Sensitive and responsive maternal caregiving behavior strengthens infant self-regulatory capacities (HL), but this regulatory role may be diminished in some mothers with second-trimester prenatal exposure to depression and/ or anxiety (MDA). This study examined maternal and infant behavior during infant heel lance (HL) when mothers had or did not have MDA. Ethological methods and micro-analytic approaches capable of distinguishing and comparing time-based patterning in maternal and infant behavior were used to clarify biological mechanisms, such as MDA, that may underlie observed behavior. Aims were to examine group differences in caregiving behavior between mothers with and without MDA 5 min Pre-HL and 5 min Post-H, and relationships between MDA, maternal caregiving behavior and infant pain behavior self-regulation, concurrently.At second trimester, mothers were assessed for symptoms of mild-severe depression or anxiety. Mothers whose scores exceeded predetermined cut-off scores on one or more of the mental health measures were allocated to the MDA-exposure group, those below to the non-MDA-exposure group. Reliable observers, blinded to MDA status and study phases, coded video records of the caregiving behavior of each study mother for the full duration of the 5 min Pre-HL and 5 min Post-HL study phases. Group differences and associations between mean measures of maternal mental health scores, time-based measures of maternal behavior, and time-based measures of infant pain behavior regulation (previously coded) were concurrently analyzed using comparative and correlational statistics.MDA-exposed mothers spent significantly more time not embracing, engaging or responding to infant cues than maternal controls Pre-HL and Post-HL. MDA was associated with atypical maternal caregiving behavior, which in turn was related to atypical infant pain behavior self-regulation during and after the HL.Our findings have implication for practice. We recommend inclusion of mothers with MDA and their infants in interventions that strengthen the early mother-infant interaction and mother’s regulatory caregiving role. MDA and maternal caregiving behavior must be considered in future infant pain studies to examine if they confound effectiveness of mother driven caregiving interventions for neonatal pain. We highlight the importance of examining maternal mental health throughout the perinatal and postnatal trajectory, and particularly the newborn period. Sensitive and responsive caregiving actions and interactions  play a crucial role in protecting, buffering and strengthening infant ability to self-regulate to everyday stressors . This isCurrently, there are no analgesics that can be safely administered to infants to relieve procedural pain . There iGrowth in the number of maternal caregiving interventions for neonatal pain and their uptake into the clinical setting provides confidence in their reported effectiveness . While iDepression and/or anxiety during pregnancy are considered some of the most serious and concerning issues confronting pregnant women and perinatal health practitioners . Both ofSystematic review studies estimate that major depression, during pregnancy, affects up to 12.7 % of pregnant women . The preStudies that have linked maternal psychopathology with maternal caregiving have mostly involved mothers with postnatal depression. Findings show that some of these mothers exhibit a range of inconsistent or atypical caregiving behaviors such as being less or overly responsive to their infant cues, intrusive, or they withdraw from interacting with their infants . CurrentThe previous findings highlight the concerning implications of 2nd-trimester exposure to maternal depression and/or anxiety (hereafter referred to as MDA) on maternal and infant outcomes as well as pointing to the potential mediating effect of maternal caregiving behavior. As it pertains to maternal caregiving interventions for neonatal pain, improved reporting and targeted recruitment of mothers with MDA and their infants into the study will help ensure the interventions are representative. However, more research is needed to clarify the significance of MDA during the neonatal period and if it underlies atypical maternal caregiving behavior as well as neonatal pain outcomes during routine pain procedures.To our knowledge, there have only been two independent studies that have examined some of the linkages between MDA, and/or postnatal maternal caregiving behavior and/or newborn pain outcomes in full-term infants , 19. BotTaken together, findings from the two studies , 19 indiThis study addresses gaps and builds on findings of the two preceding studies , 19. TheThe first aim was to examine association and group differences in caregiving behavior between mothers with and without MDA before and following infant HL. To do this behavioral data were generated from the pre-recorded videotapes of the caregiving behavior of 24 mothers that had been collected but never coded or analyzed in the Warnock et al.  study. OThe second aim was to examine associations between maternal MDA-exposure, time-based measures of maternal caregiving behavior and the three atypical patterns of infant pain behavior self-regulation previously reported  , concurrThis was an ethological micro-analytic descriptive comparative study that made use of systematic observation methods and time-based analytic approaches. We chose these methods and approaches because unlike conventional approaches, they are capable of generating precise measures of the proportion of time (Prop-T) that an individual or group spends in a particular behavior during an observation session(s), and that can be compared . FurtherMain outcomes of the current study were Prop-T time scores of maternal caregiving behavior and infant pain behavior self-regulation for the 5 min Pre-HL, 2 min infant HL (including post-HL sub-phase), and 5 min Post-HL study phases. Prop-T scores were calculated by dividing the full duration of time a mother or infant spent in a particular behavior by the entire duration of the respective study phase. MDA was operationally defined as mild to severe symptoms of prenatal depression and/or anxiety based on mean scores of prenatal mental health measures that mothers completed at the 2nd trimester. Descriptions of maternal recruitment and data collection procedures that we provide below are summarized from Warnock et al. . This isAs reported by Warnock et al. , mothersn = 1) or because of insufficient data on maternal caregiving behavior (n = 11). Of the remaining 24 mothers, six had reported taking drugs during pregnancy (SSRIs (n = 3), crack cocaine (n = 2) or SSRI + crack cocaine (n = 1). Those six mothers were not excluded from the current analysis in recognition that in our region, 5 % of pregnant women are prescribed SSRIs antidepressant medications  than during Post-HL . HAM-A and EPDS scores also accounted for up to 38 % of the variance in atypical expression Post-HL: HAM-A , EPDS .HAM-D scores accounted for a smaller proportion of variance in atypical behavior during Pre-HL  and 27 % in immobility  during the actual HL.Results of the final simple linear regressions that involved the matched subsample of 16 mothers and their infants showed the Prop-T that mothers spent in not engaging/ responding or attending to infant’s behavioral cues during the 5 min Pre-HL phase, predicted the Prop-T that infant offspring spent exhibiting strained/erratic limb movements . The Prop-T the mothers spent in not engaging or attending Pre-HL explained up to 21 % of the variance in infant weak/strained cry post-HL subphase: .We also found the Prop-T the 16 mothers spent not engaging or attending during the 5 min Pre-HL phase, predicted the Prop-T their infants spent in weak/strained cry during the post-HL sub-phase  would predict Prop-T scores of maternal atypical caregiving behavior and that maternal atypical caregiving behavior Pre-HL would be associated with the three patterns of atypical infant pain behavior self-regulation reported previously  were botWe also found that the Prop-T that the 16 mothers in the subsample spent in not embracing and in not engaging/responding or attending to infant behavioral cues during the 5 min Pre-HL phase were associated with the Prop-T their infants spent in immobility and in strained/erratic limb movement during actual HL, and in weak/strained cry during the post- HL sub-phase. These findings extend the findings by Warnock et al.  in that The dearth of similar studies in the context of neonatal pain makes it difficult to compare our findings. Other studies that did not involve infant pain have reported linkages between prenatal maternal depression and/or anxiety and postnatal alteration in maternal caregiving behavior and later child outcomes. For example, findings have shown that prenatal anxiety predicts depressive symptoms during the postpartum  and thatThe in-depth descriptions of the types and temporal qualities of maternal caregiving behavior that were elucidated in this study are clinically relevant. Clinicians with interests in mother driven interventions for neonatal pain may use the findings to consider inclusion of mothers with MDA and their infants. These mothers may represent the 36 % of women who decline participation , and it This study has limitations that necessitate cautious interpretation of study findings. Although this basic observation study yielded foundational data based on a huge amount of time-based measures, the small subject sample size limits the generalizability of the findings. Some of the study mothers had taken antidepressant medications and/or drugs  during pregnancy. Although we controlled for these effects statistically, these mothers may exhibit a unique behavioral profile due to impacts of the drugs and chronic exposures to environmental adversity and stress. It was beyond the present study to have distinguished any behavioral differences, and this may be an area for future dedicated research. Findings were based on independent measures of prenatal depression and anxiety and the study focused on maternal and infant behavior. Targeting comorbidity at recruitment, making use of multivariate analysis and obtaining physiological and other measures  in future larger sized samples will help clarify pathways of association.Sensitive and responsive maternal caregiving behavior strengthens infant self-regulatory capacities, but this powerful regulatory role may be diminished in some women with MDA. Our findings suggest that mothers and infants with 2nd trimester MDA exposure exhibit atypical behavior during a routine infant pain event and that MDA may underlie these atypical patterning in behavior. The findings are consistent with findings of studies that have examined associations between MDA, maternal caregiving behavior that did and did not involve infant pain, and that were conducted during the postpartum. We recommend early inclusion of mothers with MDA and their infants in interventions aimed to strengthen the early mother-infant interaction and a mother’s caregiving regulatory role. MDA and maternal caregiving behavior must be considered in future infant pain studies and to examine potential confounding effects of MDA on effectiveness of mother driven interventions for neonatal pain. We highlight the importance of future research on maternal mental health throughout the trajectory of the perinatal period, particularly during the early newborn period, and on development and implementation of early strength-based interventions.


There is an unprecedented rise in the prevalence of stroke in sub-Saharan Africa (SSA). Secondary prevention guidelines recommend that antihypertensive, statin and antiplatelet therapy be initiated promptly after ischemic stroke and adhered to in a persistent fashion to achieve optimal vascular-risk reduction. However, these goals are seldom realized in routine clinical care settings in SSA due to logistical challenges.We seek to assess whether a polypill containing fixed doses of three antihypertensive agents, a statin and antiplatelet therapy taken once daily per os for 12 months among recent stroke survivors would result in carotid intimal thickness regression compared with usual care (UC).The Stroke Minimization through Additive Anti-atherosclerotic Agents in Routine Treatment (SMAART) trial is a phase 2, open-label, evaluator-blinded trial involving 120 Ghanaian recent-ischemic-stroke survivors. Using a computer-generated sequence, patients will be randomly allocated 1:1 into either the intervention arm or UC. Patients in the intervention arm will receive Polycap DS®  taken as two capsules once daily. Patients in the UC will receive separate, individual secondary preventive medications prescribed at the physician’s discretion. Both groups will be followed for 12 months to assess changes in carotid intimal thickness regression – a surrogate marker of atherosclerosis – as primary outcome measure. Secondary outcome measures include adherence to therapy, safety and tolerability, health-related quality of life, patient satisfaction, functional status, depression and cognitive dysfunction.An efficacy-suggesting SMAART trial could inform the future design of a multi-center, double-blinded, placebo-controlled, parallel-group, randomized controlled trial comparing the clinical efficacy of the polypill strategy for vascular risk moderation among stroke survivors in SSA.ClinicalTrials.gov, ID: NCT03329599. Registered on 11 February 2017.The online version of this article (10.1186/s13063-018-2564-0) contains supplementary material, which is available to authorized users. There has been an astronomical rise in the prevalence of stroke in sub-Saharan Africa (SSA) which, when compared to stroke profiles in high-income countries (HIC) is characterized by a younger age of onset, higher early and long-term fatality rates, and more severe cognitive deficits, emotional and social isolation and disability among survivors –10. StroAmong stroke survivors, a major source of subsequent mortality and functional decline is from recurrent stroke and myocardial infarction (MI) –17. PrevThe use of evidence-based therapies for vascular risk reduction among stroke patients receiving conventional care in low- and middle-income countries (LMIC) is extremely low . UsuallyFixed-dose combination pills, also known as “polypills,” containing generic drugs: aspirin, a statin and blood pressure (BP)-lowering medication(s) may be a viable low-cost avenue to broadly improve medication adherence and consequently reduce further disability or death on a large scale among stroke survivors in SSA –54. The Hence, the overarching objective of the Stroke Minimization through Additive Anti-atherosclerotic Agents in Routine Treatment (SMAART) trial is to assess whether a polypill containing fixed doses of three antihypertensive agents, a statin and antiplatelet therapy taken once daily per os for 12 months would result in carotid intimal thickness regression, improved adherence, and tolerability compared with the UC group on separate individual secondary preventive medications among Ghanaian first-time stroke survivors.SMAART is a phase II, randomized, open-label, blinded-endpoint clinical trial to evaluate the effect of a polypill taken once daily per os in improving CIMT regression and adherence to secondary risk reduction drug intake vs. a UC group with separate individual secondary preventive medications among Ghanaians with recent-onset stroke or transient ischemic attack (TIA) in a single-center study. See Fig. SMAART will be conducted at the Komfo Anokye Teaching Hospital (KATH), a tertiary-referral center and teaching hospital with 1000 beds. There is a stroke unit and a neurology outpatient service . Stroke The participants will include 120 adult Ghanaian recent stroke/TIA patients (within 2 months of stroke onset) meeting the inclusion/exclusion criteria who will be randomly assigned to either the intervention or the UC arm.Above the age of 18 years; male or femaleStroke/TIA diagnosis no longer than 2 months before enrollment. Only ischemic strokes including lacunar and large-vessel atherosclerotic subtypes are eligibleLegally competent to sign informed consentUnable to sign informed consent and having no proxyContraindications to any of the components of the polypillSevere cognitive impairment/dementia or severe global disability limiting the capacity of self-careSevere congestive cardiac failure (NYHA grades III–IV)2), renal dialysis; awaiting renal transplant, or transplant recipientSevere renal disease, estimated glomerular filtration rate (eGFR) < 30 ml/min/1.73 mCancer diagnosis or treatment in the past 2 yearsNursing/pregnant mothersParticipants not agreeing to the filing, forwarding or use of their pseudonymized data2); or prior MI.The diagnosis of stroke will be defined as an acute episode of focal cerebral, spinal or retinal dysfunction caused by infarction of central nervous system tissue, not resulting in death. Patients meeting the eligibility criteria will be allocated to the experimental or active comparator arm. Subjects with stroke may also present with at least one of the following additional conditions: documented diabetes mellitus or previous treatment with an orally administered hypoglycemic agent or insulin; documented hypertension (HT) > 140/90 mmHg or previous treatment with antihypertensive medications; mild to moderate renal dysfunction  to participants at each yearly follow-up visitParticipants will be provided with contact details of the responsible researcher so that they can make contact if for any reason they are unable to continue their study medication or have missed multiple doses and are unsure whether to continuePatients allocated to the experimental arm will receive two Polycap DS®  taken per os once a day. Polycap DS® is produced by Cadila Pharmaceuticals, Ahmedabad, India. Patients assigned to the polypill will have their antihypertensive agents, lipid modifiers and antithrombotic agents withdrawn and replaced with the polypill if they are already receiving such treatments before enrollment. Since our focus is to isolate the effect of the polypill strategy itself and create equipoise, at study inception providers for patients in both study arms will receive a brief one-off training and a one-off email synopsis on guideline-recommended biomarker targets after stroke . To maxiPatients allocated to the UC arm will receive standard-of-care therapies for secondary prevention with drugs and doses left to the discretion of the treating physicians. Measures outlined to ensure adherence to study medications will be also be deployed in the UC group.If there are clear contraindications to one or more of the antihypertensive agents – ACEI, beta-blocker or thiazide; antithrombotic agent or lipid modifiers due to side effects, the polypill will be stopped and an open-label treatment with other components of the polypill excluding the class(es) for which the contraindication has developedIf there are symptoms of severe hypotension, one approach will be to stop the polypill, introduce the individual components gradually at reduced doses and consider re-starting at a later dateIndication for a high dose of a particular agent – the agent will be added as open label without the need to unblindThe following strategies will be employed for patients with side effects deemed due to study medication and sufficiently severe to warrant a change of treatment:Participants who have had the study drug discontinued for any reason other than the above should be encouraged to restart the study drug as soon as practically and medically appropriate at the discretion of the investigator.Serious adverse events (SAEs) which are, in the opinion of the investigator, related to the study treatmentThe investigator feels it is in the subject’s best interestThe investigator must not deviate from the protocol except when there is a contraindication due to the patient’s condition or the patient is intolerant of the drugs. In these cases the study treatment should be discontinued. Study treatment should also be discontinued if any of the following occurs:In the event of discontinuation of treatment, the patient should still remain in follow-up and attend study visits as scheduled. Physicians would then prescribe other medications for risk factor control and reasons for discontinuation documented.Other medications deemed necessary for the management of other comorbidities, such as diabetes mellitus, will be permitted.A total number of 120 patients will be randomized (1:1) to treatment arms. Patients with stroke meeting the inclusion/exclusion criteria will be recruited from the Family Medicine, Internal Medicine and Neurology Outpatient Clinics at KATH. Randomization will take place within 8 weeks of the index event (ischemic stroke) in a 1:1 ratio to one of the two arms: polypill versus UC.Randomization of subjects in blocks of 4 will be conducted by a statistician using a computer-generated random sequence of numbers. Consented participants will be assigned to either study arm at the baseline visit using the computer-generated randomization sequence. The randomization sequence uses a minimization algorithm to ensure balance of prognostic factors such as stroke severity, an average of three recorded baseline SBP measurements, and whether or not they are taking background BP-lowering agents, lipid modifiers and antithrombotic agents. Each sequence generated will be kept concealed in an envelope which will be opened by the research coordinator in the presence of the consenting study participant at enrollment.Physicians and sonographers who will be assessing primary outcomes and research assistants assessing feasibility and secondary outcomes will remain blinded as to patients’ group status throughout the study.All potentially eligible participants referred to, or who contact the study team directly, will undergo screening to determine their eligibility using the pre-specified criteria.C will be collected and contraindications for study medications assessed. All concomitant medications will be recorded in the Case Report Form.Information on stroke type from a cranial computed tomography (CT) scan performed within 10 days of stroke symptom onset will be reviewed by the principal investigator (PI) (FSS); stroke subtype information where available will be sought to classify ischemic stroke using the TOAST classification  into carParticipants will be followed for 12 months with scheduled visits at months 1, 3, 6, 9 and 12 for clinical assessments and primary, secondary and tertiary/feasibility study outcome evaluations as shown in Table The primary endpoint will be CIMT burden. Each study participant will undergo carotid Doppler ultrasonic evaluation at baseline and month 12 for evidence of clinical or sub-clinical carotid artery disease – a validated surrogate marker of atherosclerosis . To achiThese will include (1) medication adherence indicators assessed using the self-reported Morisky-Green Questionnaire, pill count and medication possession ratio; (2) safety and tolerability indicators such as changes in renal and liver function tests, side effects and treatment discontinuations; (3) health-related quality of life; (4) patient satisfaction; (5) changes in cognitive function; (6) depression and (7) functional status.These will include CVD risk-factor-control indices, such as change in mean SBP between baseline and month 12, as well as changes in mean LDL-C from baseline to month 12. Major adverse cardiovascular events, such as recurrent stroke, MI, CVD-related deaths and all-cause mortality, are included as tertiary outcome measures.Participants who experience side effects will be reviewed by their physicians to assess severity and appropriate measures instituted.t test. The rate of change in common CIMT in treated patients is around 0.085 mm/year with a standard deviation of 0.035 [The end-of-study CIMT value will be subtracted from the baseline CIMT value and divided by the length of follow-up. The rate of change in CIMT (mm/year) between treatment arms will be tested with a two-sided of 0.035 . We assuAll data entry will be collected onto a Case Record Form (CRF) and doubly entered into a password-protected, validated, encrypted study electronic CRF on REDCap. This web-based data management system will allow for real-time data query generation for values entered outside of pre-set valid ranges and consistency checking. This system will speed up data reporting and assist overall trial management. The Internet-based data management system is managed at the Kwame Nkrumah University of Science and Technology (KNUST) and hosted by the Medical University of South Carolina server, which has extensive experience in clinical trial data capture and security. Data entry will be performed at the KNUST/KATH using a password-protected, encrypted HTTPS connection. Only staff listed in the delegation log will be given the unique individual password to access the Internet-based data management system. The database will undergo logic checks to ensure that data are entered in mandatory fields and undergo value-range checks to ensure accuracy and to reduce the chance of missing data. Reports and data query management will also be included in the system to assist with centralized online monitoring by the data manager and statistician.Prior to addressing each hypothesis, univariate descriptive statistics and frequency distributions will be calculated as appropriate for all biological variables  comparing individuals by treatment arms (polypill arm vs. UC arm). Briefly, box plots will be used to examine the relative distribution of variables stratified by treatment arm. Non-parametric and equivalent parametric statistics will be utilized to compare groups. Appropriate regression models  will be used to estimate the association of covariates with each outcome. When building models for each specific aim, the first stage of the model building algorithm will involve testing whether the individual covariates are correlated with the main outcome variables. A liberal alpha = 0.20 will be used for these unadjusted analyses . Once thA DSMB, comprised of three experienced external experts, will meet twice per year to review the safety, ethics and outcomes of the study. The responsibilities of the DSMB will include monitoring blinded response variables and safety outcomes for early dramatic benefits or potential harmful effects and provide reports on recommendations to continue or temporarily halt recruitment to the study. The DSMB will be governed by a charter that will outline their responsibilities, procedures and confidentiality, and will review unblinded data from the study at regular intervals during follow-up and monitor BP differences between the two groups, dropout rates and event rates. The first meeting will be held within 3–6 months after the start of the study recruitment. One or two formal interim analysis will be planned to review data relating to patient safety and quality of trial conduct.All SAEs and adverse events of special interest (AESI) experienced by a participant after the informed consent document is signed and until the end of the study will be collected and reported to the Institutional Review Board and applicable regulatory guidelines. If an SAE is unresolved at the conclusion of the study, a clinical assessment will be made by the medical monitor as to whether continued follow-up of the SAE is warranted.The overarching objective of the proposed SMAART trial is to assess whether a polypill containing fixed doses of three antihypertensive agents, a statin and antiplatelet therapy taken once daily per os would result in carotid intimal thickness regression, improved adherence and tolerability among first-time stroke survivors in SSA. The ultimate objective would be the design of a future multi-center, double-blinded, placebo-controlled, parallel-group randomized controlled trial (RCT) comparing the clinical efficacy of the polypill strategy vs. UC in the African context to derive locally relevant, high-quality evidence for routine deployment of polypill for CVD risk moderation among stroke survivors in LMIC. There are six major RCTs on polypills currently on-going or planned, three of these RCTs – TIPS-3 , HOPE-3 Subject accrual and retention: multiple tactics will be used to facilitate high patient retention including obtaining full contact information for patients, caregivers and relatives at time of enrollment and updating every 3 months. Transportation support will be provided when needed and follow-up visits will be scheduled, whenever possible, on days that subjects already have scheduled clinic visitsMeeting sample size: there is a high case load of stroke patients with approximately 700 cases per year. At a recruitment rate of five cases per week, recruitment of 120 stroke patients meeting eligibility criteria is a realistic objectiveMissing data: we anticipate that there may be missing data due to loss to follow-up but we will minimize this by home visits where geographically feasible, providing transportation stipends and employing statistical methodology to account for missing dataWe anticipate the following potential challenges:Recruitment is anticipated to begin in April 2018.Additional file 1:SPIRIT 2013 Checklist: recommended items to address in a clinical trial protocol and related documents. (DOC 121 kb)


Thus we used the synthetic biology approach to create theophylline controllable RNAi-based genetic switches which silenced TINCR in a dosage-dependent manner. Both RNAi-OFF and ON switches can be used to quantitatively control the expression of TINCR in bladder cancer to suppress the progression of bladder cancer. These findings suggest that lncRNA-TINCR could promote bladder cancer development and progression and artificial control of its expression through inducible RNAi may represent a new kind of therapeutic strategy for treating human bladder cancer.TINCR is a well-known lncRNA which acts as a master regulator in somatic differentiation development. However, it is still unclear whether TINCR is also involved in caner occurrence and progression. In this study, we observed that TINCR was up-regulated in bladder cancer tissues and cells and contributed to oncogenesis and cancer progression. Silencing TINCR expression inhibited cell proliferation and promoted apoptosis  The completion of the ENCODE project led to the staggering discovery that >80% of genome is transcribed into RNAs, whereas <3% is translated into proteins15689Bladder cancer (BCa) is the most common urologic malignancy with high prevalence and recurrence rates121516Riboswitches are familiar as “functional noncoding RNA domains” that control associated gene expression by selectively binding metabolites and are involved in crucially regulatory networks at the transcriptional or post-transcriptional level in numerous types of bacteria2021222425in vitro. Theo-RNAi switches could be used to quantitatively control the expression of TINCR in BCa cell to suppress the progression of BCa.Based on the engineering principles of medical synthetic biology, here, we presented the novel RNA regulators (theo-RNAi switches) which activate or inactivate the RNAi pathway to control TINCR expression in a theophylline dosage-dependent manner. In this study, we demonstrate that TINCR is up-regulated in BCa tissues and cell lines (SW780 and 5637) and contributes to oncogenesis and cancer progression. Silencing TINCR expression inhibits cell proliferation and promotes apoptosis The relative expression level of TINCR was measured by real-time qPCR in 49 pair-matched specimens obtained from patients with BCa and 2 BCa cell lines. Overexpression of TINCR was observed in BCa tissues compared with paired non-cancerous tissues . AdditioTo test the hypothesis that TINCR functions as an oncogene in BCa, we down-regulated endogenous TINCR expression in 2 BCa cell lines 5637 and SW780 using chemically synthesized siRNAs. To ensure the RNAi efficiency in target regulation, we synthesized two different siRNAs (si-TINCRs A and B) according to the former studyWe further determined whether TINCR promotes cell proliferation in BCa. After cells were transiently transfected with si-TINCR and si-NC, EdU assay  and CCK-To further expand on this issue and to more precisely control the expression of TINCR, we set out to construct the theo-RNAi switches targeting TINCR based on the engineering principles of synthetic biology. The theo-RNAi switches can be turned off or turned on through binding of theophylline.The first and foremost, we constructed the theo-dependent RNAi-OFF switch that transcribed a pre- artificial miRNA in which the RNA aptamer for theophylline was inserted into the loop region of the pre- miRNA scaffold. The stem region of this scaffold was the sequence that encoded mature artificial miRNA targeting TINCR. The design for fusing RNA aptamer was inspired by the observations that some ligand regulators can selectively recognize the terminal loops of miRNA precursors, prevent the cleavage processes from Microprocessor complex and inhibit the generation of mature miRNA sequence27Then we constructed the theo-dependent RNAi-ON switch by connecting the RNAi-OFF switch and a shRNA targeting TINCR. The mature miRNA was designed to pair with loop region of the shRNA scaffold. This design was also inspired by the stereo-hindrance effect, where the existence of a miRNA binding site in a pre-shRNA region suppressed the processing of the transcript. So in the RNAi-ON switch, the RNAi activation and TINCR repression would be induced by the addition of theophylline .At last, we developed mathematical models to inform the design of two devices to elucidate important parameters. The models indicated the relationships between the theophylline concentration and lncRNA knockdown efficiency using a series of differential equations describing the each process. The model predicted that increasing the input concentration would lead to substantial increases in the change in target gene expression, and that the relationship was nonlinear and saturable, which were the key features of many biological phenomena.To test whether the behaviors of the switches were consistent with theory predictions, we set up various ranges of theophylline concentrations according to relative researches202122To evaluate the utility of theo-RNAi switches in BCa cells, we repeated the functional assays. BCa cells 5637 and SW780 cells were transfected with theo-RNAi switches and cultured at 0 or 2 mM theophylline, respectively.Using RNAi-OFF switch, the results of EdU assay  and CCK8Oppositely, the expression of TINCR can be “switched off” at 2 mM theophylline by RNAi-ON switch. EdU assay  and CCK8An emerging revolution has been sparked in the lncRNA field due to the novel technologies. In almost all aspects of biology, lncRNAs have served as critical regulators2931323436In this study, we found that TINCR expression was significantly up-regulated in BCa tissues and cells. Also, patients with higher TINCR levels presented the higher tumor stage. Thus, we highly speculated TINCR may also function in BCa. To further test this hypothesis, a series of functional tests were performed in 2 BCa cell lines 5637 and SW780. More assays than one were taken in every aspect in order to reduce errors. CCK8 assays and EdU assays revealed that silencing TINCR expression contributed to significant inhibition of BCa cell proliferation. Flow cytometric assay and ELISA assay demonstrated that knockdown of TINCR expression led to promotion of BCa cell apoptosis. Taken together, our data revealed that TINCR might serve as a potential oncogene in BCa. Unfortunately, the further explorations of the underlying biological mechanisms haven’t been done due to the restriction of experimental conditions.With the remarkable development of synthetic biology, the therapeutic strategies of diseases have been improved with each passing day394022232223In conclusion, our findings demonstrated that knockdown of lncRNA-TINCR could inhibit the malignant phenotypes of bladder cancer cells, and TINCR may be used as a potential therapeutic target for bladder cancer. Besides, the theo-RNAi-TINCR switches present a specific useful tool to inhibit un-controlled growth of BCa cells by controlling the expression of TINCR.Tumor samples were collected during the surgery, and non-tumor samples were cut at least 5 cm away from the tumors. A total of 49 paired BCa tissues and corresponding noncancerous tissues were included in this study. The methods were carried out in accordance with the approved guidelines. Written informed consent was obtained from all subjects. All experimental protocols were approved by the Research Ethics Committee of Shenzhen Second People’s Hospital.2 maintained at 37 °C. All the media were also supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.BCa cells  and SV-40-immortalized human uroepithelial cell line (SV-HUC-1) were obtained from the Institute of Cell Research, Chinese Academic of Sciences, Shanghai, China. 5637 cells were maintained in RPMI-1640 media; SW780 cells were maintained in DMEM media; SV-HUC-1 cells were maintained in F12K medium in a humidied atmosphere of 5% COAll siRNAs and plasmids involved in this study were synthesized by GenePharma, Shanghai, China. The sequences of siRNA oligonucleotides were listed as follow: si-TINCR A (sense sequence): 5′-GCAUGAAGUAGCAGGUAUUUU-3′; si-TINCR B (sense sequence):5′-GAUCCCGAGUGAGUCAGAAUU-3′; si-NC (sense sequence): 5′-UUCUCCGACGUGUCACGUTT-3′. The sequences of the theo-RNAi switches and the negative control system were designed and chemically synthesized. All these related sequences were inserted into siCHECK™-2 vector at the restriction sites of Xhol/Notl and pGPU6-GFP-Neo at the restriction sites of BbsI/BamHI, respectively. Cells were seeded in the plates till they reached 50–70% confluency and transiently transfected with the mixtures of siRNAs or plasmids using Lipofectamine® 3000 (Life Technologies) according to the manufacturer’s instructions.−ΔCt method was used to calculate the relative amount of TINCR compared with GAPDH expression.Total RNA from tissues or cells was isolated with Trizol reagent (Life Technologies) and quantified by NanoDrop according to the manufacturer’s instructions. cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara) according to the manufacturer’s instructions. Real-time qPCR was performed using the SYBR Premix Ex Taq II (Takara) with the follow primers. TINCR primers forward: 5′-TGTGGCCCAAACTCAGGGATACAT-3′, reverse: 5′-AGATGAC AGTGGCTGGAGTTGTCA-3′; GAPDH primers forward: 5′-CGCTCTCTGCTCCTCCTGTTC-3′, reverse: 5′-ATCCGTTGACTCCGACCTTCAC-3′. The 2et al.Cell proliferation assays were performed by the Cell Counting Kit-8, CCK-8 (Beyotime Institute of Biotechnologya) and ethynyl-2-deoxyuridine, EdU kit (Ribobio) according to the manufacturer’s instructions. Both of the two experiments were performed as previously reported in Zhan et al.Cell apoptosis assays were detected by the flow cytometry and the caspase-3 ELISA kit (Cusabio) according to the manufacturer’s protocols. Both of the two experiments were also performed as previously reported in Zhan All statistical analyses were executed by using SPSS 22.0 software (IBM). Data were presented as mean ± SD, except for How to cite this article: Chen, Z. et al. Theophylline controllable RNAi-based genetic switches regulate expression of lncRNA TINCR and malignant phenotypes in bladder cancer cells. Sci. Rep.6, 30798; doi: 10.1038/srep30798 (2016).


The photoluminescence (PL) characteristics indicate that there exists an optimal location with superior crystalline quality in the CIGS thin films obtained by one-step selenization. The energy dispersive spectroscopy (EDS) reveals that the Ga lateral distribution in the one-step selenized CIGS thin film is intimately correlated to the blue-shifted PL spectra. The surface morphologies examined by scanning electron microscope (SEM) further suggested that voids and binary phase commonly existing in CIGS films could be successfully eliminated by the present one-step selenization process. The agglomeration phenomenon attributable to the formation of MoSe2 layer was also observed. Due to the significant microstructural improvement, the current–voltage (J-V) characteristics and external quantum efficiency (EQE) of the devices made of the present CIGS films have exhibited the remarkable carrier transportation characteristics and photon utilization at the optimal location, resulting in a high conversion efficiency of 11.28%. Correlations between the defect states and device performance of the one-step selenized CIGS thin film were convincingly delineated by femtosecond pump-probe spectroscopy.In this work, aiming at developing a rapid and environmental-friendly process for fabricating CuIn CIGS possesses superior optical properties such as high absorption coefficient (α ~ 105 cm−1), long-term stability, and high radiation tolerance, due to that it belongs to the family of direct band gap chalcopyrite semiconductor compounds. Moreover, its band gap can be engineered by the partial substitution of indium by gallium, which further renders the flexibility of manipulating the optical absorption , with τ1 and τ2 being related to hot carrier relaxation and defect-related recombination, respectively. It is apparent from the results that the longest defect-related carrier lifetime (τ2) is obtained at location of P10, suggesting the minimum density of defect states in this region [τ2) are further compared with conversion efficiency in Fig. In order to investigate the defects existing in the one-step selenized CIGS thin film, the femtosecond pump-probe spectroscopy measurement was carried out, which is a powerful technique to delineate non-equilibrium carrier dynamics in the present CIGS films , 18. Upos region , which i2Se can meet the demand of high efficiency and environmental friendly method for preparing high-quality CIGS thin films. The photoluminescence intensity mapping image obtained from the one-step selenized CIGS thin film suggested that there exists an optimal location (P10) for obtaining CIGS film with superior crystalline quality. The blue-shifted photoluminescence spectra obtained from the regions at P5 and P10 are consistent with the lateral distribution of Ga element as revealed by energy dispersive spectroscopy (EDS). According to the surface morphologies examined by scanning electron microscope, it is evident that the binary phase and voids of one-step selenized CIGS film can be eliminated at the P10, with agglomeration of grain and the formation of a MoSe2 layer. Consequently, the current-voltage characteristics and external quantum efficiency of the devices made of film regions near P10 exhibited characteristics of highly efficient carrier transportation and remarkable photon utilization, which are intimately related to the improvements of grain structures and stoichiometric composition. Finally, the femtosecond pump-probe spectroscopy studies unambiguously revealed the close correlations between the defect-related carrier lifetime, density of defect states, and the conversion efficiency of the devices. The present results indicate that the one-step selenization process might serve as a viable avenue for developing the high-efficiency CIGS solar cells.In summary, we have successfully demonstrated that the one-step selenization process using selenium vapor instead of H


Neurodegenerative diseases are a clinically heterogeneous group of disorders characterized by the progressive degeneration of neurons in both central nervous system (CNS) and peripheral nervous system (PNS), among which microglia-mediated neuroinflammation is one of the shared prominent features. Microglial polarization appears to be heterogeneous with diverse functional phenotypes that range from pro-inflammatory M1 phenotypes to immunosuppressive M2 phenotypes [reviewed in Tang and Le ]. Given First, Subramaniam and Federoff provided a review on the molecules and signaling that modulate microglial polarization states in animal models and clinical trials of Parkinson's disease (PD), which supports targeting of microglial phenotypes as a novel therapeutic approach for PD. Porrini et al. used the nuclear factor-κB (NF-κB)/c-Rel deficient mice to investigate the dynamics of microglial polarization and mild inflammatory profiles that contribute to modeling late-onset Parkinsonism. Two original articles then focused on the potential effects of microglia phenotypes in Alzheimer's disease (AD). Hypoxia as a pivotal environmental factor has been demonstrated to aggravate AD via exacerbating Aβ and tau pathologies. Zhang et al. figured out that acute hypoxia promoted M1 polarization but suppressed M2 polarization in both AD transgenic mice and their wild type littermates, the process of which was associated with NF-κB induction through toll-like receptor 4 (TLR4). This study thus indicates the pathological role of hypoxia in the immunopathogenesis of AD. Mutations of DNAX-activating protein of 12 kDa (DAP12) and triggering receptors expressed on myeloid cell 2 (TREM2) have been risk genes for AD. In the study by Zhong et al. they demonstrated that TREM2/DAP12 complex suppressed the activity of JNK signaling to suppress lipopolysaccharides (LPS)-triggered M1 pro-inflammatory responses, thereby executing anti-inflammatory functions. Geloso et al. then analyzed the features and timing of M1/M2 microglial polarization in the transgenic mice of amyotrophic lateral sclerosis (ALS), and reviewed the preclinical approaches that may affect microglia phenotypes. Similarly, Yang et al. summarized current knowledge about M1/M2 microglial polarization and their corresponding signaling pathways in the pathogenesis and progression of Huntington's disease (HD).Traumatic brain injury (TBI) can also trigger neurodegeneration and is a major risk factor for the development of dementia. Microglial polarization following TBI may contribute to the restoration of homeostasis in the brain. The study by Donat et al. reviewed the effects of microglia in TBI, including the analysis of their distribution, morphology and functional phenotypes over time in multiple experimental animal models and in humans. This may give an insight into the time course of microglial polarization at the injury site and the spreading of inflammation to other brain areas. It reminds us that ameliorating the inflamed milieu would be required to facilitate neuron rescue at the injury site and achieve long-term protection and recovery in TBI. And this is also applied for ischemic stroke. Liu et al. investigated the neuroprotective functions of dietary phytochemical curcumin in experimental stroke models, and demonstrated that curcumin post-treatment has a profound regulatory effect on microglial responses over time, promoting M2 polarization and suppressing pro-inflammation.Sc. The cytokine profile is associated with both pro- and anti-inflammatory reactions, suggestive of a mixed inflammatory profile in the prion-diseased brain. The fact that different prion strains, disease stages, and detection techniques are also added into the complexity of inflammatory profiles in prion diseases.As an extension of the brain, retina is also affected in several neurodegenerative diseases. Ramirez et al. described the contribution in retina to microglia polarization in AD, PD, and glaucomatous neurodegeneration. Notably, those neuroinflammation in the retinal tissue might be of great use for the early diagnosis and monitoring of neurodegeneration. Obst et al. then reviewed the current understandings on the role of microglia in the pathogenesis of prion disease, which is a chronic neurodegenerative disorder characterized by the accumulation of scrapie prion protein PrPDespite the simplified division of M1/M2 functionally polarization, modulation of microglial phenotypes has become a potential therapeutic approach for the treatment of neurodegenerative diseases. In light of this, Song and Suk reviewed recent studies of pharmacological modulators, especially the small-molecule compounds and their intracellular target proteins that participate in microglial polarization. Moreover, Pena-Altamira et al. also focused on nutritional approaches by the intake of food bioactive compounds such as carotenoids, phytosterols, and so forth, which may intervene microglial polarization, contributing to neuron survival and therefore improve cognitive aging impairment. Specifically, Roser et al. described how the inhibition of Rho kinase (ROCK) pathway can induce the microglia phenotype towards the beneficial “M2” and explored ROCK inhibition as a promising treatment option for PD and ALS. Atypical chemokine receptors (ACKRs), which belong to a small subset of chemokine receptors, were shown to limit inflammation through chemokine scavenge at the inflammatory sites. The review by Salvi et al. summarized the potential role of the ACKRs in neuroinflammation, particularly focusing on the microglial polarization and function. Moreover, Labandeira-Garcia et al. described the effects of the brain renin-angiotensin system (RAS) on microglial polarization. Angiotensin II (Ang II) acts through its type 1 receptor (AT1), leading to the NADPH-oxidase complex activation and giving rise to pro-oxidative and pro-inflammatory effects. In the opposite way, these effects are counteracted by Ang II/AT2 receptor signaling and Angiotensin 1-7/Mas receptor (MasR) signaling, which produce anti-inflammatory effects and promote M2 polarization.It then comes to the technology section of studying microglia functions. The advancement of experimental cell-specific depletion methods in recent years has greatly boosted the microglial research. In the study by Lund et al. they reviewed and compared the different depletion approaches that have been used to eliminate microglia or monocyte-derived macrophages in a range of neurodegenerative disease such as ALS and AD, and discussed their prospects for immunotherapy. In the technology report by Noristani et al. the authors combined magnetic resonance imaging (MRI) and multiphoton to study microglia/monocytes alterations after spinal cord injury (SCI). Ex vivo diffusion MRI allows detailed examination of CNS tissues, while advances in clearing procedures allow detailed imaging of fluorescent-labeled microglia/monocytes at high resolution. Another critical method for studying microglia is definitely transcriptomes. Increasing studies have investigated microglial transcriptome remodeling over the course of pathogenesis and figured out the microglial response diversity to different diseases. Hirbec et al. therefore critically reviewed and compared the different methods developed to isolate microglia, and decipher microglial transcriptomes as well, including RNA-Seq and microarray, in multiple neuropathological conditions.Aging is one of the most important risk factors for the onset and progression of neurodegenerative diseases, during which age-dependent and senescence-driven microglia function impairments have played essential roles. Spittau summarized the current knowledge of microglia phenotypes and functions in aged CNS, and also discussed the implications of age-related changes in microglia functions for the development of PD and AD. Specifically, Caldeira et al. investigated key-aging associated responses in microglia stimulated by Aβ in cell culture, with a reduced phagocytosis, migration and lower expression of inflammatory miRNAs over time. This study thus manifests the heterogeneous microglial responses along the progression of AD, and improves our understanding that therapeutic targeting microglia might differ from early to late stages. What is more, the crosstalk among glia, neural progenitor cells, and neurons also becomes dysfunctional during aging, along with harmful effects to dopaminergic neuron plasticity and repair. As reviewed by L'Episcopo et al. the aging-induced M1 pro-inflammatory responses disrupt the subventricular zone (SVZ) plasticity via PI3K-Wnt/β-catenin dysregulation, thereby impairing the neurogenic process.The study by Walker et al. investigated the potential functions of colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34) in microglia derived from human autopsy AD brains. They showed that IL-34 induced primarily M1 responses similar to that of CSF-1, which is contrary to that in earlier work with rodent microglia. This discrepancy might come from either species difference or age difference, but it indeed reminds us that further studies shall also make the most use of human subjects to recapitulate the real AD pathology. Finally, Au and Ma described another type of microglia, bipolar/rod-shaped microglia, which is previously less studied mainly due to the lack of a well-established in vitro culture system. The spatial arrangement of bipolar/rod-shaped microglia is important for the reorganization and remodeling of neuronal and synaptic circuitry after CNS injuries. They then discussed the potential neuroprotective roles of bipolar/rod-shaped microglia, and the possibility of transforming ramified/amoeboid microglia into bipolar/rod-shaped microglia to promote CNS repair. This study will broaden our knowledge on microglia polarization at a very different angle.in vivo. With the advent of single-cell RNA-Seq, studies have shown that microglial transcriptome profiles are varied and context dependent, with regard to microglial polarization . When dOverall, this collection is plenty of interesting articles that brought comprehensive understandings of microglial polarization in a range of neurodegenerative diseases, linking with aging-related alterations in microglia and technologies developed for microglial studies. We hope that this topic would give exciting insights into therapeutic approaches in the treatment of neurodegeneration, through targeting microglial polarization.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Progress made in peptide-based vaccinations to induce T-cell-dependent immune responses against cancer has invigorated the search for optimal vaccine modalities. Design of new vaccine strategies intrinsically depends on the knowledge of antigen handling and optimal epitope presentation in both major histocompatibility complex class I and -II molecules by professional antigen-presenting cells to induce robust CD8 and CD4 T-cell responses. Although there is a steady increase in the understanding of the underlying mechanisms that bridges innate and adaptive immunology, many questions remain to be answered. Moreover, we are in the early stage of exploiting this knowledge to clinical advantage. Several adaptations of peptide-based vaccines like peptide-adjuvant conjugates have been explored and showed beneficial outcomes in preclinical models; but in the clinical trials conducted so far, mixed results were obtained. A major limiting factor to unravel antigen handling mechanistically is the lack of tools to efficiently track peptide vaccines at the molecular and (sub)cellular level. In this mini-review, we will discuss options to develop molecular tools for improving, as well as studying, peptide-based vaccines. Recent breakthroughs in immunotherapy of cancer have unveiled that clinical responses correlate with activation and expansion of tumor-specific T lymphocytes that mostly target mutation-based neo-antigens –6. Alongin vivo processing routes of peptides is based on murine models and little data are available in humans. Additionally, the sequence and amino acid composition may alter the physical properties and immunological behavior of individual peptides. Therefore, mechanisms of intracellular routing and processing of administered peptides in APC require in depth examination.Immunogenicity of synthetic peptide-based vaccines can be significantly influenced by the mode of delivery –17. For The processing machineries for peptide loading in MHC class I or -II for presentation is characterized by distinct protease systems. While MHC class I processing pathways involves cytosolic proteasomes, peptidases and ER-resident trimming aminopeptidases, MHC class II peptide production takes place in endolysomal compartments involving cathepsin-like proteases , 22. Thein vivo.This mini-review encompasses our current knowledge of peptide-based vaccine modalities, the possibilities to properly target them through defined alterations, novel options for rational design, and the development of (bio)chemical visualization tools to improve our understanding of peptide-based vaccine behavior Peptide vaccination is based on the biological concept that induction of a T-cell response relies on the specificity of the T-cell receptor to recognize a presented oligopeptide-epitope. This epitope corresponds to only a fraction of the entire protein (polypeptide) antigen. Therefore, to initiate a T-cell response against a specific protein, a vaccine essentially needs to include only the minimal immunogenic peptide sequence which can be produced synthetically.Vaccination with minimal epitopes in form of synthetic peptides was shown to raise antigen-specific T-cell responses  and reprin vivo studies have revealed that, rather than the exact epitope, peptides consisting of a termini-extended sequence (long peptides) promotes higher quality T-cell responses , such as C-type lectin- (CLR), toll-like- (TLR), and NOD-like-receptors. These receptors are highly expressed by professional APCs and are essential for pathogen sensing and immune-stimulation. Different ligands have been identified for these receptors which can be employed for targeting and immune-stimulation. This approach can also modulate the internalization routing of endocytosed antigen . For exa in vivo .3CSK4 have been shown to strongly improve T-cell priming in vivo thanks to the combined effect of increased uptake of long peptides and co-delivery with the immune-stimulatory signal . This represents a promising platform for potentiating neo-epitope-based personalized peptide vaccines.A second approach that has resulted in improved T-cell activation has been the direct conjugation of long peptides to TLR ligands. TLR-mediated trafficking was described to impact antigen presentation. A study shows that the presence of antigen and TLR ligand in the same endosomes determines entrance to the presentation pathways, suggesting that TLRs or other PRRs might have an important role in determining efficient presentation after antigen uptake . Conjugay signal , 50. Fur cancers , 50 presentation of antigens. Peptide-based vaccination studies using antigen-bound fluorophores show co-localization with endosomal markers in DC, which correlated with a robust antigen-specific T-cell immune response. However, the strong influence of the relatively large fluorophore on the physico-chemical properties of the antigenic peptide to gain trustworthy physiological information and the limitations to detect peptide intermediates makes interpretations of this complex process difficult. To unravel how activation of APCs orchestrate molecular and cellular mechanisms of antigen processing and presentation operate Several technologies to visualize antigens in APCs, other than using T-cell readouts, have been developed in the last decades. Most of these have relied on tracking the activity of an enzyme through a cell. Examples of enzymes used for this are ß-lactamase, luciferase, and horseradish peroxidase. Using these approaches, the endocytic compartments involved in (cross-)presentation could be observed, as well as the cytosolic location of proteins during this event –90. Fluob-SIINFEKL pMHC complex antibody 25-D1.16 presentation: during antigen presentation any protein must be degraded into peptides to allow for its MHC-loading. As enzyme activities are reliant on largely intact proteins, this means that later stages of the pathway will be invisible using this approach. The use of fluorophore-modified antigens partially solves this by making the detectable signal independent of the intactness of the protein. However, the physicochemical properties of fluorophores must also be considered. Due to their bulky and hydrophobic structure compared with relatively small peptides, fluorophores could strongly influence the behavior of the antigenic peptide and mask epitope residues as well as proteolytic cleavage sites. Moreover, the size of conjugated fluorophores may hamper these peptides to pass through the proteasomal α-annulus of several ångström wide . Similar25-D1.16 ], translbioorthogonal chemical group—a chemical group which can be selectively ligated within the context of the living cell or organism—to form a covalent linkage to a detectable group after the biological time course has been completed. It is relatively easy (in Escherichia coli) to produce bioorthogonally labeled recombinant proteins (in vivo fate of chemically defined peptide vaccines. By ex vivo secondary staining of relevant cell types or tissues using fluorescent microscopy or histological analysis, the presence and location of the peptide vaccine can be determined. This could be valuable information to improve peptide vaccine design.One field of chemistry of which we are currently exploring the potential is click chemistry . This typroteins –97 usingproteins , 99. Thiproteins  to allowproteins –103. Oncin vivo. A major limitation to this understanding is the lack of techniques that allow the study of late stages of antigen processing, and presentation on a cellular and molecular level. Fundamental questions about transfer of peptides within and between cells are currently troublesome since tags or fluorophores are lost and prone to altering essential physicochemical properties due to their bulkiness. The introduction of novel types of chemistry may in future circumvent these problems, which in turn may lead to novel insight in the complex cellular and molecular interactions in immune response induction.Our current knowledge on innate and adaptive immune system allows us to design molecularly well-defined vaccine moieties. Adjuvant molecules that bind PRR can be synthetically coupled to antigenic peptide sequences. Even though these defined peptide vaccines have strong vaccination capacity, the mechanisms underlying these improvements are only understood to a basal level. To improve the design of peptide-based vaccines, we need to better our understanding of chemically altered vaccines on the events unfolding during vaccination BH and ET have contributed equally to the manuscript in writing and figure design. SK and FO have been responsible for scientific input and revisions of the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The feasibility of chronic uneventful LP retrievals, if confirmed by subsequent trials or follow-up studies, will demonstrate that there is an effective end-of-life device strategy, which makes LP therapy a viable alternative to standard lead-based pacing. Considering the increasing incidence of patients in whom the Nanostim battery fails to meet their projected longevity, this information is highly relevant for all physicians implanting these devices.The potential inability to retrieve chronically implanted leadless pacemakers (LP) at end-of-life limits the application of this novel technology. Data on safe long-term LP retrieval is lacking, yet is of paramount importance. We present the case of an 80-year-old patient in whom a Nanostim LP was successfully implanted for chronic atrial fibrillation with symptomatic slow ventricular response in January 2013. Background history was notable for rheumatic aortic and mitral stenosis and status post mechanical aortic and mitral valve replacement. At a regular follow-up visit 4 years and 9 months after LP implantation, the initial normal communication with the device was lost during impedance measurement, a known trigger for battery failure. The patient had 73% right ventricular pacing. Strategies to replace LPs reaching end of service remain an unsettled concern. Suggested strategies comprise the following: placing an additional leadless device adjacent to the LP or retrieving the non-functioning LP and subsequently implanting a new device. We decided to extract the LP in order to limit the amount of non-functioning intracardiac hardware and mitigate the potential device-device interference and unknown long-term risks of multiple intracardiac devices. In addition, retrieval may result in a more accessible right ventricle (RV) in case re-implantation of a new device is indicated. The procedure was performed in the catheterization laboratory under fluoroscopic guidance and local anesthesia


Zero index materials where sound propagates without phase variation, holds a great potential for wavefront and dispersion engineering. Recently explored electromagnetic double zero index metamaterials consist of periodic scatterers whose refractive index is significantly larger than that of the surrounding medium. This requirement is fundamentally challenging for airborne acoustics because the sound speed  in air is among the slowest. Here, we report the first experimental realization of an impedance matched acoustic double zero refractive index metamaterial induced by a Dirac-like cone at the Brillouin zone centre. This is achieved in a two-dimensional waveguide with periodically varying air channel that modulates the effective phase velocity of a high-order waveguide mode. Using such a zero-index medium, we demonstrated acoustic wave collimation emitted from a point source. For the first time, we experimentally confirm the existence of the Dirac-like cone at the Brillouin zone centre. et al. utilize the Dirac-like dispersion in a double-zero-index material to overcome this problem and to collimate sound.Impedance mismatch between acoustic metamaterials and a surrounding medium hinders efficient applications, especially for zero-index materials. Here, Dubois  An acoustic double zero refractive index metamaterial with simultaneously zero density and infinite bulk modulus achieving finite impedance overcomes such an obstacle .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes and Supplementary ReferencesThe acoustic field envelope merges into the point source at 18.3 kHz because of the double negative refractive index below the Dirac-like pointThe acoustic field envelope is breathing with a uniform phase distribution at 18.7 kHz because of the double zero refractive index at the Dirac-like point.The acoustic field envelope propagates away from the point source at 19.1 kHz because of the double positive refractive index above the Dirac-like point.


Rapid and accurate cancer staging following diagnosis underpins patient management, in particular the identification of distant metastatic disease. Current staging guidelines recommend sequential deployment of various imaging platforms such as computerised tomography (CT) and positron emission tomography (PET) which can be time and resource intensive and onerous for patients. Recent studies demonstrate that whole body magnetic resonance Imaging (WB-MRI) may stage cancer efficiently in a single visit, with potentially greater accuracy than current staging investigations. The Streamline trials aim to evaluate whether WB-MRI increases per patient detection of metastases in non-small cell lung and colorectal cancer compared to standard staging pathways.The Streamline trials are multicentre, non-randomised, single-arm, prospective diagnostic accuracy studies with a novel design to capture patient management decisions during staging pathways. The two trials recruit adult patients with proven or highly suspected new diagnosis of primary colorectal (Streamline C) or non-small cell lung cancer (Streamline L) referred for staging. Patients undergo WB-MRI in addition to standard staging investigations. Strict blinding protocols are enforced for those interpreting the imaging. A first major treatment decision is made by the multi-disciplinary team prior to WB-MRI revelation based on standard staging investigations only, then based on the WB-MRI and any additional tests precipitated by WB-MRI, and finally based on all available test results. The reference standard is derived by a multidisciplinary consensus panel who assess 12 months of follow-up data to adjudicate on the TNM stage at diagnosis. Health psychology assessment of patients’ experiences of the cancer staging pathway will be undertaken via interviews and questionnaires. A cost (effectiveness) analysis of WB-MRI compared to standard staging pathways will be performed.We describe a novel approach to radiologist and clinician blinding to ascertain the ‘true’ diagnostic accuracy of differing imaging pathways and discuss our approach to assessing the impact of WB-MRI on clinical decision making in real-time. The Streamline trials will compare WB-MRI and standard imaging pathways in the same patients, thereby informing the most accurate and efficient approach to staging.ISRCTN43958015 (registered 25/7/2012). Streamline L ISRCTN50436483 (registered 31/7/2012).Streamline C  Lung and colorectal cancer are the second and third most common malignancies in the UK, each accounting for 16% of all new cancer diagnoses; approximately 87,000 patients diagnosed per year [In colorectal cancer, 50% of patients undergoing primary surgery with curative intent, relapse subsequently with metastatic disease, often within 12 months .Cancer staging depends on high technology imaging platforms such as computerised tomography (CT), positron emission tomography (PET) and magnetic resonance imaging (MRI), together with standard scintigraphy, plain X- rays and ultrasound. Staging pathways are complex because these various modalities have differing diagnostic accuracies across different tumour types and body organs. The UK National Institute for Health and Care Excellence (NICE) guidelines outline an integrated but step-by-step sequential deployment of various imaging modalities before tumour staging is deemed complete and the first treatment decision is made . It is nRecent technological advances allow whole body MRI scanning within one hour; sensitivity for metastatic disease is reported to be high –10. MRI This paper outlines the protocols of two multicentre studies comparing WB-MRI to standard NICE-approved diagnostic pathways for staging, (1) colorectal cancer –Streamline C  and, (2) non-small cell lung cancer -Streamline L . The studies are pragmatic and incorporate a variety of MRI platforms and interpreting radiologists, and assess the impact of WB-MRI on clinical decision making in real-time. In addition, the trials incorporate a health psychology assessment of patients’ experiences of WB-MRI, investigating potential impact on the physical and psychological burden of the cancer staging pathway, and predictors of patient preference. A detailed analysis of the cost and the cost-effectiveness of WB-MRI versus standard NICE-approved staging pathways will also be conducted. These trials are funded by the UK National Institute of Health Research (NIHR) Health Technology Assessment (HTA) programme, sponsored by University College London, and coordinated by the Cancer Research UK and UCL Cancer Trials Centre.To evaluate whether initial staging with whole body magnetic resonance Imaging (WB-MRI) increases per patient sensitivity for metastasis in colorectal and lung cancer compared to standard NICE-approved diagnostic pathways.To determine how WB-MRI influences time to, and nature of, the first major treatment decision compared to standard investigations, and to determine whether early WB-MRI could reduce or replace standard investigations based on its diagnostic accuracy.To assess the accuracy of WB-MRI and standard diagnostic pathways for local and distant cancer staging. WB-MRI will be evaluated both as an additional test to standard pathways, and as a replacement test based per organ and per metastasis analysis.To determine the lifetime incremental cost and cost-effectiveness of staging using WB-MRI compared to standard diagnostic pathways.To evaluate patients’ experiences of staging using WB-MRI and to determine the priorities they place on differing attributes offered by competing staging pathways, including impact of reducing time to first treatment.To determine the inter-observer variability of WB-MRI diagnosis of metastatic disease by different radiologists and their confidence in diagnosis.To evaluate the diagnostic accuracy of WB-MRI protocols limited to diffusion and T1 weighted imaging only, and to assess the incremental benefit on diagnostic accuracy of intravenous gadolinium contrast enhancement.Streamline C and Streamline L are parallel multi-centre prospective cohort studies comparing the staging accuracy of WB-MRI with standard pathways for newly diagnosed colorectal (Streamline C) and non-small cell lung cancer (Streamline L) conducted at 25 English National Health Service (NHS) hospitals. Data investigating the therapeutic impact of WB-MRI on patient management compared to standard pathways will be collated. The trial design compares two different staging strategies in the same patients ensuring comparable data are collected for both pathways while simultaneously increasing trial power to meet endpoints by using paired data.Both qualitative and quantitative assessments will be employed to determine the psychological burden and acceptability of WB-MRI versus standard pathways, and to identify those attributes that influence patient preference most strongly. Health-related quality of life data is being collected, which will be used to inform cost-effectiveness modelling.Inclusion and exclusion criteria are outlined below. A series of imaging hubs will perform the WB-MRI according to the trial protocol (see below). Patients will be recruited from local hospitals (recruitment sites) and referred to the closest imaging hub for the WB-MRI. Some hospitals will act as both recruitment sites and imaging hubs.In the colorectal cancer trial (Streamline C), eligible patients are aged 18 or older, able to give written informed consent, with histologically proven or strongly suspected colorectal cancer referred for staging .Patients with polyp cancer will be excluded because metastatic disease in these patients is exceedingly rare.In the lung cancer trial (Streamline L), eligible patients are aged 18 or older, able to give written informed consent, with histologically proven or clinically diagnosed primary non-small cell lung cancer (NSCLC) with potentially radically treatable disease. Clinically diagnosed NSCLC cancer is defined as radiological diagnosis of lung cancer on chest CT with sufficient confidence to trigger staging investigations. Potentially radically treatable disease is defined as stage IIIb or less on diagnostic CT . Patients should have a performance status 0 to 2 inclusive . Patients with unequivocal metastatic or N3 disease on diagnostic CT chest and abdomen  will be excluded as will those in whom further staging work up is not indicated due to poor performance status or patient choice. Those with histology other than non-small cell lung cancer will also be excluded given the differing biological behaviour of small cell lung cancer.For both Streamline C and Streamline L, patients will be excluded if they have any psychiatric or other disorder likely to impact on informed consent, or evidence of severe or uncontrolled systemic disease, which make it undesirable for the patient to participate in the trial. Pregnant patients or others with contraindications to MRI  are also excluded.The Streamline trials were approved by the London – Camden and King’s Cross Research Ethics Committee on 3rd October 2012 and are being conducted in accordance with the principles of ICH guidelines on good clinical practice in clinical trials and the Research Governance Framework for Health and Social Care (England). All patients give written informed consent prior to inclusion in the trials.2) and contrast enhanced T1 images through the liver, lungs and brain.The choice of MRI platform (i.e. manufacturer and Tesla (T) strength) will be decided by the local hub radiologist according to scanner availability and their usual clinical practice; either 1.5 T or 3 T platforms can be used. WB-MRI will be performed no later than 3 weeks after the final standard staging investigation. A minimum sequence dataset will be acquired including standard T1, T2 axial sequences supplemented by diffusion weighted  will be recorded, along with the presence and location of any metastatic disease based on the radiological report, for later comparison.WB-MRI will be reported by radiologists blinded to the results of standard imaging tests and other clinical information (other than the cancer diagnosis and site of the primary tumour). A novel approach employing a secure central imaging server (3Dnet™) provided by Biotronics3D  has been employed to ensure the required level of blinding. Specifically, WB-MRI datasets will be anonymised by radiographer/technologists before upload from each hub to the secure central imaging server (3Dnet™). This solution enables rapid and simple upload of complex imaging datasets via a standard internet connection. Unanonymised WB-MRI images will only be released to the hospital picture and archiving system (PACS) after their revelation at the multi-disciplinary team (MDT) meeting (see below) so those reporting standard staging investigations are blinded to the WB-MRI images and findings.WB-MRI will be interpreted by designated trial radiologists who have prior experience in interpretation, defined as at least 20 validated WB-MRI cases in patients with lung or colon cancer. Radiologists with experience of less than 100 WB-MRI datasets will in addition undergo a period of “buddy” reporting with a more experienced radiologist (>100 WB-MRI datasets), and will only report alone once deemed competent by the more experienced radiologist. Interpretation may be performed using the 3Dnet™ viewing software or local workstations after download of the WB-MRI images, according to radiologist preference. A sequential viewing paradigm with ordered reading of selected sequences will used to examine the contribution of individual MRI sequences to radiologist diagnostic confidence and accuracy.A free text clinical report will be produced based on all available sequences containing information relating to the local tumour (T), its nodal (N) stage, together with the presence, location, number and size of any metastatic deposits (M), as well as clinically important incidental findings, for example aortic aneurysm. The radiologist may recommend additional tests for equivocal findings as per their routine clinical practice.As per usual clinical practice, the MDT meeting will review all standard staging imaging initially blinded to the WB-MRI findings. MDT meetings will occur at least fortnightly, and usually weekly in a dedicated meeting room and as a minimum will include a radiologist, oncologist, histopathologist and respiratory physician and/or surgeon (Streamline L) or colorectal surgeon (Streamline C). It is anticipated that most MDT meetings will include more than one representative of these specialities. The MDT will have access to the full clinical record and histopathological data via the patient notes or electronic patient record, together with all standard imaging and reports via PACS. Based on this alone, the MDT will document the first major treatment decision , as per usual care, along with the TMN stage.The WB-MRI report and images will then be revealed to the MDT members by the MDT coordinator or designated representative via 3Dnet™ using an internet enabled PC (or via written paper copies in cases of IT failure). MDT members will evaluate the WB-MRI report and document any additional tests they deem the WB-MRI result generates (if any). If these additional tests have already been performed as part of the standard investigations, the MDT will then document the theoretical first major treatment decision based on the WB-MRI and results of the tests it would have generated. Finally, the MDT will then document their definitive first major treatment decision based on all available information (including the WB-MRI).If the additional tests generated by the WB-MRI result have not already been performed as part of standard investigations and are deemed necessary by the MDT, these will be performed and the patients re discussed at the next scheduled MDT meeting when the process described above will be followed.Occasionally a patient may commence definitive treatment before the date of a scheduled MDT such that the WB-MRI  will not yet have been revealed to the MDT. In this scenario, an ad hoc MDT will be convened consisting of all specialities relevant to the patient’s clinical care, and the MDT process described above will be followed.The WB-MRI images and reports may be released early by reporting radiologists (ie before controlled revelation at the MDT meeting) if a diagnosis is made that potentially requires urgent intervention , or on request of the patient’s lead clinician for the patient’s clinical care in an emergency clinical situation. If early release precludes unbiased MDT assessment of the standard staging investigations, then the patient will be replaced.The total time taken to fully stage the patient using standard staging pathways will be calculated from the request date of the first staging investigation to the date of the MDT meeting’s first major treatment decision based on the standard imaging pathway. For Streamline L, the start of the staging process will be defined as the request date of the first staging investigation following a proven or highly suspected diagnosis of lung cancer (for example the date of requested PET/CT after diagnostic chest CT). For Streamline C, the start of the staging process will be defined as the request date of the first staging investigation following a proven or highly suspected diagnosis of colorectal cancer . Because revelation of WB-MRI is deferred until after standard staging is complete, precise measurement of time to full staging using WB-MRI will not be possible. The theoretical time to complete staging using WB-MRI will be thus modelled taking into consideration the time from recruitment date to the date of the WB-MRI, plus the number and type of additional staging investigations generated by WB-MRI.http://www.euroqol.org/) every three months for subsequent health economic analysis.Patients will be followed for a period of 12 months from the date of consent. Patients are asked to complete a resource use diary every 3 months  and complete a validated general health questionnaire (EQ-5D) , and will elicit any factors that patients feel would have made staging easier. These qualitative data will inform the design of a patient experience questionnaire and, thereafter, a discrete choice experiment (DCE). It is planned to administer the questionnaire until 50 patient responses are collated both before and after the WB-MRI, by which point we anticipate saturation will have occurred. The DCE will be targeted at 50 patients for each cancer type and aims to elicit preferences for staging pathways by estimating the relative importance of different attributes, and the trade-offs between them. WB-MRI and standard staging pathways differ in associated attributes, not only related to physical experience but also rate of adverse events, time to diagnosis, and overall accuracy etc. The most important attributes will be identified in the interview and questionnaire studies and appropriate levels assigned to each based on accumulating data from the trials, together with appropriate literature review. A DCE questionnaire will be developed whereby patients state their preference between two choices, with each choice containing different levels of the identified attributes. Comparisons will be repeated a number of times depending on the number of attributes and levels identified.Sample size for the discrete choice experiment should be greater than (500*c)/(t*a) where t = the number of sets of choices, a = the number of scenarios to choose between in each choice, and c = the largest number of levels for any one attribute. Assuming each patient will undertake 15 sets of choices, there are two scenarios in each choice, and the largest number of levels for any one attribute is 3, the required sample size is (500*3)/(15*2) = 50 per cancer type.Further questionnaires including patient satisfaction, the positive and negative affect schedule (PANAS)  and the MDT records and hospital data repositories will be used over the 12-month follow-up period to collate primary histological stage (where surgery performed), the results of any biopsy procedure, and details and findings of follow-up imaging investigations (in particular the presence or absence of metastasis). The date and cause of patient death and post mortem findings (if performed) will also be recorded.Multi-disciplinary consensus panel review is standard methodology for diagnostic test accuracy studies where an independent reference standard does not exist, or is impossible because of incorporation bias. Consensus panels will convene regularly to derive the reference standard for tumour stage at diagnosis for recruited patients completing 12-months of follow-up. The panels will consider all available clinical information including the results of all original standard staging investigations, WB-MRI, histopathology , follow up imaging, post-mortem reports (where available) and MDT meeting records. Each panel consists of at least an oncologist, and/or a colorectal surgeon, and a minimum of two radiologists, one external to the recruitment site and one internal. At least one radiologist must have specific expertise in WB-MRI and in PET-CT. It is not necessary for this radiologist be interpreting WB-MRI as part of the trials. The panel will have access to a histopathologist if required. The purpose of the panel is to adjudicate on the TNM stage of the cancer at diagnosis, including the organ specific sites and burden of metastatic spread against which the accuracy of WB-MRI and standard staging pathways are compared.Resource use data for the main drivers of hospital costs will be collected from patient records, notably number of outpatient visits, inpatient hospital stays, interventional procedures including surgery, repeat imaging investigations and treatment with chemotherapy or radiotherapy. In addition, patients are requested to complete resource use diaries at baseline, and at 3, 6, and 9 months. The diaries will be used to collect data on primary and community care contacts during the 1-year period of follow up period. Additionally, health-related quality of life score, measured according to the EQ-5D, will be measured at baseline and at 3, 6, 9 and 12 months for all surviving patients.Per patient sensitivity for metastasis detection by WB-MRI compared to standard staging pathways in newly diagnosed colorectal and non-small cell lung cancer.The time and test number taken to reach, and the nature of, the first major treatment decision based on WB-MRI compared to standard staging pathwaysDiagnostic accuracy of WB-MRI and conventional staging pathways for local tumour staging and detection of metastasis. WB-MRI will be evaluated both as an additional test to standard pathways per patient, and as a replacement test based on per organ and per metastasis analysis.The lifetime incremental cost and cost-effectiveness of staging using WB-MRI compared to standard diagnostic pathwaysComparative patient experiences of staging using WB-MRI and standard investigations, and the average relative importance weighting of attributes ascribed to standard versus WB-MRI staging pathways.Inter-observer variability in WB-MRI accuracy and effect of radiologist diagnostic confidence on staging accuracy.Diagnostic accuracy of WB-MRI limited to T1 and diffusion weighted sequences compared to full WB-MRI protocols.For both trials, study power is based on a difference in sensitivity for metastases detection, with the WB-MRI pathway replacing the standard staging pathway. Both trials are powered at 80% for type II error and 5% for type 1 error. The sample size was calculated assuming WB-MRI is more sensitive than standard staging pathways, and accounting for the expected prevalence of metastasis in study cohorts .For Streamline C, assuming WB-MRI has 10% greater sensitivity for metastasis than standard pathways (85% vs. 75%), a 40% prevalence of metastasis, 73% concordance between WB-MRI and standard staging in patients deemed to have metastatic disease by the consensus panel, and assuming 10% withdrawal rate, we need to recruit 322 patients, giving 290 evaluable patients (i.e. patients with a cancer diagnosis and completing WB-MRI with sufficient follow-up to assign a consensus reference stage).The initial recruitment target for Streamline L was 250 patients including 200 evaluable patients, based on 24% greater WB-MRI sensitivity for metastasis , 25% prevalence of metastasis and 53% concordance between WB-MRI and standard staging in patients deemed to have metastatic disease by the consensus panel. A withdrawal rate of 20% was assumed.During trial recruitment, and based on advice from the independent data monitoring committee, recruitment targets were increased to 360  for Streamline C and 353  for Streamline L because of higher than expected withdrawal rates of 19% and 43% respectively. The higher withdrawal rate was predominantly due to either a final non-cancer diagnosis, or failure to undergo or complete the WB-MRI scan.A detailed statistical analysis plan will be produced and finalised prior to data lock and transfer to trial statistician. Analysis will consider all patients in the study using multiple imputation to account for missing data with a sensitivity analysis based on complete case analysis.p-values <0.05 will be considered statistically significant. A similar approach will be used for secondary outcomes.Analysis for the primary outcome will use multivariate logistic regression of paired binary outcomes for comparison of sensitivity and specificity of WB-MRI and standard investigations within patients. 95% confidence intervals will be calculated and p-values for secondary outcomes for multiple testing. STATA  statistical software will be used.There will be no adjustment of Health economic analysis will be performed by calculating  the lifetime costs and the quality-adjusted life-years (QALYs) experienced. Costs extrapolated beyond the time horizon of one-year follow-up will be deduced by development of a de novo cost-effectiveness model for the disease pathways, which will be populated via available evidence observed from the trials. Individual patients will then be grouped according to the specific disease path and the accuracy of the staging result. We will calculate mean costs and QALYs for each group and for WB-MRI versus standard staging algorithms.Analysis of health psychology interviews will unearth the potential options for the health psychology questionnaire and the discrete choice experiment. Comparative patient experience between WB-MRI and standard staging investigations, identification of important staging pathway attributes, comparative anxiety, expectations and attribute importance before and following the staging process will be analysed using appropriate comparative and regression statistics.At the time of writing, the Streamline trials are the largest prospective multicentre trials to directly compare WB-MRI with standard imaging pathways for cancer staging. The trials will be particularly informative because they compare comprehensive staging pathways rather than binary comparison of single imaging test platforms. Pathway comparisons are made at relevant points in the patient’s trajectory and recruits are representative of patients undergoing WB-MRI in clinical practice should it be introduced into the NHS. These trials will therefore provide valuable information to guide implementation of high technology imaging platforms for cancer staging.The trial design includes two important features: methods to ensure blinding of those interpreting trial imaging; and evaluation of the impact on therapeutic management decisions made real time in the context of the MDT meeting. The latter in particular is unusual in imaging trials of this size. Furthermore, the trials include a discrete choice experiment for patient preferences, again an infrequent component of imaging technology trials. We employ a reference standard based on consensus panel meetings, to allow assessment of potential incorporation bias, as discussed below.Ascertaining the true standalone diagnostic accuracy of a novel imaging test demands removal of certain external influences on radiological decision making. Interpretation of WB-MRI is likely to be influenced by knowledge of other competing staging tests, to which the reporting radiologists will therefore be blinded. However, to mimic normal clinical practice, those interpreting WB-MRI will not be blinded to the cancer diagnosis nor the site of the primary tumour . The WB-The PACS is the usual repository for imaging studies in the NHS and accessible to many hospital staff. To send un-anonymised WB-MRI images immediately to the local PACS for reporting would potentially unmask blinding. Instead, anonymised image data will be uploaded at each imaging hub to a secure central imaging server (3Dnet™) provided by Biotronics3D. A computer based internet gateway will be installed in each imaging hub to facilitate automated transfer of WB-MRI from the scanner/workstation to 3Dnet™ for reporting and storage, and thereafter automatically back to PACS at the appropriate time point.The 3Dnet™ server will also facilitate simple and timely release of the WB-MRI report and images to the MDT meeting without the need for CD transfer.The trials are designed to compare the impact of the WB-MRI staging pathway on patient management with that based on standard imaging in the context of an MDT. Impact is captured in real-time, contemporaneous within the patients’ actual clinical care pathway determined at the MDT. This design also facilitates health economic analysis as it provides prospective data on patient management decisions made as a direct result of the staging test results.Specifically, WB-MRI imaging and reports are only revealed once the MDT has committed itself to a full staging and treatment decision based on standard investigations, but prior to any treatment being instigated. After WB-MRI has been revealed the MDT will state whether, based on WB-MRI alone, the patient had been adequately staged or whether additional tests are required to clarify potential pathology indicated by WB-MRI findings.Treatment decision based on standard investigations alone .Treatment decision based on WB-MRI and any additional staging investigations generated by the WB-MRI .Final treatment decision incorporating information from all available tests.The final treatment plan for each patient will be based on all available information, incorporating both standard imaging pathways and WB-MRI. This design is necessary because it would be unethical to not act on potentially important WB-MRI findings. It will therefore be possible to capture the following:In addition, record will be made of any complications attributable to staging pathways, notably contrast reactions, biopsy complications , and delays in commencing definitive treatment.There is no single reference standard test for the initial staging of lung and colorectal cancer. In such cases it is acceptable design to convene a consensus to judge the presence or absence of the target condition based on multiple sources of information. In Streamline, panels will consist of a variety of personnel from different disciplines allowing all imaging modalities and clinical data to be discussed with equal emphasis informed by strict guidance for the classification of new lesions identified during the follow-up period, or indeterminate lesions.To standardise panel decisions, a member of the central trial team will attend each individual consensus meeting to ensure similar criteria are used across centres to define disease extent. The comparative findings of all staging and follow up investigations  will be documented to allow estimation of any potential incorporation bias in the final consensus panel decision..Discrete choice experiments (DCE) will be designed to elicit patient preferences for the different attributes of WB-MRI and conventional staging pathways  . DCE desThe Streamline colorectal and lung cancer trials are multi-centre prospective cohort studies comparing the diagnostic accuracy of WB-MRI with standard staging tests for initial tumour staging. The trials include important design features such as real time capture of patient management decisions based on staging test results, allowing impact comparisons for therapeutic decision making. Novel approaches for blinding of image interpretation are employed and in depth patient experience information will be collected to analyse the acceptability of the two separate pathways.The Streamline trials have completed recruitment. Streamline C opened on 8th March 2013 and closed to recruitment on 19th August 2016, recruiting 370 patients. Streamline L opened on 7th February 2013 and closed to recruitment on 5th September 2016 recruiting 353 patients. The analysis plan described in this protocol will be implemented in due course after data retention, follow up and cleaning.