, educated patients who regularly use email.Finally, implications for both future research and practice using patient-centered, Web-based technologies include improved communication of study design and workflow between the design and technical implementation teams and more rigorous quality checks on intervention integrity. Communication of research goals may be facilitated via improved use of shared artifacts such as models of study data and workflows . SoftwarThe intended effects of a Web-based, self-care education, monitoring, and communication coaching intervention on cancer symptom distress were modified by intervention use frequency. The voluntary, remote use of ESRA-C was most frequent in working participants with higher education levels and those receiving radiation therapy.


Inflammatory bowel diseases (IBDs) accompany a critical loss of the frontline barrier function that is achieved primarily by intestinal epithelial cells (IECs). Although the gene-regulation pathways underlying these host-defense roles of IECs presumably are deranged during IBD pathogenesis, the quantitative and qualitative alterations of posttranscriptional regulators such as microRNAs (miRNAs) within the cells largely remain to be defined. We aimed to uncover the regulatory miRNA–target gene relationships that arise differentially in inflamed small- compared with large-IECs. Whereas IBD significantly increased the expression of only a few miRNA candidates in small-IECs, numerous miRNAs were upregulated in inflamed large-IECs. These marked alterations might explain why the large, as compared with small, intestine is more sensitive to colitis and shows more severe pathology in this experimental model of IBD. Our in-depth assessment of the miRNA–mRNA expression profiles and the resulting networks prompts us to suggest that miRNAs such as miR-1224, miR-3473a, and miR-5128 represent biomarkers that appear in large-IECs upon IBD development and co-operatively repress the expression of key anti-inflammatory factors. The current study provides insight into gene-regulatory networks in IECs through which dynamic rearrangement of the involved miRNAs modulates the gene expression–regulation machinery between maintaining and disrupting gastrointestinal homeostasis. IBD is a chronic relapsing disorder of the gastrointestinal tract that is due to intestinal inflammation and epithelial injury and includes Crohn’s disease and ulcerative colitis. Although IBD is thought to arise through interactions among genetic, immunologic, and environmental factors, the etiology underlying the pathophysiology of IBD remains largely unknown. In particular, Crohn’s disease and ulcerative colitis appear to result from distinct pathologic mechanisms that have a shared origin in the deterioration of the gastrointestinal barrier function, which is supported primarily by IECs and their secreted productsmiRNAs are a class of endogenous, small noncoding RNA molecules that control gene expression at the posttranscriptional levelThe phenotypic and anatomic differences between ulcerative colitis and Crohn’s disease are thought to reflect the intestinal region affected: ulcerative colitis is limited to the colon, whereas Crohn’s disease can occur throughout the gastrointestinal tractHere, we used IECs isolated from mice with and without DSS-induced colitis to: 1) determine the hierarchical profiles of miRNAs and mRNA expression in small- compared with large-IECs, and 2) propose associations between miRNAs and their target genes that explain the differing sensitivities and pathologic outcomes of the small compared with large intestine to DSS-induced colitis. Finally, we suggest gene regulatory networks in IECs that link miRNAs and targets in IBD and provide a strategy for identifying biomarkers and targets that might improve IBD diagnosis and treatment.We used the DSS-induced colitis model, in which the disease symptoms of affected mice are similar to those in human patients with ulcerative colitisTo obtain comprehensive profiles of the miRNAs that are expressed differentially in the epithelial cells of the small intestine compared with the large intestine under physiologic conditions, we first isolated IECs from the intestinal tissue of DSS-untreated mice. Because the isolation protocol yields heterogeneous cell populations, we used flow cytometry to sort the IECs according to cell size. Using large-IECs as an example, the cell population indicated as “iii”  largely miRNAs were selected according to their expression intensity in normal small- compared with large-IECs. Of approximately 1,900 miRNAs on the microarray, normal small-IECs contained 86 miRNAs whose expression was markedly increased in terms of both fold change (>2-fold) and fluorescence intensity (>100 units above background), whereas 33 miRNAs in normal large-IECs showed increased expression . In addiUsing methodology similar to that for assessing miRNA expression under physiologic conditions, we evaluated the miRNA profiles of small- and large-IECs harvested from DSS-treated mice. The miRNA expression profiles of inflamed small- and large-IECs exhibited striking differences from those of normal small- and large-IECs . In partBy contrast, 39 miRNAs in the large-IECs of mice with DSS-induced colitis showed increased expression . InteresWe next examined overall numbers of miRNAs highly expressed in each experimental group . We set the expression threshold as the median expression value of all four miRNA arrays and analysed the population overlaps. Overall, 648, 844, 541, and 514 miRNAs were expressed in normal small-IECs, normal large-IECs, inflamed small-IECs, and inflamed large-IECs, respectively , and 401We then compared the global miRNA expression level between small- and large-IECs from DSS-treated mice. Numerous highly expressed miRNAs in the large-IECs were increased in the inflamed, compared with normal, condition . ConversWe attempted to acquire a profile of the IEC miRNAs by measuring their fold change after DSS administration. The criteria for choosing miRNAs of interest was identical to that used to compare miRNAs between small- and large-IECs in Again using the same selection criteria , we analyzed mRNA microarrays with the small- and large-IEC samples from DSS-treated and untreated mice to acquire global gene-expression profiles in terms of fold change. Among the mRNAs of approximately 24,000 genes (Methods), the expression of 24 mRNAs was increased and that of 326 was decreased in inflamed small-IECs . By contThe miRNAs whose expression changed dramatically after DSS-induced colitis may contribute to pathogenesis by controlling the expression of their target genes. We therefore defined networks of miRNAs and their putative target genes in inflamed IECs in which the ‘direction’ of miRNA expression was opposite to that of the target gene. Considering that changes in miRNA-induced transcription repression might lead to altered gene expression, we created putative networks that comprised the miRNAs markedly up- or down-regulated in inflamed small- or large-IECs  and theiThis process revealed that small-IECs harbored only one up-regulated miRNA (miR-223-3p)  and one Next, we further analyzed biological pathways possibly associated with the target genes of significantly altered miRNAs in inflamed large-IECs, by using a bioinformatics tools (TargetMine)We then examined the putative target genes of miRNAs that were down- or up-regulated in inflamed IECs. In the microarray, the expression of miR-223-3p was augmented in inflamed, compared with normal, small-IECs. Consistent with this finding, miR-223-3p is one of the main miRNAs increased during IBD pathogenesismiR-223-3p might control the expression of its 13 putative targets, which were decreased in expression in inflamed small-IECs . Among tInflamed large-IECs contained 9 putative target genes for miR-223-3p whose expression was decreased in the same cells : GOLGB1,The population of miRNAs in inflamed large-IECs whose expression was markedly changed from that in normal IECs was largely different from those in inflamed small-IECs. To validate the results from the microarray analysis , we usedWe next sought to identify candidate targets for the miRNAs with significantly changed expression levels in both microarray and quantitative PCR analyses. Among the target genes whose expression was inversely proportional to that of miRNAs in the inflamed large-IECs, we selected genes of interest on the basis of their mention in previous studies. As shown in Next we analyzed the target genes that were down-regulated in inflamed large-IECs and the miRNAs whose expression was up-regulated; the expression levels of these miRNAs were validated through quantitative PCR analysis . The tarBased on the results from the correlations between miRNAs and their targets significantly altered selectively in inflamed large-IECs, we propose models for the intracellular pathways, which are initiated with significant alterations in miRNAs. The scenario is proposed in In summary, through functional categorization of the target genes markedly up- or down-regulated in inflamed large-IECs, our study has revealed potentially important gene-regulation loops. As a result, the novel miRNAs revealed in the current study contribute to our integrated understanding of the miRNA-orchestrated manipulation of gene expression integral to the maintenance or disruption of gastrointestinal homeostasis.Guidelines for Use and Care of Experimental Animals and approved by the Animal Committee of the Institute of Medical Science, the University of Tokyo.Balb/c mice  were purchased from Clea Japan . All of the mice were maintained with free access to food and water on a standard 12-12 h light-dark cycle. Mice were acclimated for at least 1 wk before they entered the study. All experiments were performed according to the To induce experimental colitis, mice had free access to 3% DSS  in drinking water for 8 days. Healthy control mice received DSS-free drinking water. Clinical symptoms, including body weight, rectal bleeding, and diarrhea, were monitored daily. Mice were euthanized on day 8, and their intestines were removed.To address the degree of inflammation, samples of intestinal tissues were fixed in 4% paraformaldehyde  for 24 h, embedded in paraffin, sectioned at 5  μm, stained with hematoxylin and eosin, and evaluated by light microscopy .g for 20 min, the interface between the 25% and 40% layers was collected to obtain IECs. The cells were stained with antibodies to epithelial cell adhesion molecule (EpCAM) , or CD45 (Biolegend) and nucleic acid dye (Via-Probe) . The EpCAM+ cells were further validated to be IECs via intracellular staining with antibody to pan-cytokeratin  and Fix and Perm reagents . The IECs were sorted . Because the efficiency of this method for isolating highly purified (>98%) viable cells from gut tissues has been established previously in our laboratory, we used the sorted IECs immediately, without additional evaluation, in subsequent experiments. The data were analyzed by using FlowJo software .A standard protocol was used, with minor modifications, to isolate IECs. Briefly, the small and large intestines were harvested individually from 8 to 10 mice and rinsed extensively with RPMI-1640  after Peyer’s patches were removed . The rinsed intestines were opened longitudinally and macerated; the tissue pieces were shaken gently in RPMI-1640 containing 2 mM EDTA and 10% FCS. The tissue preparations were passed through 70-μm mesh filters, and the resulting single-cell suspensions were applied to Percoll  density gradients of 25%, 40%, and 75%. After centrifugation at 2,000 × http://microrna.sanger.ac.uk/sequences/). After stringent washes of the sample, fluorescent signals were scanned  and analyzed by using 3D-Gene Extraction software (Toray). The relative expression level of a given miRNA or mRNA was calculated by comparing the signal intensities of valid spots throughout the microarray experimentsThe live IECs sorted from the intestines of 8 to 10 mice per group  were pooled; total RNA was isolated from each pool by using Trizol (Invitrogen) and used to create microarrays to obtain their miRNA and mRNA expression profiles. Total RNA was labeled  according to the manufacturer’s instructions; labeled RNAs were hybridized onto 3D-Gene oligo chips for miRNA  or mRNA . The oligonucleotide sequences of the probes were confirmed by the miRBase miRNA database  as described earlier. cDNA was synthesized from approximately 800 ng to 1 μg of total RNA (NCode miRNA First-strand cDNA Synthesis and qRT–PCR (Invitrogen) or PrimeScript  kits and applied to quantitative real-time PCR analysis using a Power SYBR Green PCR Master Mix (Invitrogen). All reactions were repeated at least three times, and U6 and β-actin were used as endogenous controls to normalize the expression of miRNAs and mRNAs, respectively. Gene expression was analyzed by using a StepOnePlus Real-Time PCR System . The forward primers for miRNAs were designed according to the manufacturer’s instructions , and the universal qPCR primer supplied in the kit was used as the reverse primer. The primer sequences used in this study are shown in t-test was used to compare the groups with control values (DSS-untreated mice).The data are presented as mean ± 1 standard deviation for at least three independent experiments. Student’s We have shown here that the IECs in the large intestine contain numerous miRNAs that actively participate in inflammation. The putative networks among miRNAs and their targets in large-IECs from mice with DSS-induced colitis were nearly unique compared with those in similarly obtained small-IECs. Moreover, we disclosed several novel miRNAs that might modulate the expression of key molecules involved in either ameliorating or aggravating IBD pathology. Thus our study provides an initial step toward understanding, at the molecular level, the specific susceptibility of the large intestine to DSS-induced inflammation and identifying candidate miRNA biomarkers associated with either promotion or suppression of the inflammatory disorder.It is worthy to focus on IECs in DSS-induced colitis model in two important aspects; alteration of barrier function and trigger of inflammatory stimulation. In the model the sulfated polysaccharide of DSS causes fatal injury of IECs and alter their mucosal barrier functionIt is generally recognized that the small and large intestines differ in their susceptibilities to gut inflammation and that environmental differences including resident microflora might be the root cause56The miR-223-3p was the only miRNA that showed increased expression in both inflamed small- and large-IECs in both microarray and quantitative PCR analyses  and 6c. The microarray data did not agree perfectly with the results from the quantitative PCR analysis, presumably due to technical differences between the methodsOf the 18 mRNA targets up-regulated in inflamed large-IECs, 9  were revealed to correlate with results from previous studies of IBD 1718192021718192021222324172025in-vivo models that possess enterocyte-specific, overexpressed miRNAs in the near future. It is also pivotal to elucidate any clinical relevance of current study by analyzing intestinal samples from patients with ulcerative colitis or Crohn’s disease.Among the 17 target genes down-regulated in inflamed large-IECs , only 3 3536in vitro and in vivo approaches, the current study is an initial step toward the creation of a global platform in the IEC miRNAs for understanding an implicate solution to identifying new biomarkers not only for miRNAs and mRNAs but also for their correlation loops. Finally, this work advances our understanding of how IECs rearrange their genetic components to cope with chaotic situations such as IBD progression.In conclusion, we expect that the miRNA database profile we obtained by using sorted IECs will be instrumental in determining, at the genetic and molecular levels, the source of the different susceptibilities of the small and large intestine to inflammation and their differences in inflammation-induced pathology. Whereas the expression of few miRNAs was significantly increased or decreased in small-IECs upon DSS-induced inflammation, large-IECs in the experimental IBD model exhibited markedly altered patterns (both up- and down-regulation) in the expression of mRNAs and miRNAs; our study may be the first to compare these genetic profiles between normal and inflamed IECs. Furthermore, this immense shift in the genetic components of large-, but not small-, IECs under the situation of IBD might represent the quality differences between both intestines. Although the biological significance of these biomarker candidates needs to be validated through How to cite this article: Lee, J. et al. Profiles of microRNA networks in intestinal epithelial cells in a mouse model of colitis. Sci. Rep.5, 18174; doi: 10.1038/srep18174 (2015).


ST statistics, and the three-population test were used to investigate the underlying genetic structure and ancestry of the different ethno-linguistic groups. Our analyses revealed a genetic component for Sudanese Nilo-Saharan speaking groups (Darfurians and part of Nuba populations) related to Nilotes of South Sudan, but not to other Sudanese populations or other sub-Saharan populations. Populations inhabiting the North of the region showed close genetic affinities with North Africa, with a component that could be remnant of North Africans before the migrations of Arabs from Arabia. In addition, we found very low genetic distances between populations in genes important for anti-malarial and anti-bacterial host defence, suggesting similar selective pressures on these genes and stressing the importance of considering functional pathways to understand the evolutionary history of populations.East Africa is a strategic region to study human genetic diversity due to the presence of ethnically, linguistically, and geographically diverse populations. Here, we provide new insight into the genetic history of populations living in the Sudanese region of East Africa by analysing nine ethnic groups belonging to three African linguistic families: Niger-Kordofanian, Nilo-Saharan and Afro-Asiatic. A total of 500 individuals were genotyped for 200,000 single-nucleotide polymorphisms. Principal component analysis, clustering analysis using ADMIXTURE, F East Africa is a strategic region to study human genetic diversity due to the presence of ethnically, linguistically, and geographically diverse populations. North African and sub-Saharan African populations in East Africa are connected by the Nile River. It has been proposed that the Nile River Valley acted during history as a genetic corridor allowing gene flow between North and South in Eastern Africa1East Africa complexity can be seen in the fact that all families of continental African languages are represented in the region. Continental African languages have been classified into four major language families: Afro-Asiatic, Nilo-Saharan, Niger-Kordofanian (or Niger-Congo), and Khoisan. Afro-Asiatic, spoken predominantly by northern and eastern African pastoralists and agro-pastoralists, covering North Africa, includes the Semitic, Cushitic, and ancient Egyptian (Coptic) languages. Nilo-Saharan, spoken predominantly by eastern and central African pastoralists, includes in its main Chari-Nile branch the Central Sudanic and Eastern Sudanic  languages. Niger-Kordofanian, spoken predominantly by agriculturalist populations across western, eastern, central, and southern Africa, includes the Bantu languages3et al.6In an extensive and detailed study, Tishkoff Here, we focus on the region of Sudan and South Sudan with some other external related populations ; we refer as Sudanese Region to the ensemble see . The gen156ST to identify putative signals of adaptive selection in these populations, with a focus on immunological adaptation in anti-malarial, anti-bacterial and anti-fungal defence genes.The first aim of this study was to provide new insights into the genetic history of East African populations by analysing six Sudanese ethnic groups belonging to the main African linguistic families spoken in the region , in addition to ethno-linguistic neighbouring groups . We assessed the genetic diversity and relationships between these different ethno-linguistic groups to clarify the genetic history of East Africa. The second aim of the study was to use genetic distance estimated as FWe applied a principal component analysis (PCA) to investigate the population structure of the new populations genotyped in this study from the Sudanese region . PC1  with previously studied populations of special interest for this analysis: QatarTo test whether these particular sets of Immunochip SNPs (140 K and 14 K data sets) can recover population structure, we extracted 1000 Genome data from world-wide populations and observed that the genetic structure between them is maintained across the different data sets of SNPs used . In addiST statistic, a measure of global population differentiation, confirmed the PCA clustering  was found both in the pair Arabs and Nubians, located at the Nile River Valley, and in the pair Beja and Ethiopians, located at the coast. Among North-East populations, Nubians had the highest FST values when compared with Beja and Ethiopians (average FST of 0.006 and 0.007 respectively). South-West populations showed higher population differentiation among themselves than North-East populations. When comparing North-East populations with South-West populations, all comparisons have a high FST (between 0.044 and 0.054). Copts, with a strong individual heterogeneity, are more similar to Arabs (FST = 0.019) than to any other East African population. Copts and South-West populations are the most distant populations (FST > 0.1). Fulani had on average lower FST values when compared to South-West  than to North-East populations . These values show a complex situation beyond the simple North African versus Sub-Saharan Africa main differentiation.Pairwise Fustering . Populatoutliers . The lowST) between populations are correlated. We found a significant positive correlation between genetic and geographic distance .To test the hypothesis that geographically close populations are genetically similar, we performed a Mantel test to determine to which extent geographic and genetic distances , whereas the South-West cluster  share an ancestry component (light blue) with sub–Saharan samples. The North-East cluster  shows both components, although the main component (~70%) is that detected in North Africa and Middle East , supporting a common origin with Egypt (or other North African/Middle Eastern populations). They are known to be the most ancient population of Egypt and at k = 4 , they shk = 5 show their own component  than North African (<40%) or sub–Saharan (<15%) and at omponent . They ha3 statistics)−5): those of the North-East cluster  and Fulani. Populations from the North-East cluster: Beja, Ethiopians, Arabs and Nubians  between populations using the genetic variation in genes involved in defence against different agents. We selected among the genes genotyped in the Immunochip those associated with resistance/susceptibility to malariaST of those genes was compared to the mean FST of a set of randomly selected SNPs from genic regions with the same sample size and similar MAF, using a permutation test (10.000 permutations). All pairwise comparisons showed that the mean FST score of malaria-related genes was significantly lower than the mean FST score of the sampling distribution  and Afro-Asiatic Ethiopians . Y-chrom1et al.k = 6, which is different from the Sudanese component  related to Nilotes of South Sudan, but not to other Sudanese populations or sub-Saharan populations . This anomponent  and do nThe presence of the core of Nilo-Saharan languages in the confluence of the two Nile rivers suggests that the Sudanese region is the place of origin of the Nilo-Saharan linguistic family despite their fragmented distribution, as shown by the location of the Nubian language21Population displacement, whether it is followed with cultural or genetic exchange with local populations, would explain why not every Nilo-Saharan speaking group has this genetic component (as is the case of Nubians) and not every population that has it is mainly formed by Nilo-Saharan speakers (as is the case of Niger-Kordofanian speaking Nuba).k = 4  language and occupy a large area in Africa’s Sahel. Their origin is still controversial, as mitochondrial DNA indicates a West African and traces of North African origina-priori hypothesis was that selective pressures on host defence genes induced by similar infections would determine lower genetic distances between populations, as compared with a genome-wide distribution: a de facto convergent evolution in host defence. Similar signals of convergent evolution in the TLR1/2/6/10 cluster were recently reported between European and Rroma populations living in the same geographic areaThe second objective of our study was to analyse how infectious pressures affected the genetic variation of East African populations. The In this work, we analyse genotyping data for almost 140,000 SNPs in nine East African populations from Sudan, South Sudan and Ethiopia. Our main results add new and interesting features to the North East African genetic complexity, with new populations that define a genetic component in southern Nilo-Saharan speakers that cannot be related to a North-African or other sub-Saharan components. These populations should be included in further population genetics and epidemiological studies to have a representative sample of the genetic diversity of the region of East Africa. Moreover, a functional analysis shows similar genetics signals related to genes involved in antimalarial and antibacterial immune response. These findings suggest convergent evolution of the immune system of various ethnic groups in East Africa due to the major common selective pressures attributable to parasitic and bacterial infections acting on these populations.Saliva samples were collected from 500 individuals belonging to nine east African populations based on self-reported ethnicity. The samples used in the present research were collected and studied with ethical approval and informed consent. All experimental protocols were approved by the IRB of University of Medical Sciences and Technology in Khartoum and that of Universitat Pompeu Fabra  in Barcelona and were carried out in accordance with the approved guidelines. The population samples from the Sudan belonged to: the Afro-Asiatic ; the Nilo-Saharan ; and the Niger-Kordofanian linguistic families . In addition to these populations, we also collected samples from neighbouring populations: Nilo-Saharan speaking Nilotes (n=50) from South Sudan, and Afro-Asiatic speaking Ethiopians (n=40) currently living in Khartoum (Sudan). Samples from Niger-Kordofanian speaking Fulani (n=40), a nomadic group that usually traverse Africa’s Sahel, were also analysed. These samples were genotyped on the Immunochip (Illumina Infinium single-nucleotide polymorphism microarray), a custom-made, high-density genotyping array containing 195,806 single-nucleotide polymorphisms (SNPs) and 718 small insertion-deletionshttp://d-maps.com/carte.php?num_car=737&lang=en and http://d-maps.com/carte.php?num_car=740&lang=en, using Adobe Photoshop 7.0.1. software . Data are publicly available at the web page of JB (http://biologiaevolutiva.org/jbertranpetit/wp-content/uploads/2015/02/SudanImmunochip.zip) or under request.Nine samples were identified as being second or third-degree relatives by identity by descent (IBD) matrix (IBD > 0.185) and were removed from the analyses. SNPs with a call rate below 99% and on sexual chromosomes were also removed. After these steps, a total of 143,602 SNPs and 447 samples remained: 27 Copts, 36 Beja, 39 Ethiopians, 112 Arabs, 79 Nubians, 35 Darfurians, 37 Nuba (18 Nilotic and 19 Niger-Kordofanian speakers), 49 Nilotes, and 33 Fulani. This data set is further referred to as the “140 K” data set.For comparative studies, a Middle Eastern population (Qatar)To study the genetic relationships among East African ethno-linguistic groups, we used principal components analysis (PCA) as implemented in the Eigensoft packageST valuesST between populations. Mantel test was calculated using the R ADE4 packagePopulation differentiation was estimated using classical pairwise Fk = 2 through 10) were tested successively and the optimal value of k was estimated by ten-fold cross-validation. Clustering results were visualized with DistructPopulation admixture was analysed using ADMIXTUREthree-population test implemented in the ADMIXTOOLS software package3 , where a negative value of the f3 statistic implies that population X (target population) was the result of an admixture event between the two ancestral populations of A and B (source populations). We tried every combination of source populations for each of our nine target populations and estimated the mixing coefficient (α) with Yoruba as the outgroup population. It is the proportion of the admixture of the target population given by the source population A, while 1 − α is the proportion given by the source population B. For each comparison we kept the results with a significantly negative value of the f3 statistic after multiple testing correction .To formally test whether admixture happened within the studied populations, and to measure its extend, we used the To take advantage of the particular design of the array used, groups of functionally related genes were analysed to look for particular signals in a given population. Genes related with resistance/susceptibility to malariaST statistics. For each SNP we computed the FST between each pair of groups or populations using the BioPerl module PopGenST index is correlated with heterozygosity3435ST score of those genes was compared to the sampling distribution of the average FST value of a subset of randomly selected genomic SNPs with the same sample size and similar MAF values than those of the functional categories. P values were calculated using a permutation test (10.000 permutations).For every pair of groups  we detected unusual allele frequency differentiation among them by FHow to cite this article: Dobon, B. et al. The genetics of East African populations: a Nilo-Saharan component in the African genetic landscape. Sci. Rep.5, 9996; doi: 10.1038/srep09996 (2015).Supplementary Figures 1-6