In premenopausal women with advanced breast cancer the luteinising hormone-releasing hormone agonist goserelin  will produce serum levels of oestradiol equivalent to those following surgical oophorectomy or the menopause. This paper reports our further experience of using this drug in 75 premenopausal patients with advanced breast cancer. In addition to response rates, duration of response is reported. An objective response was seen in 25 patients (33%), the median duration of which was in excess of 15 months. Seven patients (9%) showed a complete response to therapy; median duration greater than 37 months. There was no significant difference in time to disease progression  and probability of survival  between those patients assessed as having either static disease, or those showing a partial response at six months. Response to therapy correlates significantly with the oestrogen receptor status of the primary tumour . The modest side-effects, ease of administration and reversibility make this approach to therapy very attractive. This is to be remembered in that 53% of patients had disease progression whilst receiving goserelin. These patients thus avoided the unnecessary and irreversible morbidity associated with surgical oophorectomy. With the proven efficacy and minimal morbidity associated with goserelin we believe there is no current role for surgical oophorectomy in the management of premenopausal patients with advanced breast cancer.


Sixty patients with advanced breast cancer unresponsive to tamoxifen have been randomised to receive four course of mitozantrone, 14 mg m-2 (n = 30) intravenously every 3 weeks  or megesterol acetate, 160 mg bd (n = 30). One in three patients (11 from each group) had substantial disease control for a minimum period of 6 months i.e., lack of progression; seven patients (23%) showed objective response to mitozantrone compared to four (13%) receiving megesterol. Non-progressive disease occurred in all sites, including visceral metastases and receptor negative patients. There were no significant differences between treatment groups in the median time (5 months each) to disease progression response duration or survival  from commencing second-line therapy. Toxicity was considerably higher in the mitozantrone group. Second-line hormonal therapies can produce similar therapeutic results as those achieved from a short course of a 'short option' single agent cytotoxic in patients who were previously thought hormone insensitive. Provided that the patient does not have life threatening disease a trial of megesterol acetate is worth consideration in that it does not prejudice subsequent response to combination cytotoxic chemotherapy.


The molecule is folded about the ethylene bridge, adopting a staggered conformation such that the benzene ring of the tosyl group and the five-membered ring of the phthalimide moiety have a face-to-face orientation with a centroid-to-centroid separation of 3.7454 (12) Å. The crystal structure is stabilized by weak intermolecular π–π interactions between symmetry-related five-membered rings of the phthalimide groups, with a centroid-to-centroid distance of 3.3867 (11) Å. The compound is used for the attachment of a suitable chelate functionality for radiolabeling purposes.In the title molecule, C Å                           b = 12.5642 (12) Å                           c = 19.3194 (19) Å                           β = 107.121 (2)°V = 3173.8 (5) Å3                                                   Z = 8                           Kα radiationMo −1                        μ = 0.23 mmT = 90 (2) K                           0.40 × 0.35 × 0.30 mm                                 Bruker APEX CCD area-detector diffractometerSADABS; Brucker, 2007T                           min = 0.913, T                           max = 0.934Absorption correction: multi-scan (16184 measured reflections3865 independent reflectionsI > 2σ(I)3773 reflections with R                           int = 0.020                                                            R[F                           2 > 2σ(F                           2)] = 0.052                           wR(F                           2) = 0.122                           S = 1.25                           3865 reflections218 parametersH-atom parameters constrainedmax = 0.48 e Å−3                        Δρmin = −0.41 e Å−3                        Δρ                                 SMART  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008CrystalMaker (Palmer, 2006SHELXTL (Sheldrick, 2008Data collection: 10.1107/S1600536808037951/lh2734sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536808037951/lh2734Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Tuberculosis (TB) is the second leading cause of mortality associated with infectious diseases globally and the leading cause of death among people living with HIV. In 2010, there were an estimated nine million people with TB – a third of whom do not have access to TB services – and approximately 1.5 million deaths were attributed to this disease [This year, the STOP TB PARTNERSHIP is focusing on diagnosis, treatment and cure for all .Comparing the pathogenesis of TB with other infections, most infected people can remain asymptomatic for decades. The clinical definition of a latent infection is limited to a positive response to an injection of purified mycobacterial components. The spectrum of active disease is complex. Adult pulmonary TB is considered a public health problem, and extrapulmonary disease occurs in a significant proportion of co-infected patients. Also, paediatric manifestations are difficult to diagnosis. Moreover, many of the 3 million undiagnosed people live in the world's poorest and most vulnerable communities , 3.Treatment is complicated. The combination of drugs requires sequenced administration, unlike other antibacterial treatments that have to be administered for a minimum of six months. The complex interplays between the genetic strain of the bacteria, immune response of the host, and pathogenicity and acquisition of resistance increase the challenges posed by the global TB epidemic and require the development of appropriate approaches for the discovery of drugs and diagnostic techniques applicable in these settings. More research is needed in order to increase our understanding of, and evaluate, the natural history of TB in the context of co-infections, comorbidities and risk behaviours .New England Journal of Medicine in 2012 and based on findings from 4000 patients monitored by the Chinese Center for Disease Control and Prevention showed that in newly diagnosed TB cases, 34.2% had some form of drug resistance, 5.7% had MDR TB and 0.5% had XDR TB. Among those previously treated, 54.5% had some form of drug resistance, 25.6% had MDR TB and 2.1% had XDR TB [Microbial resistance has existed since the dawn of the antibiotic era. Multidrug-resistant (MDR) TB requires treatment for up to two years, with success rates of about 65–75%. Extensively drug-resistant (XDR) TB involves additional resistance to two second-line drugs; it is more difficult to treat and may be incurable. One study published in the The sputum smear misses diagnosis in a half of those infected and provides no information about drug resistance. In low-income and middle-income countries, where the global burden is heaviest, sputum, solid culture and chest radiography are scarcely available for diagnosis. However, these tests do not have sufficient sensitivity and specificity, and they are usually not used or available at the periphery of the health system.The ideal test for TB is a true point-of-care test that enables accurate diagnosis and the detection of drug resistance within the time of clinic consultation.The biggest challenge today is to progress in the development of new diagnostic assays.Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance, was endorsed by the World Health Organization (WHO) in December 2010. This assay was specifically recommended for use as the initial diagnostic test for suspected drug-resistant or HIV-associated pulmonary TB. Although the development of the Xpert MTB/RIF assay is undoubtedly a landmark, the clinical and programmatic effects and cost-effectiveness remain to be defined [Rapid progress has been made in the development of new diagnostic assays for TB in recent years. New technologies have been developed. The Xpert MTB/RIF assay, which enables simultaneous detection of  defined .Future advances in molecular diagnosis should build on this success. Despite recent developments, progress in nucleic acid amplification is still weak and should be strengthened.A rapid, accurate point-of-care diagnostic test that is affordable and can be readily implemented is urgently needed. Investment in the TB diagnostics pipeline should remain a major priority for funders and researchers.Another objective of the World TB Day is treatment and cure for all. New treatments and regimens for patients with drug-resistant as well as drug-sensitive TB are needed. The long duration of treatment and poor tolerability represent important challenges.As stated, the incidence of MDR in certain regions, such as eastern Europe, southern Africa and central Asia, is a current problem, as is retreatment as compared with new cases. Shorter regimens are essential. With new drugs for TB, multidrug regimens will require considerations of interactions, pharmacokinetics, toxicity, drug metabolism and the thresholds for the development of resistance. The recognition that patients with TB frequently present with co-infections and comorbidities, such as HIV or other viral infections , must be assessed at the earliest possible stage in clinical development .There are new drugs in a pipeline that is more robust than ever. Advancing the planning and coordinated assessment of treatment combinations is a high priority. Trials of a novel nitro-dihydro-imidazooxazole (delamanid), together with a background regimen of second-line drugs for MDR TB, resulted in culture conversion at eight weeks, increasing from 30% in placebo versus 45% in the delamanid group. A study of another novel agent, bedaquiline, showed similar results to the delamanid study, although the placebo group showed substantially lower results (9% efficacy). This may be due to the inclusion of more potent fluoroquinolones in the delamanid study [The REMoxTBtrial13 replaced either isoniazid or ethambutol with the fluoroquinolone moxifloxacin (M) in two experimental, four-month regimens (2HRZM/2HRM and 2MRZE/2MR); results are expected shortly. If the findings from the REMoxTB trial are positive, regulatory approval will be sought in 2014 and a national launch could start as early as 2015 . MoxifloReduce sexual transmission of HIV.Halve the rate of HIV infection among persons who inject drugs.Eliminate new HIV infections among children.Increase the number of persons on life-saving treatment to 15 million.Reduce by half the number of TB-related deaths in persons living with HIV.Finally, eliminating TB and TB/HIV in the 21st century is a priority for various high-level partners. This has been delineated by UNAIDS, world leaders and the United Nations General Assembly, who are committed, by 2015, to [In summary, World TB Day on 24 March 2014 brought with it the vision and possibility of achieving “a TB test, treatment and cure for all.”


Although brief alcohol intervention (BI) is widely studied, studies from psychiatric outpatient settings are rare. The aim of this study was to investigate the effects of two variants of BI in psychiatric outpatients. By using clinical psychiatric staff to perform the interventions, we sought to collect information of the usefulness of BI in the clinical setting.Psychiatric outpatients with Alcohol Use Disorders Identification Test (AUDIT) scores indicating hazardous or harmful drinking were invited to participate in the study. The outpatients were randomized to minimal  or BI . Measurements were performed at baseline and at six and 12 months after the intervention. The primary outcome was change in AUDIT score at the 12-month follow-up.In all, 150 patients were enrolled and received either a minimal intervention (n = 68) or BI (n = 82). At 12 months, there was a small reduction in AUDIT score in both groups, with no significant differences in outcome between groups. At 12-month follow-up, 21% of participants had improved from a hazardous AUDIT score level to a nonhazardous level, and 8% had improved from a harmful level to a hazardous level (8%).Brief alcohol interventions may result in a reduction of AUDIT score to a small extent in psychiatric patients with hazardous or harmful alcohol use. Results suggest that BI may be of some value in the psychiatric outpatient setting. Still, more profound forms of alcohol interventions with risky-drinking psychiatric patients need elaboration. Over the past two decades, numerous investigations of the effectiveness of brief intervention (BI) for hazardous or harmful drinking have been performed,2. BriefBrief intervention is a method of addressing alcohol problems in an early stage. The method usually comprises a screening procedure , brief feedback on the results, and personalized information about possible consequences. Five to 15 minutes is the average length of a BI . WrittenThe effectiveness of BI can be studied from different aspects. When measured by reduction of weekly alcohol consumption, BI delivered in primary and emergency care has been found to be effective at reducing hazardous drinking at 12 months or longer. In primMost studies on BI have been performed in primary care, in-hospital care, or emergency care settings,10,11. AHulse et al.,16 invesIn this study, we sought to investigate the effects of two variants of BI  in psychiatric outpatients. We hypothesized that patients who received the more intensive BI  would have lower AUDIT scores at 12-moThis study was part of a project aiming to determine the need for and suitability of BI in a psychiatric outpatient setting. The project was conducted at the Clinic of General Psychiatry at Uppsala University Hospital, Sweden . Study participants were adult outpatients aged ≥18 years who were treated at the clinic. Patients with psychosis or substance abuse disorders (SUDs) were treated at other clinics in the division of psychiatry and, therefore, were not included in this study.An important feature of the project was its naturalistic approach: interventions as well as the distribution of questionnaires were carried out by ordinary psychiatric clinical staff. Involving ordinary staff and using well-established questionnaires gives a realistic picture of effects and allow other psychiatric clinics to readily adopt the design. At the beginning of the project, all psychiatric treatment staff  received information and training for three hours. PhysiciThe AUDIT was developed in 1982 from a WHO collaborative project as a simple method to screen and identify individuals at risk of developing alcohol- and alcohol-related problems. The 10-The AUDIT-C is a condensed version of the full AUDIT. It consists of the three consumption items (items 1–3) from the full AUDIT. Scoring corresponds to that of the full AUDIT, ranging from 0–4. The AUDIT-C is frequently used in busy settings and for follow-up measurement-23.In a drinking diary, the number of standard drinks consumed each day per week is reported retrospectively.The RCQ is a 12-During a three-month period in the autumn of 2009, a questionnaire package that included the AUDIT as well as drug, tobacco and gambling items was administered to patients visiting seven outpatient units at the Uppsala University Hospital general psychiatric clinic. At each outpatient unit, receptionists were instructed to distribute a questionnaire package to each patient. After filling out the questionnaires, the patient handed them to his or her caregiver , who forwarded them to the first author of this paper to calculate AUDIT scores. The AUDIT score of every patient was communicated on paper to the patient’s caregiver. If the patient met the inclusion criterion for the study (AUDIT score of 6–19 for women or 8–19 for men), specific instructions to the caregiver were added. (Only three patients were enrolled by a physician).The only exclusion criterion was ongoing treatment for SUD. If patients fulfilled the inclusion criterion, caregivers were instructed to inform them that their drinking habits were considered hazardous and to invite them to participate in the study. The caregiver provided the patients with written information about the study. No incentives were offered. Patients who agreed to participate gave written consent. For every risky-drinking patient, the caregiver was instructed to return a form to the first author that included either the written consent or the information that the patient was not interested in participating. After randomization of consenting patients, the caregivers were handed written instructions on how to proceed at the forthcoming consultation. For patients indicating dependence (AUDIT score ≥20), the caregiver was advised to take further action.For the randomization process, a person from outside the research group, blinded from information of patient characteristics, created an allocation sequence by drawing pieces of paper out of a basket. Patients were assigned to intervention groups in the order the signed consent forms reached the principal author, following the allocation sequence.The interventions took place during the patient’s ordinary consulting time with their caregiver. Estimated time taken was 10 minutes for the minimal intervention and 15–20 minutes for the BI.Patients in the minimal intervention group were asked to complete the questionnaire package (RCQ and drinking diary) and were given an information leaflet. The leaflet included facts about risky drinking and practical advice on how to reduce alcohol consumption. Caregivers were instructed to avoid any further discussions on drinking habits.Patients in the BI group were asked to complete the same questionnaire as the patients in the minimal intervention group. The one difference from minimal intervention was that they also received a face-to-face intervention with their psychiatric caregiver. The intervention, for which a template was used, took the form of brief advice designed to meet the needs of a person with psychiatric problems, with the aim to enhance motivation to reduce drinking (see Appendix). The principles of MI were considered in the design of the intervention. Finally, BI patients were offered the information leaflet previously described.Staff training in how to perform interventions took place before baseline data collection and at follow-up two months later. During training sessions, intervention fidelity was stressed and discussed. The staff training course is described in detail elsewhere.Measurements were performed at baseline and at six and 12 months. The primary outcome was changes in AUDIT score at 12 months as measured by the full 10-item AUDIT. To measure changes in alcohol habits at six months, the AUDIT-C was used. Changes in AUDIT-C score were measured by extracting AUDIT items 1–3 from the baseline AUDIT and comparing them with the AUDIT-C score at six months. At both the six- and 12-month follow-ups, participants were asked to complete the RCQ and a drinking diary for the preceding week. Participants were contacted by the research team by mail and, if needed after one reminder, by telephone. See FigureInformation on the patient’s diagnosis and employment status at baseline was collected from medical records. To estimate psychiatric severity, the number of psychiatric consultations during the 12-month period after the initial screening was collected for each patient. The study was approved by the Research Ethics Committee at Uppsala University Hospital (DNR 2009/042).χ2 –test. Fisher’s test was used when applicable. Independent samples t-tests were used to test differences in AUDIT scores at baseline between dropouts and responders. All statistical analyses were performed using SPSS , version 19.A conservative intention-to-treat (ITT) analysis was applied in which all patients included had their last rank carried forward in the analysis in order to compensate for dropouts. Although data on alcohol habits may be defined as nonparametric, we chose to treat AUDIT scores as parametric data to make our results comparable with those of previous studies. For the outcome analysis, ANOVA was used . Effect sizes are denoted by d. Comparison of baseline characteristics between participants in each group were performed with Pearson’s Of the 559 eligible risky-drinking patients, 167 patients agreed to participate in the study. One hundred forty-one declined, and another 249 patients made no return visit during the specified period and were therefore not invited. Two patients were not invited because of ongoing SUD treatment elsewhere. After randomization, 150 of the 167 patients who agreed to participate were still willing and eligible. Of the 150 patients, 68 received a minimal intervention, and 82 received BI. Baseline demographic data are presented in TableThe mean AUDIT score at baseline in the enrolled group was 10.9 ± 3.5 points and was 10.6 ± 3.6 points in patients who declined to participate. Mean age in the enrolled group was 28.4 ± 9.9 years for women and 34.9 ± 13.6 for men; the mean age was 28.6 ± 11.0 years for women and 31.7 ± 11.8 years for men among patients who declined to participate. Gender proportions in the enrolled group were 65.3% women and 34.7% men. In the group that declined, the proportions were 73.8% women and 26.2% men. Interventions took place a mean of 21 ± 22.3 days from baseline AUDIT screening . The attrition rate was 15% (n = 22) at six months and 28% (n = 43) at 12 months. The results reported are based on the whole group of 150 participants.At 12 months, there was an overall reduction in AUDIT scores from 10.9 to 9.8 . Participants in the BI group reduced their score from 10.7 to 9.4 points. Participants in the minimal intervention group reduced their score from 11.2 to 10.3 points. There was no interaction effect between groups. The BI did not affect AUDIT scores more than the “minimal” intervention. In all, 21% of participants reduced their drinking to below hazardous level, regardless of intervention assignment. Another 8% reduced their drinking from the harmful to the hazardous level. There were no statistically significant differences in readiness to change stage over time in either group. No changes in number of drinks consumed per week could be discerned over time. At the six-month follow-up, there was a reduction in AUDIT-C scores from 5.0 ± 1.5 points to 4.7 ± 2.0 points  in the entire sample.χ2 = 9.4, p < 0.01). The improved group had a reduction in AUDIT-C scores already at the six-month follow-up .Further analysis was undertaken to compare participants who improved their drinking habits (the improved group) with those who did not improve their drinking habits (the nonimproved group). In the improved group, participants were significantly more motivated to change their drinking habits at baseline than participants in the nonimproved group. At baseline, 58.5% (n = 24) in the improved group were contemplating change versus 31.1% (n = 32) in the nonimproved group . No gender differences or differences in the number of psychiatric consultations were found between the improved and the nonimproved groups.In the improved group, the AUDIT consumption item with greatest reduction at 12-month follow-up was item 2: To our knowledge, this is the first study of BI in the psychiatric setting using clinical psychiatric staff. The major finding is that BI has a positive  12-month effect on alcohol habits in psychiatric outpatients. The minimal intervention produced similar results as the more intense BI: i.e., assessment, feedback, and a leaflet, with or without brief advice, reduced AUDIT score in risky-drinking patients. The reported major strategy among patients for reduced drinking was cutting down on number of drinks per drinking day. Gender or psychiatric severity as measured by number of psychiatric consultations had no impact on results.Investigations of BI in a psychiatric outpatient setting are rare. In one such study, by Eberhard et al., the desIn one other study conducted targeting inpatients in a psychiatric setting, Hulse et al. comparedPatients with psychiatric disorders were included in a recent review of BI for substance use. Except The results of our study are in line with those of Hulse et al. and Eberhard et al., suggesting that BI may be of some value also in the psychiatric setting. Our results imply that the intervention may be very brief and still be effective. Addressing the issue through assessment, feedback, and information leaflet may be sufficient. Intervention effects are relatively small, which indicates that regression to the mean as well as natural recovery processes cannot be ruled out.In this study we preferred the label “minimal intervention group” rather than control group. Patients in the minimal intervention group underwent assessment, were given feedback on drinking habits, gave consent, and received a leaflet. Intrinsic as they are to intervention studies, such factors are known to reduce drinking significantly over time,31.In general, the mechanisms that lead the risky-drinking person to change drinking habits after BI are unclear. It is plausible that BI works simply by pointing out a potential problem, which would stimulate risky-drinking persons to contemplate change. Most excessive drinkers who change, even those with more advanced alcohol problems or comorbid psychiatric disorders, change their drinking patterns without treatment,33. The Psychiatric outpatient units are busy; therefore, secondary alcohol assessment and prevention is not commonly given high priority. Consequently, such measures need to be short and easy to use but still efficient. In this study, BI was administered in a clinical psychiatric setting with caregivers of all categories performing both the screening and the intervention. In previous studies conducted in psychiatric settings, interventions were conducted by research staff. Physicians in primary care commonly are considered the most suitable professional group to perform BI, although nurses may assist,35. PsycMethodological limitations of this study need to be addressed. One limitation is the reliance on self-reported data. To engage all categories of psychiatric staff, we chose to use questionnaires only and not biological markers. However, biomedical markers have disadvantages: their use requires medical staff, and they are not reliable enough on their own. FurtherAnother limitation of our study is the small sample size. If a larger sample was used, conclusions could be more strongly supported. The dropout rate is another limitation. Women dropouts (n = 28) had higher AUDIT scores at baseline than women whom completed follow-up . No other systematic attrition could be identified. With the use of a conservative ITT analysis, we eliminated the risk of overestimating intervention effects. Also, follow-up data were collected by the research team only and not by the patient’s psychiatric caregiver, thus lowering the risk that the patient underestimated his or her drinking to please the caregiver.The major strength of this study is that it adds knowledge to the field of secondary alcohol prevention in a group of patients who are in urgent need of such strategies. By addressing alcohol habits in a simple, time-efficient manner, psychiatric staff of any profession may initiate small but measurable improvements in risky drinking behavior.Brief alcohol interventions in psychiatric care may result in a reduction of AUDIT score to a small extent in patients with hazardous or harmful alcohol use. Brief intervention can easily be performed by different psychiatric health professionals within their normal schedule and may be of some value in the psychiatric outpatient setting. Still, the modest effects of very brief interventions suggest that more profound forms of alcohol interventions with risky-drinking patients need to be elaborated in psychiatric care.Brief (approximately 10-minute) alcohol intervention for psychiatric patients with hazardous or harmful drinking (AUDIT score of 6–19 points for women and 8–19 points for men).Instructions:1. Inform the patient that her/his AUDIT score is above the limit usually considered low-risk; i.e., if she/he continues drinking at this level, there is a risk of harmful effects or dependence, even if that is not the case today.2. Alcohol often affects how one feels. What does the patient know about that? How does she/he use alcohol, and how does alcohol make her/him feel? Listen.3. Offer information; e.g., “Would you like some information about how alcohol affects mental health?”4. Describe why a person with psychiatric problems should be especially observant of drinking habits; for example:If you are having or have had psychiatric problems, you are more sensitive to the adverse effects of alcohol. This is the case even if you are a moderate drinker.Alcohol use can prolong periods when you feel more affected by your psychiatric symptoms.Alcohol may help you fall asleep more easily, but it also disturbs your sleep. It is common to wake up too early the morning after drinking with a feeling of anxiety.Alcohol may induce depression. Many research studies have shown this connection.If you’re on medication, alcohol interacts with medicines in an unpredictable way. Your medicine may fail to be effective, or you may get unexpected side effects.5. What are your patient’s thoughts when hearing this information? Listen.6. Ask if she/he would like some advice on how to cut down on drinking. If yes, introduce the tips presented in the leaflet, giving examples of how they can be used.7. Ask the patient to take the leaflet home to study it more thoroughly.The authors declare that they have no competing interests.CN carried out the collection of data, performed the statistical analysis, and drafted the manuscript. LG, AF, and LJ helped draft the manuscript. All authors took part in the design of the study and in interpreting the results. All authors have read and approved the final manuscript.


Oxygen tensions and gradients play essential roles in modulating biological systems in both physiologic and pathologic events. Thus, controlling oxygen tension is critical for mimicking physiologically relevant in vivo environments for cell, tissue and organ research. We present a new approach for on-demand generation of various oxygen tensions for in vitro hypoxia models. Proof-of-concept prototypes have been developed for conventional cell culture microplate by immobilizing a novel oxygen-consuming biomaterial on the 3D-printed insert. For the first time, rapid (~3.8 minutes to reach 0.5% O2 from 20.9% O2) and precisely controlled oxygen tensions/gradients (2.68 mmHg per 50 μm distance) were generated by exposing the biocompatible biomaterial to the different depth of cell culture media. In addition, changing the position of 3D-printed inserts with immobilized biomaterials relative to the cultured cells resulted in controllable and rapid changes in oxygen tensions (<130 seconds). Compared to the current technologies, our approach allows enhanced spatiotemporal resolution and accuracy of the oxygen tensions. Additionally, it does not interfere with the testing environment while maintaining ease of use. The elegance of oxygen tension manipulation introduced by our new approach will drastically improve control and lower the technological barrier of entry for hypoxia studies. Since the biomaterials can be immobilized in any devices, including microfluidic devices and 3D-printed tissues or organs, it will serve as the basis for a new generation of experimental models previously impossible or very difficult to implement.The development of  By innt years , 5. Oxygnt years , 7. Hypoin vivo or control at the microscale. Moreover, oxygen scavenging agents alter the cell growth environment and may affect cellular responses [Many technologies and devices have been developed for controlling oxygen in both macro and micro environments. Current techniques for global oxygen control of macro-environments include hypoxic chambers/workstations/perfusion chambers or directly adding oxygen scavenging agents. Critical challenges remain for these approaches including only providing a single condition of a hypoxic level at a time, slow equilibration time  and beinesponses . To fulfesponses –12. Theyesponses –20. Whilin vitro hypoxia models to overcome these challenges. Proof-of-concept prototypes have been developed for conventional cell culture microplates (96-well and 24-well cell culture microplates) by immobilizing a novel oxygen-consuming biomaterial on the 3D-printed insert. First, the effects of different biomaterial compositions were investigated. Oxygen tensions at different distance from the optimized biomaterial and the response time to reach each steady state oxygen tension were then measured. The dynamic modulation of oxygen tensions was further evaluated for the ischemia-reperfusion injury model. After that, the cytotoxicity tests of the biomaterials were performed. Finally, the developed prototypes were evaluated by examining the release of hypoxia-inducible factor 1α (HIF-1α) and tumor necrosis factor α (TNF-α) by macrophages under different hypoxic levels.In this study, we developed a new approach for on-demand generation of various oxygen tensions for 2 to 15 U/cm2 and from 1:0 to 1:50, respectively. The glucose oxidase and catalase enzymes were diluted with distilled water to have the density of 25 U/μl and 625 U/μl, respectively. A 1% chitosan solution was prepared by dissolving 1.0 g of chitosan in 100 ml of 1.0% acetic acid. For the biomaterial with 0 U/cm2, 10 μl glutaraldehyde solution (1.25%) was added into the 100 μl 1% chitosan solution. For the biomaterial with glucose density from 1.5 U/cm2 to15 U/cm2, 1–10 μl glucose oxidase solution and 2–20 μl catalase solution were dissolved in 100 μl 1% chitosan solution, and then 10 μl glutaraldehyde solution (1.25%) was added. The biomaterials were coated on the targeted surface and allowed to cure at 4°C for 24 hours prior to use.The biomaterial is a hydrogel comprising glucose oxidase  and catalase  enzymes cross-linked with chitosan (1% (w/v) chitosan acetic acid solution; 448877, Sigma) and glutaraldehyde . Glucose oxidase density and the ratio of glucose oxidase to catalase were adjusted from 0 U/cmGlucose oxidase (GOX) enzyme oxidizes D-glucose molecule, which is one of major components of the cell culture medium (most cell culture media have glucose concentrations between 5 and 25 mM), into gluconic acid and hydrogen peroxide. Catalase (CAT) enzyme decomposes hydrogen peroxide into water and oxygen. Based on the following chemical equations, two oxygen molecules are consumed and only one is produced, resulting in a net loss of oxygen .Net reaction:The insert device was designed to integrate with the multiwell cell culture microplates and fabricated through 3D printing technology. The insert was designed using CAD tools, and ordered by mail . The devOxygen tensions were measured using a precision temperature compensated fiber-optic oxygen measurement system . A 21 gauge hole was drilled on the bottom plate in order to insert the oxygen sensing probe. The tip of the oxygen microsensor was aligned to the bottom surface of the 96-well plate. The 3D-printed insert for 96-well was repositioned using a precision XYZ manipulator to control the distance between the biomaterial and oxygen microsensor.All experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the Feinstein Institute for Medical Research. Male Sprague-Dawley (SD) rats (250–350 g) purchased from Charles River Laboratories  were used. Rats were housed in a temperature-controlled room on a 12-h light/dark cycle and fed a standard Purina rat chow diet. At the day of harvesting peritoneal macrophages, rats were anesthetized by isoflurane inhalation, and the abdomen was shaved and washed with 10% povidone iodine. One hundred ml of HBSS were injected to the peritoneal cavity and the rats were gently shaken for 2 min. A small incision on the abdomen was performed to insert a 20-gauge needle to collect all the peritoneal fluid. Then, the rats were rapidly and painlessly euthanized using carbon dioxide exposure followed by exsanguination, as recommended by the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals: 2013 Editions. All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering.6 cells/ml RPMI 1640 media in 96-well and 24-well plate [2 incubator. After replacing the overnight media with fresh one, the sterilized inserts that can generate various oxygen tensions were placed into the pate.Extracted peritoneal macrophages were cultured at 1 X 10ll plate . The plaHydrogen peroxide assay kit  and glucose (GO) assay kit  were used to measure the levels of hydrogen peroxide and glucose. MTS viability assay  for mammalian cells was performed to test the biocompatibility of the biomaterials. TNF-α ELISA assay set  was used to evaluate the effect of hypoxia on peritoneal macrophages.The levels of HIF-1α protein from hypoxia induced peritoneal macrophage cells were determined by western blot analysis using HIF-1 alpha antibody . The β-actin western blot results from each individual line were used as the internal controls. The band densities from each line were digitalized using Bio-Rad densitometry image system. The ratio of HIF-1α / β-actin from each group were calculated, averaged and graphed in the histogram. The control group (cells only) and matrix group (no enzymes) were included as the background controls.All data were expressed as mean ± SD (n = 4–12) and compared by one-way ANOVA and the Student Newman-Keuls methods. P<0.05 was considered statistically significant.2 and the ratio of glucose oxidase to catalase was adjusted to 1:0, 1:1, 1:5, 1:10 and 1:50. As shown in 2, 3 U/ cm2, and 15 U/ cm2, the D-glucose concentration was decreased to 89.99±0.26%; 88.05±0.76% and 69.98±1.35% respectively after 24 hours incubation. With 48 hours incubation, the D-glucose concentration was further decreased to 86.82±0.31%; 85.89±1.27% and 54.77±2.18% respectively. Many classical media are supplemented with approximately 5.5 mM D-glucose which approximates normal blood sugar levels in vivo. RPMI 1640 media contains 11.1 mM D-glucose. With 15% glucose consumption, the glucose level decreases from 11.1 mM to 9.4 mM which is still much higher than normal glucose level (5.5 mM). Hence, there was acceptable glucose consumption for the coating density of 1.5 U/ cm2 and 3 U/ cm2. For the optimized biomaterial, glucose oxidase density and glucose oxidase to catalase ratio were fixed to 1.5 U/cm2 and 1:50, respectively.The working principle of the biomaterial indicates that it not only consumes oxygen but also consumes D-glucose and produces hydrogen peroxide and gluconic acid. First, hydrogen peroxide levels from different glucose oxidase and catalase combinations were measured. Biomaterials were coated on 96 well flat bottom plates and 100 μl of RPMI 1640 medium was incubated for 24 hours. Glucose oxidase density was fixed to 1.5 U/cm2 incubator with accurate pH meter . We found that the gluconic acid by-product accumulated in the medium did not affect the pH significantly . Most cell culture media contain a buffer system in their original formula to maintain the pH of the media at a certain range for a period of time. There are also supplemental buffer systems  available to minimize the pH changes for those pH sensitive cell lines. The RPMI 1640 Medium we used in this study contains a sodium bicarbonate buffer system (at 2.0 g/L). In addition, cell culture media is normally replaced by a fresh one twice a week. Therefore, the pH changes caused by the accumulation of gluconic acid will not be a factor for the cell growth in our hypoxia model.The production of gluconic acid may cause acidification of culture media. The pH of the cell culture media was measured after 48 hours incubation at 37°C in a humidified, 5% CO2 and glucose oxidase to catalase ratio of 1:50 were coated on the bottom plate of the 3D-printed insert for 96-well plate. After aligning the oxygen microsensor to the bottom surface of the well plate, the insert was repositioned using a precision XYZ manipulator. 100 μl of RPMI 1640 medium containing 11.1 mM D-glucose was added. First, the response time to reach the steady state oxygen tension was measured at the difference distance from the biomaterial. The response time was hereby defined as the time required reaching 90% of the steady-state value. As shown in Biomaterials with glucose oxidase density of 1.5 U/cm2 for this experiment), oxygen tensions were quickly recovered to almost baseline levels (<0.3% O2 deviation). 1.2% O2 level was generated when the distance between biomaterial and oxygen microsensor was 200 μm. The oxygen level was increased to 4.1%, 7.6%, 9.2% and 12.6% when the biomaterial was moved away from the oxygen microsensor 600 μm, 900 μm, 1000 um and 1200 μm distance, respectively.The dynamic changes of oxygen tensions were evaluated by modulating the distance from the biomaterial. Again, the oxygen microsensor was aligned to the bottom surface of the well plate and the insert with biomaterial was repositioned using a precision XYZ manipulator. As shown in 6 cells/ml) were incubated in supernatant for another 24 hours. 2. However, the biomaterial without catalase enzyme showed toxic. It indicates that the toxicity of biomaterials is mainly determined by the hydrogen peroxide levels and not other factors such as glucose, gluconic acid etc.The biocompatibility of the biomaterials with different glucose oxidase enzyme densities was evaluated. RPMI 1640 medium was exposed to the biomaterials for 24 hours and the supernatant was collected. Rat macrophages  to defend the host. In addition, macrophages also are capable of responding rapidly to hypoxia (low oxygen conditions) by altering their gene expression patterns. This way, macrophages are able to switch metabolism to anaerobic glycolysis, to produce more ATP and to increase their supply of oxygen and nutrients. It is well known that hypoxia and inflammatory signals share very similar transcriptional events in macrophages, including the activation of members of both the hypoxia-inducible factor (HIF-1α and HIF-2α) and nuclear factor κB  families . We, the6 cells/ml RPMI 1640 media in 24-well plates. Two hypoxic levels, 0.84% and 2.21%, were generated by placing two inserts with different pillar lengths  as shown in 2) and two different hypoxic conditions. Consequently, the experimental results demonstrate that oxygen tensions can be precisely controlled using the developed biomaterials.Primary peritoneal macrophages were cultured at 1 X 10In vivo physiological oxygen tension ranges from 1 to 11%, which is well below ambient atmospheric oxygen level of 20.9% [in vitro cell culture system is needed in which the oxygen level can be maintained at a low level. In this study, a novel device has been developed to generate rapid, highly accurate and controllable oxygen tensions by manipulating the distance between the immobilized biomaterial and cultured cells through 3D-printed inserts. Our experimental results have shown that it took around 3.8 minutes to reach 0.5% hypoxia from normoxia (20.9%). Compared to the conventional hypoxic chambers / incubators which require more than 3 hours for the medium inside the cell culture plate to equilibrate to the gas outside of it [Oxygen is critical in a wide variety of cellular signaling pathways. of 20.9% , 30. HypOxygen dissolves from air into the cell culture medium and oxygen consumption occurs on the surface of biomaterial. The oxygen equilibrium forms when oxygen consumption rate by biomaterial equals oxygen supply rate by air diffusion. By adjusting the distance between the cultured cells and biomaterial, various stable oxygen tensions can be achieved. The oxygen tension was controlled precisely with respect to the distance from the biomaterial, e.g., 2.68 mmHg per 50 μm increment in the range of 0 to 500 μm along the Z-axis. Our approach doesn’t require the cumbersome components of its forebears, such as syringe pumps or unwieldy interconnections. In addition, it can be reliably used into the cell incubators. 3D printing technology enables rapid, one-step fabrication of complex geometries not possible with planar lithography with high resolution , 32. It 2 deviation) hypoxic conditions (from 12.6% to 1.2% oxygen tension) can be induced by changing the distance between the cells and biomaterials from 200 μm to 1200 μm. The results highlighted the potential of the developed hypoxia insert to be used for in vitro hypoxia / reoxygenation model of ischemia-reperfusion injury [Dynamic test results further showed that rapid (<130 seconds) and reversible (<0.3% On injury , 34.In this study, we only used the biomaterial as an oxygen consuming material. However, the biomaterial also consumes the D-glucose in the cell culture media and generates hydrogen peroxide. All the chemical reactions happen on the biomaterial surface and diffuse into the media. It implies that hypoxic conditions with various D-glucose and hydrogen peroxide gradients can also be developed for different applications with different combinations of glucose oxidase and catalase enzymes –37. The in vivo environment not yet seen in tissue culture studies. This biomaterial will serve as the basis for a new generation of experimental models previously impossible or very difficult to implement.The developed biomaterial was applied to conventional cell culture microplates to control global oxygen of macro-environments as a proof of concept prototype. However, it can be applied to microfluidic devices to control local and complex oxygen of micro-environments that can closely mimic the microenvironment of living tissue . The elein vitro hypoxia models. Compared to the current technologies, this approach allows enhanced spatiotemporal resolution and accuracy of the oxygen tensions. Additionally, it does not interfere with the testing environment while maintaining ease of use. This biomaterial can be applied for both conventional cell culture microplates and microfluidic devices to control global oxygen of macro-environments and local oxygen of micro-environments. Furthermore, advanced disease models can be developed by coating the biomaterial on the 3D-printed tissues or organs.Taken together, we have developed a novel approach for on-demand generation of various oxygen tensions or gradients for


Macrostomum lignano, a basal flatworm with excellent regeneration capacity. We demonstrate that microinjection of DNA constructs into fertilized one-cell stage eggs, followed by a low dose of irradiation, frequently results in random integration of the transgene in the genome and its stable transmission through the germline. To facilitate selection of promoter regions for transgenic reporters, we assembled and annotated the M. lignano genome, including genome-wide mapping of transcription start regions, and show its utility by generating multiple stable transgenic lines expressing fluorescent proteins under several tissue-specific promoters. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model organism.Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. However, the lack of transgenesis methods considerably hampers their wider use. Here we report development of a transgenesis method for  Macrostomum lignano, as well as a new annotated genome sequence.Regeneration capable flatworms have emerged as powerful models for studying stem cell biology and patterning, however their study has been hindered by the lack of transgenesis methods. Here, the authors describe a transgenesis method for  Significant biological insights on stem cell biology and body patterning were obtained using free-living regeneration-capable flatworms (Platyhelminthes) as models4. The most often studied representatives are the planarian species Schmidtea mediterranea2 and Dugesia japonica5. Many important molecular biology techniques and resources are established in planarians, including fluorescence-activated cell sorting, gene knockdown by RNA interference, in situ hybridization, and genome and transcriptome assemblies4. One essential technique still lacking in planarians; however, is transgenesis, which is required for in-depth studies involving e.g., gene overexpression, dissection of gene regulatory elements, real-time imaging and lineage tracing. The reproductive properties of planarians, including asexual reproduction by fission and hard non-transparent cocoons containing multiple eggs in sexual strains, make development of transgenesis technically challenging in these animals.Animals that can regenerate missing body parts hold clues to advancing regenerative medicine and are attracting increased attentionMacrostomum lignano (Macrostomorpha) emerged as a model organism that is complementary to planarians9. The reproduction of M. lignano, a free-living marine flatworm, differs from planarians, as it reproduces by laying individual fertilized one-cell stage eggs. One animal lays ~1 egg per day when kept in standard laboratory conditions at 20 °C. The eggs are around 100 microns in diameter, and follow the archoophoran mode of development, having yolk-rich oocytes instead of supplying the yolk to a small oocyte via yolk cells10. The laid eggs have relatively hard shells and can easily be separated from each other with the use of a fine plastic picker. These features make M. lignano eggs easily amenable to various manipulations, including microinjection11. In addition, M. lignano has several convenient characteristics, such as ease of culture, transparency, small size, and a short generation time of three weeks7. It can regenerate all tissues posterior to the pharynx, and the rostrum12. This regeneration ability is driven by stem cells, which in flatworms are called neoblasts13. Recent research in planarians has shown that the neoblast population is heterogeneous and consists of progenitors and stem cells15. The true pluripotent stem cell population is, however, not identified yet.More recently, a basal flatworm M. lignano using microinjection of DNA into single-cell stage embryos and demonstrate its robustness by generating multiple transgenic tissue-specific reporter lines. We also present a significantly improved genome assembly of the M. lignano DV1 line and an accompanying transcriptome assembly and genome annotation. The developed transgenesis method, combined with the generated genomic resources, will enable new research avenues on stem cells and regeneration using M. lignano as a model organism, including in-depth studies of gene overexpression, dissection of gene regulatory elements, real-time imaging and lineage tracing.Here we present a method for transgenesis in M. lignano is an obligatorily non-self-fertilizing simultaneous hermaphrodite  within 3 h after injection  gene and made a transcriptional GFP fusion in the Minos transposon system  gene as described previously22. The injection of the transgene fragment followed by irradiation demonstrated 5% transgenesis efficiency  errors within contigs compared to 1871 in the ML2 assembly , compared to 94.88% of transcripts mapped on the ML2 genome assembly, and among the mapped transcripts more have intact open reading frames in the Mlig_3_7 assembly than in ML2 . The non-redundant transcriptome has TransRate scores of 0.4360 and 0.4797 for transcriptional units and gene sequences, respectively, positioning it among the highest quality transcriptome assemblies32. The transcriptome is 98.1% complete according to the Benchmarking Universal Single-Copy Orthologs33, with only 3 missing and 3 fragmented genes . For genes tested in this study, the regions up to 2 kb upstream of the transcription start sites are sufficient to faithfully reflect tissue-specific expression patterns of these genes , filled with nutrient enriched artificial sea water (Guillard’s f/2 medium). Worms were fed ad libitum on the unicellular diatom Nitzschia curvilineata  . Climate chamber conditions were set on 20 °C with constant aeration, a 14/10 h day/night cycle.The DV1 inbred M. lignano EFA promoter sequence was obtained by inverse PCR. Genomic DNA was isolated using a standard phenol-chloroform protocol; fully digested by XhoI and subsequently self-ligated overnight (1 ng/μl). Diluted self-ligated gDNA was used for inverse PCR using the EFA specific primers Efa_IvPCR_rv3 5′-TCTCGAACTTCCACAGAGCA-3′ and Efa_IvPCR_fw3 5′-CAAGAAGGAGGAGACCACCA-3′. Subsequently, nested PCR was performed using the second primer pair Efa_IvPCR_rv2 5′-AAGCTCCTGTGCCTCCTTCT-3′ and Efa_IvPCR_fw2 5′-AGGTCAAGTCCGTCGAAATG-3′. The obtained fragment was cloned into p-GEM-T and sequenced. Later on, the obtained sequence was confirmed with the available genome data. Finally, the obtained promoter sequence was cloned into two different plasmids: the MINOS plasmid (using EcoRI/NcoI) and the I-SceI plasmid (using PacI/AscI).The 8 and codon weight matrices were calculated using the 100 most abundantly expressed non-redundant genes. C. elegans Codon Adapter code48 was adapted for M. lignano (http://www.macgenome.org/codons) and used to design codon-optimized coding sequences (Supplementary Data ). Gene fragments  containing codon-optimized sequences, EFA 3′UTR and restriction cloning sites, were inserted into the pCS2+ vector to create optiMac plasmids used in the subsequent promoter cloning.Highly expressed transcripts were identified from RNA-seq dataM. lignano genome assemblies and MLRNA1509 transcriptome assembly8. RAMPAGE signal was used to identify the transcription start site and an upstream region of 1–2.5 kb was considered to contain the promoter sequence. An artificial ATG was introduced after the presumed transcription start site. This ATG was in-frame with the GFP of the target vector. The selected regions were cloned into optiMac vector using HindIII and BglII sites. Primers and cloned promoter sequences are provided in Supplementary Data Promoters were selected using Mlig_3_7, as well as several earlier Worms used for egg laying were kept in synchronized groups of roughly 500 per plate and transferred twice per week to prevent mixing with newly hatching offspring. The day before microinjections, around 1000 worms from 2 plates were combined (to increase the number of eggs laid per plate) and transferred to plates with fresh f/2 medium and no food . On the day of the injections, worms were once again transferred to fresh f/2 without food to remove any debris and eggs laid overnight. Worms were kept in the dark for 3 h and then transferred to light. After 30 min in the light, eggs were collected using plastic pickers made from microloader tips , placed on a glass slide in a drop of f/2 and aligned in a line for easier handling.Needles used in the microinjection procedure were freshly pulled using either borosilicate glass capillaries with filament  or aluminosilicate glass capillaries with filament  on a Sutter P-1000 micropipette puller  with the following settings: Heat = ramp-34, Pull = 50, Velocity = 70, Time = 200, Pressure = 460 for borosilicate glass and Heat = ramp, Pull = 60, Velocity = 60, Time = 250, Pressure = 500 for aluminosilicate glass. The tips of the needles were afterwards broken and sharpened using a MF-900 microforge . Needles were loaded using either capillary motion or microloader tips . Embryos were kept in position using glass holders pulled from borosilicate glass capillaries without a filament  using P-1000 puller with the following settings: Heat = ramp + 18, Pull = 0, Velocity = 150, Time = 115, Pressure = 190. The holders were broken afterwards using a MF-900 microforge to create a tip of ~140 µm outer diameter and 50 µm inner diameter. Tips were heat-polished to create smooth edges and bent to a ~20° angle.M. lignano embryos. An AxioVert A1 inverted microscope  equipped with a PatchMan NP2 for the holder and a TransferMan NK2 for the needle  was used to perform all of the micromanipulations. A FemtoJet express , with settings adjusted manually based on the amount of mucous and debris surrounding the embryos, was used as the pressure source for microinjections. A PiezoXpert  was used to facilitate the penetration of the eggshell and the cell membrane of the embryo.All microinjections were carried out on fresh one-cell stage γ-radiation within 1 h post injection.Irradiation was carried out using a IBL637 Caesium-137 source . Embryos were exposed to 2.5 Gy of 0) were selected based on the presence of fluorescence and transferred into single wells of a 24-well plate. They were then crossed with single-wild-type worms that were raised in the same conditions. The pairs were transferred to fresh food every 2 weeks. Positive F1 animals from the same P0 cross were put together on fresh food and allowed to generate F2 progeny. After the population of positive F2 progeny grew to over 200 hatchlings, transgenic worms were singled out and moved to a 24-well plate. The selected worms were then individually back-crossed with wild-type worms to distinguish F2 animals homozygous and heterozygous for the transgene. The transgenic F2 worms that gave only positive progeny in the back-cross (at least 10 progeny observed) were assumed to be homozygous, singled out, moved to fresh food and allowed to lay eggs for another month to purge whatever remaining wild-type sperm from the back-cross. After the homozygous F2 animals stopped producing new offspring, they were crossed to each other to establish a new transgenic line. The lines were named according to guidelines established at http://www.macgenome.org/nomenclature.html.Positive hatchlings  and 43HE (DsRed), an Axio Scope A1 with a MRc5 digital camera or an Axio Imager M2 with an MRm digital camera.2, overnight hybridization at 68 °C.Southern blots were done using the DIG-System (Roche), according to the manufacturer’s manual with the following parameters: vacuum transfer at 5 Hg onto positively charged nylon membrane for 2 h, UV cross-linking 0.14 J/cmThe Universal GenomeWalker 2.0 Kit  with restriction enzymes StuI and BamHI was used according to the manufacturer’s protocol. Sanger sequencing of PCR products was performed by GATC Biotech (Germany).49 (20 ng/µl), and stored at −80 °C. The final concentration of the probe and optimal hybridization temperature were optimized for every probe separately. Whole mount in situ hybridization was performed following a published protocol49. Pictures were taken using a standard light microscope with DIC optics and an AxioCam HRC  digital camera.cDNA synthesis was carried out using the SuperScript III First-Strand Synthesis System , following the protocol supplied by the manufacturer. Two micrograms of total RNA were used as a template for both reactions: one with oligo(dT) primers and one with hexamer random primers. Amplification of selected DNA templates for ISH probes was performed by standard PCR with GoTaq Flexi DNA Polymerase . Amplified fragments were cloned into pGEM-T vector system  and validated by Sanger sequencing. Primers used for amplification are listed in Supplementary Data 24. Head fragments were collected and treated with 0.2% colchicine in f/2  for 4 h at 20 °C to arrest cells in mitotic phase. Head fragments were then collected and treated with 0.2% KCl as hypotonic treatment for 1 h at room temperature. Fragments were then put on SuperfrostPlus slides  and macerated using glass pipettes while being in Fix 1 solution . The cells were then fixed by treatment with Fix 2 solution  followed by Fix 3 solution , before mounting by using Vectashield with Dapi . At least three karyotypes were observed per worm and 20 worms were analyzed per line.DV1 and NL10 worms were cut above the testes and left to regenerate for 48 h to increase the amount of dividing cells50. In order eliminate the residual diatoms present in the gut, animals were starved for 24 h. For each sample 100 worms were collected in an Eppendorf tube. Excess f/2 was aspirated and worms were macerated in 200 µl 1× Accutase  at room temperature for 30 min, followed by tissue homogenization through pipetting. 800 µl f/2 was added to the suspension and cells were pelleted by centrifugation at 4 °C, 1000 r.p.m., 5 min. The supernatant was aspirated and the cell pellet was resuspended in the nuclei isolation buffer . The cell suspension was passed through a 35 µm pore size filter  and treated with RNase A and 10 mg/ml PI for 15 min prior to measurement. Drosophila S2 cells (gift from O. Sibon lab) and chicken erythrocyte nuclei  were included as references. The S2 cells were treated in the same way as Macrostomum cells. The CEN were resuspended in PI staining buffer . Fluorescence was measured on a BD FacsCanto II Cell Analyzer first separately for all samples and then samples were combined based on the amount of cells to obtain an even distribution of different species. The combined samples were re-measured and genome sizes calculated using CEN as a reference and S2 as positive controls  according to manufacturer’s instructions. For the lysis step worms were kept in the supplied lysis buffer (with Proteinase K added) at 55 °C for 30–40 min and mixed by inverting the tube every 5 min. DNA was ethanol-precipitated once following the extraction and resuspended in TE buffer (for making 454 libraries Qiagen EB buffer was used instead). Concentration of DNA samples was measured with the Qubit dsDNA BR assay kit .454 shotgun DNA libraries were made with the GS FLX Titanium General Library Preparation Kit , and for paired-end libraries the set of GS FLX Titanium Library Paired-End Adaptors  was used additionally. All the libraries were made following the manufacturer’s protocol and sequenced on 454 FLX and Titanium systems.Illumina paired-end genomic libraries were made with the TruSeq DNA PCR-free Library Preparation Kit  following the manufacturer’s protocol. Long-range mate-pair libraries were prepared with the Nextera Mate Pair Sample Preparation Kit  according to manufacturer’s protocol. Libraries were sequenced on the Illumina HiSeq 2500 system.25 v. 1.4 with default parameters, except the errorRate was set to 0.04. The resulting assembly was polished with Pilon51 v. 1.20 using Illumina shotgun data mapped by Bowtie52 v. 2.2.9 and RNA-seq data mapped by STAR53 v. 2.5.2b. Next, scaffolding was performed by SSPACE26 v. 3.0 using paired-end and mate-pair Illumina and 454 data. Mitochondrial genome of M. lignano was assembled separately from raw Illumina reads using the MITObim software54 and the Dugesia japonica complete mitochondrial genome (acc. NC_016439.1) as a reference. The assembled mitochondrial genome differed from the recently published M. lignano mitochondrial genome55 (acc. no. MF078637) in just 1 nucleotide in an intergenic spacer region. The genome assembly scaffolds containing mitochondrial sequences were filtered out and replaced with the separately assembled mitochondrial genome sequence. The final assembly was named Mlig_3_7. Genome assembly evaluation was performed with REAPR27 and FRCbam28 software using HUB1_300 paired-end library and DV1-6kb-1, HUB1-3_6 kb, HUB1-3_7 kb, ML_8KB_1 and ML_8KB_2 mate-pair libraries (Supplementary Table PacBio data (acc. SRX1063031) were assembled with CanuM. lignano RNA-seq data31  and the de novo transcriptome assembly MLRNA150904 (ref. 8) were used to generate an improved genome-guided transcriptome assembly. First, trans-splicing and polyA-tail sequences were trimmed from MLRNA150904 and the trimmed transcriptome was mapped to the Mlig_3_7 genome assembly by BLAT56 v. 36 × 2 and hits were filtered using the pslCDnaFilter tool with the parameters “-ignoreNs -minId = 0.8 -globalNearBest = 0.01 -minCover = 0.95 –bestOverlap”. Next, RNA-seq data were mapped to genome by STAR53 v. 2.5.2b with parameters “--alignEndsType EndToEnd --twopassMode Basic --outFilterMultimapNmax 1000”. The resulting bam files were provided to StringTie29 v. 1.3.3 with the parameter “--rf”, and the output was filtered to exclude lowly expressed antisense transcripts by comparing transcripts originating from the opposite strands of the same genomic coordinates and discarding those from the lower-expressing strand (at least fivefold read count difference). The filtered StringTie transcripts were merged with the MLRNA150904 transcriptome mappings using meta-assembler TACO30 with parameters “--no-assemble-unstranded --gtf-expr-attr RPKM --filter-min-expr 0.01 --isoform-frac 0.75 --filter-min-length 100” and novel transcripts with RPKM <0.5 and not overlapping with MLRNA150904 mappings were discarded. The resulting assembled transcripts were termed ‘Transcriptional Units’ and the assembly named Mlig_RNA_3_7_DV1.v1.TU. To reflect closely related transcripts in their names, sequences were clustered using cd-hit-est from the CD-HIT v. 4.6.1 package57 with the parameters “-r 0 -c 0.95 -T 0 -M 0”, and clustered transcripts were given the same prefix name. Close examination of the transcriptional units revealed that they often represented precursor mRNA for trans-splicing and contained several genes. Therefore, further processing of the transcriptional units to identified boundaries of the encoded genes was required. For this, we developed computational pipeline TBONE , which utilizes exclusively experimental data to determine precise 5′ and 3′ ends of trans-spliced mRNAs. Raw RNA-seq data were parsed to identify reads containing trans-splicing sequences, which were trimmed, and the trimmed reads were mapped to the genome assembly using STAR53. The resulting wiggle files were used to identify signal peaks corresponding to sites of trans-splicing. Similarly, for the identification of polyadenylation sites we used data generated previously8 with CEL-seq library construction protocol and T-fill sequencing method. All reads originating from such an approach correspond to sequences immediately upstream of poly(A) tails and provide exact information on 3′UTR ends of mRNAs. The generated trans-splicing and poly(A) signals were overlapped with genomic coordinates of transcriptional units by TBONE, ‘cutting’ transcriptional units into processed mRNAs with exact gene boundaries, where such experimental evidence was available. Finally, coding potential of the resulting genes was estimated by TransDecoder58, and transcripts containing ORFs but missing a poly(A) signal and followed by transcripts without predicted ORF but with poly(A) signal were merged if the distance between the transcripts was not >10 kb and the spanning region was repetitive. The resulting assembly was named Mlig_RNA_3_7_DV1.v1.genes and includes alternatively spliced and non-coding transcripts. To comply with strict requirements for submission of genome annotations to DDBJ/ENA/GenBank, the transcriptome was further filtered to remove alternative transcripts with identical CDS, and to exclude non-coding transcripts and transcripts overlapping repeat annotations. This final transcriptome assembly was named Mlig_RNA_3_7_DV1.v1.coregenes and used in annotation of the Mlig_3_7 genome assembly for submission to DDBJ/ENA/GenBank.Previously published 59 v. 1.2.1 was used to assemble transposable element models directly from the repeated fraction of raw Illumina paired-end sequencing reads with the parameters “-k 31 -i 300 -m 200 -t 37 --big-graph = 1000”. To mine repeat models directly from the genome assembly, RepeatModeler package (http://www.repeatmasker.org) was used with the default settings. Identified repeats from both libraries were automatically annotated using RepeatClassifier perl script from the RepeatModeler package against annotated repeats represented in the Repbase Update – RepeatMasker edition database60 v. 20170127. Short (<200 bp) and unclassified elements were filtered out from both libraries. Additional specific de novo screening for full-length long terminal repeats (LTR) retrotransposons was performed using the LTRharvest tool61 with settings “-seed 100 -minlenltr 100 -maxlenltr 3000 -motif tgca -mindistltr 1000 -maxdistltr 20000 -similar 85.0 -mintsd 5 -maxtsd 20 -motifmis 0 -overlaps all”. Identified LTR retrotransposons were then classified using the RepeatClassifier perl script filtering unclassified elements. Generated repeat libraries were merged together with the RepeatMasker60 library v. 20170127. The resulted joint library was mapped on the genome assembly with RepeatMasker. Tandem repeats were annotated and masked with Tandem Repeat Finder62 with default settings. Finally, to estimate overall repeat fraction of the assembly, the Red de novo repeat annotation tool63 with default settings was applied.Two methods were applied to identify repetitive elements de novo both from the raw sequencing data and from the assembled scaffolds. Tedna software64, and aligned non-self blocks longer than 500 nt and at least 95% identical were calculated.To identify duplicated non-repetitive fraction of the genome, repeat-masked genome assembly was aligned against itself using LAST softwarehttp://gb.macgenome.org/downloads/Mlig_3_7.All raw data have been deposited in the NCBI Sequence Read Archive under accession codes SRX2866466 to SRX2866494. Annotated genome assembly has been deposited at DDBJ/ENA/GenBank under the accession NIVC00000000. The version described in this paper is version NIVC01000000. The genome and transcriptome assembly files are also available for download at Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Movie 1Supplementary Movie 2


Community pharmacy increasingly features in global strategies to modernise the delivery of primary healthcare. Medicine Use Reviews (MURs) form part of the English Government's medicines management strategy to improve adherence and reduce medicine waste. MURs provide space for patient–pharmacist dialogue to discuss the well-known problems patients experience with medicine taking. However, ‘underserved’ communities , who may benefit the most, may not receive this support. This study aims to develop, implement and evaluate an e-learning education intervention which is coproduced between patients from underserved communities and pharmacy teams to improve MUR provision.This mixed-methods evaluative study will involve a 2-stage design. Stage 1 involves coproduction of an e-learning resource through mixed patient–professional development (n=2) and review (n=2) workshops, alongside informative semistructured interviews with patients (n=10) and pharmacy staff (n=10). Stage 2 involves the implementation and evaluation of the intervention with community pharmacy staff within all community pharmacies within the Nottinghamshire geographical area (n=237). Online questionnaires will be completed at baseline and postintervention (3 months) to assess changes in engagement with underserved communities and changes in self-reported attitudes and behaviour. To triangulate findings, 10 pharmacies will record at baseline and postintervention, details of actual numbers of MURs performed and the proportion that are from underserved communities. Descriptive and inferential statistics will be used to analyse the data. The evaluation will also include a thematic analysis of one-to-one interviews with pharmacy teams to explore the impact on clinical practice (n=20). Interviews with patients belonging to underserved communities, and who received an MUR, will also be conducted (n=20).The study has received ethical approval from the NHS Research Ethics Committee (East Midlands–Derby) and governance clearance through the NHS Health Research Authority. Following the evaluation, the educational intervention will be freely accessible online. The involvement of underserved communities in the e-learning development process is central to the coproduction model. This provides a platform for their voices to be heard. It also incorporates the views of community pharmacy staff to ensure the intervention is sensitive to the context in which pharmacy teams deliver Medicine Use Reviews (MURs).There is a risk of social desirability bias through the implementation of self-reported questionnaires. We will seek to minimise this using self-completion online questionnaires and not through face-to-face completion.To triangulate findings, self-reported pharmacy staff behaviour change will be compared with the numbers of MURs performed with patients from underserved communities at baseline and postintervention. Pharmacy staff and patient interview accounts will further inform the interpretation of questionnaire findings and any impact on practice.Globally, community pharmacy increasingly features in delivering new services to modernise primary healthcare, often in response to the growing demand on general practitioners (GPs) and the shifting of care from resource impoverished hospital settings to comparatively cheaper care in the community.5To support medicine taking and to tackle problems of non-adherence and medicine waste, community pharmacies are increasingly being commissioned to deliver new services to support patient medicine taking.local needs and patient priorities’.Despite over 3 million MURs being conducted in 2015–2016,However, importantly, there is no obligation to target patients from underserved or ‘seldom heard voice’ communities . Although patients from these communities will have unique needs, they are all less inclined to participate in health or screening services and have poorer health outcomes.The coproduction philosophy, where health professionals and service users work in partnership to improve the patients' experience, is increasingly being used to improve services to patients.An e-learning intervention was identified as the most appropriate and cost-efficient method to deliver the training. E-learning tools have a track record in educating health professionals and are improving health-related behaviours.Coproduce, with patients from underserved communities and pharmacy teams, an e-learning resource designed to change the attitudes and behaviour of pharmacy staff to improve the provision of MURs to underserved communities.Evaluate the impact of the e-learning resource on pharmacy staff's reported behaviour scores on providing MURs to underserved communities.Characterise pharmacy staff's experience, perceived impact on practice and to investigate barriers and facilitators to successful implementation of the e-learning.This study aims to investigate whether an educational intervention coproduced with patients and professionals can change pharmacy staff attitudes and behaviour to improve the provision of MURs to underserved communities. The primary objectives are to:Explore underserved patient experiences of medicines and levels of support received from healthcare professionals.Investigate patients' experiences of community pharmacy service, their feelings of being offered and undertaking an MUR and how this has affected their knowledge of medicines and their use and perceptions of pharmacy services.The secondary objectives are to:This study will be conducted in two stages  at multiThe pharmacist e-learning educational intervention will take the form of a series (2–4) of reusable learning objects (RLOs). RLOs are small, pedagogically designed, ‘bite-sized’ chunks of e-learning that focus on a particular topic.Community pharmacy staff .Superintendent pharmacists, community pharmacy owners.Pharmacy representatives/professional leadership bodies/educational bodies , Royal Pharmaceutical Society, Pharmacy Voice, Centre for Pharmacy Postgraduate Education (CPPE)). EA is a CPPE tutor who has experience running pharmacy education workshops and will facilitate recruitment.Box 1Inclusion criteria1. Community pharmacy staff who have active involvement (on a day-to-day basis) in identifying, inviting or undertaking Medicine Use Reviews (MURs) within a community pharmacy2. Willing to provide consent to take part in the workshop activities/interviewExclusion criteria1. Pharmacy staff who are not actively involved with identifying, inviting or undertaking MURs within a community pharmacy2. Unwilling to provide consent to take part in the workshop activities/interviewPharmacy teams and patient groups from underserved communities will be recruited to workshops to coproduce the e-learning resources to inform the intervention. ‘Pharmacy teams’ will include participants from the following groups  identified as belonging to (or representing) an underserved community. For the purposes of this study, the term ‘underserved’ is used to describe patients who are eligible to receive the Medicines Use Review (MUR) service, but who, for whatever reason, not receive the service. We define patients from ‘underserved’ communities as including, but not limited to people with:People whose first language is not EnglishPeople with physical, visual, hearing or learning impairments, or any other disabilityPatients taking multiple medicines for more than one illness or conditionPeople who are housebound or their carersBlack and minority ethnic communitiesPeople with mental illnessPeople who are homeless or have no fixed addressPeople from refugee, asylum and traveller communitiesPeople from Lesbian, Gay, Bisexual and Transgender communitiesMen aged 16–252. Able to understand study information, willing and have capacity to consent to take part in the workshops/interviewExclusion criteria1. Not belonging to (or representing) an underserved community2. Unable to understand study information, unwilling or unable to provide informed consent to the workshops/interview Patients from underserved communities will be recruited via local organisations (n=15 participants) using the eligibility criteria detailed in In addition, one-to-one interviews will be undertaken with representatives from pharmacy teams (n=10) and patients from underserved communities (n=10) to further inform the e-learning intervention. The inclusion/exclusion criteria will be as above  and 2.Two patient and public involvement (PPI) representatives from undeserved communities, who are eligible themselves for an MUR, will be recruited to form an advisory panel for the study. PPI representatives will be recruited via the East Midlands Academic Health Science Network (AHSN) ‘Public Face’ newsletter. The AHSN was established by NHS England in 2013 to improve health and social care by bringing patients, carers and communities together with health and social care providers, industry and researchers.The PPI advisory panel will meet regularly to advise and challenge where necessary on the study design and information included in the e-learning educational intervention. Their involvement in the project will include attending research meetings, workshops, providing feedback on the development of the RLOs and study findings. They will bring their experience and expertise of the world outside academia and their time living with a long-term condition to ensure the patient voice is heard.The implementation and evaluation of the e-learning intervention will be assessed through three work streams.All community pharmacies (n=237) in Nottinghamshire will be approached to take part in the study. An information sheet and invitation letter will be sent to pharmacies which are currently providing the MUR service to patients. Frequently, pharmacy support staff are actively involved (on a day-to-day basis) in identifying and inviting patients to the MUR service. Pharmacists and their support staff will therefore be invited to participate in this study. The study is not powered to detect differences as there is no prior study on which to base a power calculation. It is assumed that 90% of the pharmacies in Nottinghamshire (n=237) are offering the MUR. With an estimated one pharmacist and one support-staff expressing interest to be involved, a response rate of 50% will give a minimum sample of 213 participants.Following completion of the postintervention questionnaire, a sample of pharmacists and support staff (n=20) who have completed the online questionnaire will be invited to take part in a semistructured interview to further explore their experience of using the e-learning educational resource and its impact on practice.Following their MUR, a purposive sample of patients from underserved communities that have had an MUR, will be provided with information about the study by the pharmacist. If the patient agrees to take part, a one-to-one, face-to-face or telephone interview (n=20) will be arranged. Interviews will explore their overall perceptions of MURs, in particular, their experience of how they were approached and engaged.This study will involve two stages that will be undertaken between August 2016 and March 2019 .The purpose of the workshops will be to help codevelop an e-learning training package for pharmacists to improve the provision and number of MURs to underserved communities. Initial development workshops (n=2) will focus on capturing themes through stories and experiences, including design aspects such as preferred media, on ‘storyboards’ to elicit the contents of the RLOs prior to production and enable an RLO specification to be written. The workshop will begin by providing participants with an outline of the day and reminder that there are no right/wrong answers. An ‘appreciative’ method will be used throughout the workshop to promote inclusivity. Examples of existing RLOs will be provided as well as an explanation of how they are developed. Small groups will work to explore (on flip-charts) what they think is important to include in a pharmacy staff e-learning intervention. Anticipated RLO topics may include: ‘identifying and learning about underserved communities’, ‘effective targeting and invitation strategies’ and ‘tailoring consultation skills to meet the needs of underserved communities’.Following initial concept ideas gathered from the development workshops and interviews, an iterative e-learning development process will be conducted with two ‘review’ workshops with patients from underserved communities and pharmacy teams. These workshops will be identical to the development sessions, but will be used to further develop and refine the e-learning specification. This will enable the refinement of initial ideas into a detailed specification  to be developed. Workshops will be held at the University of Nottingham and will be facilitated by AL (chief investigator (CI)), HW, other members of the University's HELM  team and PPI panel representatives will be present. A £200 inconvenience allowance will be made available to all participants.A review of the literature will ensure the contents are factually correct. The specification will be peer reviewed by experts in the field for clarity, factual content and appropriateness of any animations. For example, the peer review may identify errors in the content, and/or suggest content changes to improve the e-learning. These comments will be fed back to the author for revision of the specification and improvement.Software development and further peer review of the final RLOs will be undertaken before release.One-to-one semistructured interviews will be undertaken by the CI with patients and pharmacists to further inform the e-learning intervention. These interviews will take place at a time and place convenient to the participant and, with consent, will be audio recorded. Pharmacy staff will be invited to an interview to explore existing practice and experience of providing MURs to underserved groups and to identify knowledge gaps that will help create the contents for the e-learning materials. Patients from underserved communities who may be unable or unwilling to attend a workshop  will be invited. These interviews will explore medicine use, support from other health professionals , perceptions of the pharmacy and views on what would the patient like to see emphasised in an e-learning intervention.The e-learning resource and online questionnaires will be piloted on 10 undergraduate pharmacy students from the University of Nottingham to test the functionality of the RLOs and face validity of the data collection instruments. This will be performed by asking students to work through the e-learning and checking ease of use. Face validity of the questionnaire will also be assessed.Following permission from the pharmacy owner/manager, the pharmacist in charge of the pharmacy will be contacted and will inform their team and provide them with the study information. A poster will also be sent to each pharmacy to be displayed in staff areas to raise awareness. Assistance in recruiting pharmacies will be sought from LPCs, other pharmacy bodies and the NIHR Comprehensive Local Research Network (CLRN).All pharmacy staff will be invited to complete an online, baseline questionnaire which will be accessible over 3 weeks. Following baseline data collection (with a reminder after 2 weeks), the online e-learning educational intervention will be made available to participants for 30 days. A postintervention questionnaire will then be sent at 3 months to those who completed the baseline questionnaire (with one reminder). Consent to take part will be implied through the completion of the questionnaires.The questionnaire will collect the following data: (1) participant characteristics, that is, age, gender, educational achievement and details of any MUR training received, years qualified as a registered pharmacist; (2) pharmacy characteristics, for example, geographical area, ownership type  and economic deprivation  data for each pharmacy using the postcode as the lookup reference); (3) pharmacist and support-staff attitudes will be assessed through attitudinal statements developed from the ‘inequalities imagination’ frameworkWhereas self-reporting is a practical and efficient method of collecting data, there are limitations such as relying on respondents' honesty and accuracy of reporting. In order to triangulate and support questionnaire findings of self-reported behaviour with actual practice, ∼10 of the pharmacies taking part will be invited to record the number of MURs performed and how many were from underserved communities over a 2-week period. The sample size for the number of pharmacies included will be guided by the feasibility of data collection and resource constraints.This will occur at baseline and at 3 months following the e-learning.A purposeful sample of community pharmacy staff will be invited to a one-to-one interview about their experience of the e-learning, its usability (barriers and facilitators) of the RLOs and its effect on daily practice. Written consent will be taken before the start of the interview and permission sought for the interview to be audio recorded. It will be explained to the potential participant that entry into the study is entirely voluntary. The interviews will last for ∼30 min and will be conducted at the place of work or at any other convenient location according to participant preferences.A purposeful sample of at least 10 community pharmacies will be recruited to help identify patients from underserved groups. The pharmacist will sequentially invite every eligible patient at the end of their MUR to see if they are interested in taking part in the study. If the patient expresses interest, the pharmacist will hand them a study information sheet and seek consent for their details to be passed onto the CI. The patient will then be contacted by phone by the CI and the study will be fully explained. They will then be given time to decide whether to take part. If they agree, they will be invited to interview. The purpose of the interview will be to explore how the patient felt about being approached and engaged for the MUR, whether the strategies that were included in the e-learning were used by pharmacy staff and what benefit the MUR had to improve medicine understanding and use.2 test or Fisher's exact test as appropriate. Continuous data will be analysed using the within-group t-test or Wilcoxon signed rank-test as appropriate. Statistical significance will be assessed at the 5% (two-sided) level. All statistical analyses will be conducted using IBM Statistical Package for Social Sciences (SPSS) 22.Descriptive statistics will be used to describe participants' demographic and baseline characteristics. Continuous data will be presented using means and SDs if approximately normally distributed, and medians and IQR if non-normally distributed. Categorical data will be described using frequencies and percentages. To assess the effect of the e-learning, baseline and postintervention data scores will be compared. Categorical variables will be analysed using the χAll interviews will be transcribed verbatim and the data imported into qualitative analysis package NVivo; QSR International Pty for the purpose of coding and thematic analysis.24Initial reading and rereading of the transcribed data will be undertaken, with feedback and checking by several members of the qualitative research team, to identify common codes and categories. Actively searching for disconfirming data will be undertaken as well as regular detailed discussions among the qualitative researchers and PPI advisory panel. Consideration will then be given to how these issues group together in broader themes related to the research objectives. The principle of constant comparison will be used to test and refine the empirical conceptual consistency of codes and themes, which have been synthesised and narrated.27The study will be conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki, 1996; the principles of Good Clinical Practice and the Department of Health Research Governance Framework for Health and Social Care, 2005. All participants will be informed that participation (in any stage of the study) will be voluntary and that they may withdraw at any time.Central to the development of the e-learning educational intervention are the views of patients from underserved communities. A coproduction methodology will be used to enable patients and professionals to work in partnership to codesign the pharmacy e-learning intervention. By working alongside each other, a better understanding of the barriers to approaching people from underserved communities can be achieved. The collaborative approach can lead to long-lasting change that genuinely makes a difference to patients' experience.28However, patient participants from underserved communities may be more vulnerable and harder to reach or recruit than patients from the wider public who are not from these communities. We will ensure patients are fully informed about what is involved prior to the workshops. It is not anticipated that any participants will feel marginalised during the workshops as we will be guided by our PPI panel and use an ‘appreciative approach’ where all views will be valued. There will be sufficient facilitators during the workshops to accommodate participant needs. HW  has extensive experience in developing RLOs and will help facilitate the workshops.Face-to-face interviews will take place in the patient's home, pharmacy or any other location according to the patient's wishes. A telephone interview (or Skype/FaceTime) option will also be available. Patients will receive at least 24 hours or as long as is needed to decide whether to take part in an interview. Interviews are not expected to raise any distress or make the patient upset as they will be centred on the patient's medicines, the levels of support they receive and how services such as the MUR can help them.Should the researcher become aware that the patient requires urgent medical care, an intervention or is on the wrong medicines, they will be referred to their pharmacist, their GP or other health professional as appropriate.Following evaluation, the educational intervention will be made freely available online and accessible to pharmacy teams globally. The pharmacy workforce in the UK is made up of ∼50 000 registered pharmacists with 70% working within the community pharmacy sector.The Pharmaceutical Journal and Chemist & Druggist) and social media  to promote awareness.To support this study's contribution to wider knowledge, the research findings will be disseminated to regional, national and international audiences through conference and peer-reviewed publications targeted at service users, health professionals, academics, service commissioners and policymakers. We will also disseminate to pharmacy specific journals (eg, This paper is an important step in the dissemination process as it outlines the study's background, aims and details of methods that will be used. As well as providing pharmacy staff with knowledge and practical skills to engage patients from underserved communities, the study will also contribute to addressing the significant gap in the literature on how effective an intervention of this nature will be in improving pharmacy practice. In this respect, the study is novel and will provide information on the feasibility of the educational intervention, alongside the barriers and facilitators to implementation. Collectively, the findings from this study will act as the first stage in developing the coproduction methodology within community pharmacy and how this can be extended to address inequalities in other areas of pharmacy practice.