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Supplementary Figure from Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma

Supplementary Table from Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma

Supplementary Data from Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma

Ovarian high-grade serous carcinoma (HGSC) prognosis correlates directly with presence of intratumoral lymphocytes. However, cancer immunotherapy has yet to achieve meaningful survival benefit in patients with HGSC. Epigenetic silencing of immunostimulatory genes is implicated in immune evasion in HGSC and re-expression of these genes could promote tumor immune clearance. We discovered that simultaneous inhibition of the histone methyltransferases G9A and EZH2 activates the CXCL10–CXCR3 axis and increases homing of intratumoral effector lymphocytes and natural killer cells while suppressing tumor-promoting FoxP3+ CD4 T cells. The dual G9A/EZH2 inhibitor HKMTI-1–005 induced chromatin changes that resulted in the transcriptional activation of immunostimulatory gene networks, including the re-expression of elements of the ERV-K endogenous retroviral family. Importantly, treatment with HKMTI-1–005 improved the survival of mice bearing Trp53−/− null ID8 ovarian tumors and resulted in tumor burden reduction. These results indicate that inhibiting G9A and EZH2 in ovarian cancer alters the immune microenvironment and reduces tumor growth and therefore positions dual inhibition of G9A/EZH2 as a strategy for clinical development.

Objective To compare the effectiveness of an animation against two leaflets with and without images, in educating young people about genome sequencing (GS). Methods An experimental survey with three assessment points (pre- intervention [T1], post – intervention [T2], 6-week follow-up [T3]). Participants (N = 606) were randomly assigned to receive one of three educational interventions; animation (n = 212); leaflet with images (n = 197); or leaflet with text only (n = 197). Measures of objective and subjective knowledge were completed at T1 (N = 606), T2 (N = 606) and T3 (N = 459). Measures of attitudes, intentions and beliefs towards GS and satisfaction with intervention were completed at T2 only. Results The type of educational intervention young people received had no significant impact on their objective or subjective knowledge at both T2 and T3 (all p > .05), nor did the educational intervention type affect their attitudes, intentions and beliefs towards GS at T2 (p > .05). However, participant satisfaction was significantly higher in the animation group than the leaflet groups (p Conclusion Animations and leaflets are both effective ways to deliver genomic education to young people, but the animations lead to higher satisfaction. Practice implications Different individuals may find different modes of educational resources more accessible than others. Therefore a range of resources should ideally be made available to patients.

To reduce the spread of COVID-19, governments initiated lockdowns, limiting mobility and social interaction of populations. Lockdown is linked to health issues, yet the full impact on health remains unknown, particularly in more vulnerable groups. This study examined the impact on frailty and outcomes in high and low COVID-19 risk older adults. We examined health-related behaviours and support resources participants used during lockdown(s). Lockdown impacts in two countries were compared across four time points to examine impacts of different rules. We recruited 70 participants (aged70 years) in England and Spain. Participants were allocated to higher or lower COVID-19-risk groups based on UK NHS guidelines. They completed assessments for frailty, quality-of-life, loneliness, exercise frequency and social interaction, coping resources and perception of age-friendliness of their environment. The four assessments took place over a 7-month period. Frailty was highest at Time 1 (most severe lockdown restrictions) and significantly higher in the Spanish group. It was lower at Time 3 (lowest restrictions), but did not continue to reduce for the English participants. Perceptions of the age friendliness of the environment matched these changes. Coping resources did not mitigate changes in frailty and outcomes over time, but more frequent physical activity predicted more reduction in frailty. Lockdown had a negative impact on frailty, increasing risk of adverse events for older people, but recovery once lockdowns are eased is evidenced. Further research is required to consider longer term impacts and methods to mitigate effects of lockdown on health.

ObjectivesTo explore the experiences of, and impact on, staff working in palliative care during the COVID-19 pandemic.DesignQualitative multiple case study using semi-structured interviews between November 2020 and April 2021 as part of the CovPall study. Data were analysed using thematic framework analysis.SettingOrganisations providing specialist palliative services in any setting.ParticipantsStaff working in specialist palliative care, purposefully sampled by the criteria of role, care setting and COVID-19 experience.Main outcome measuresExperiences of working in palliative care during the COVID-19 pandemic.ResultsFive cases and 24 participants were recruited (n=12 nurses, 4 clinical managers, 4 doctors, 2 senior managers, 1 healthcare assistant, 1 allied healthcare professional). Central themes demonstrate how infection control constraints prohibited and diluted participants’ ability to provide care that reflected their core values, resulting in experiences of moral distress. Despite organisational, team, and individual support strategies, continually managing these constraints led to a ‘crescendo effect’ in which the impacts of moral distress accumulated over time, sometimes leading to burnout. Solidarity with colleagues and making a valued contribution provided ‘moral comfort’ for some.ConclusionsThis study provides a unique insight into why and how healthcare staff have experienced moral distress during the pandemic, and how organisations have responded. Despite their experience of dealing with death and dying, the mental health and well-being of palliative care staff was affected by the pandemic. Organisational, structural, and policy changes are urgently required to mitigate and manage these impacts.

Background: Palliative rehabilitation involves multi-professional processes and interventions aimed at optimising patients’ symptom self-management, independence and social participation throughout advanced illness. Rehabilitation services were highly disrupted during the Covid-19 pandemic. Aim: To understand rehabilitation provision in palliative care services during the Covid-19 pandemic, identifying and reflecting on adaptative and innovative practice to inform ongoing provision. Design: Cross-sectional national online survey. Setting/participants: Rehabilitation leads for specialist palliative care services across hospice, hospital, or community settings, conducted from 30/07/20 to 21/09/2020. Findings: 61 completed responses (England, n = 55; Scotland, n = 4; Wales, n = 1; and Northern Ireland, n = 1) most frequently from services based in hospices (56/61, 92%) providing adult rehabilitation. Most services (55/61, 90%) reported rehabilitation provision becoming remote during Covid-19 and half reported reduced caseloads. Rehabilitation teams frequently had staff members on sick-leave with suspected/confirmed Covid-19 (27/61, 44%), redeployed to other services/organisations (25/61, 41%) or furloughed (15/61, 26%). Free text responses were constructed into four themes: (i) fluctuating shared spaces; (ii) remote and digitised rehabilitation offer; (iii) capacity to provide and participate in rehabilitation; (iv) Covid-19 as a springboard for positive change. These represent how rehabilitation services contracted, reconfigured, and were redirected to more remote modes of delivery, and how this affected the capacity of clinicians and patients to participate in rehabilitation. Conclusion: This study demonstrates how changes in provision of rehabilitation during the pandemic could act as a springboard for positive changes. Hybrid models of rehabilitation have the potential to expand the equity of access and reach of rehabilitation within specialist palliative care.

Lockdowns and social distancing have been important and successful strategies to limit the spread of the coronavirus disease 2019 (COVID-19) virus. However, excess deaths related to non-COVID-19 causes have been reported, suggesting issues around availability and use of health services, particularly for people with conditions needing ongoing medical support. In addition, evidence indicates that a range of age-related diseases and frailty are impacted by physical activity and social engagement, both limited in lockdown situations. It is therefore important to learn from the effects of lockdowns in order to limit any impacts, while still protecting people from the infection. This editorial summarizes two research themes at the Centre for Ageing Research at Lancaster University in the UK, one assessing impacts of lockdown for people living with a long-term neurodegenerative condition, Parkinson's disease, and one assessing longitudinal impacts on frailty and wellbeing, with older adults aged over 70, including those living with at least one long-term condition. Uncertainty related to Parkinson's disease and to COVID-19 amplified each other, and cancelled clinical appointments and limitations on physical activity had very significant impacts on wellbeing for this group. In the longitudinal study, frailty was more severe during lockdown periods. While lockdowns reduce spread of the virus, becoming frailer could make older adults more vulnerable to the effects of the virus during these periods. Regular exercise during lockdown had beneficial effects aiding recovery once restrictions relaxed. These studies suggest factors that could lessen negative impacts of future lockdowns. Maintaining physical activity and providing access to health services during periods of lockdown are suggested as priorities.

Background COVID-19 has placed huge stress on healthcare systems and services, often impacting the well-being of staff across all settings (Mehta, Machado, Kwizera, et al., 2021). Little is known about the pandemic’s impact on hospice staff. Aims Identify how responding to COVID-19 has impacted hospice staff, whether and why this resulted in experiences of moral distress, and how hospices have responded in supporting staff well-being. Methods Qualitative multiple case study (Yin, 2017) (n= five cases), as part of the CovPall study which explored the multinational response of specialist palliative services to the pandemic. Cases were hospices in England providing specialist palliative care services in any setting. Data collection involved individual interviews with hospice professionals and analysed using framework analysis (Ritchie, Lewis, Nicholls, et al., 2013). Results 24 participants sampled by role, experience, and setting. Themes demonstrated how infection control constraints (i.e., visiting restrictions) prohibited and diluted staff’s capacity to provide care that reflected their professional values. This caused moral distress. Despite organisational, team, and individual support strategies to address moral distress, continually managing these constraints led to a ‘crescendo effect’ with cumulative effects of moral distress (e.g., sadness, guilt, frustration, and fatigue) sometimes leading to burnout. Solidarity with colleagues and the feeling of making a valued contribution provided ‘moral comfort’ for some. Conclusions Despite their experience of dealing with death and dying, the well-being of hospice staff has been, and continues to be, affected by experiences of moral distress during the pandemic. How innovative or of interest is the abstract We provide an in-depth insight into why and how hospice staff experienced moral distress during the pandemic, alongside how voluntary organisations responded. Given that prolonged experiences of moral distress has detrimental effects on staff and the quality of patient care, (Burston & Tuckett, 2013) national and organisational changes need to be implemented to alleviate and manage the short and long-term impact of moral distress (Jameton, 2017). Funding statement The CovPall study is jointly funded by UKRI and NIHR [COV0011; MR/V012908/1]. Additional support was from the National Institute for Health Research (NIHR) Applied Research Collaboration, South London, hosted at King’s College Hospital NHS Foundation Trust, and Cicely Saunders International (Registered Charity No. 1087195).

BackgroundVolunteers are common within palliative care services, and provide support that enhances care quality. The support they provided, and any role changes, during the COVID-19 pandemic are unknown.AimsTo understand volunteer deployment and activities within palliative care services, and to identify what may affect any changes in volunteer service provision, during the COVID-19 pandemic.MethodsMulti-national online survey disseminated via key stakeholders to specialist palliative care services, completed by lead clinicians. Data collected on volunteer roles, deployment, and changes in volunteer engagement. Analysis included descriptive statistics, a multivariable logistic regression, and analysis of free-text comments using a content analysis approach.Results458 respondents: 277 UK, 85 rest of Europe, and 95 rest of the world. 68.5% indicated volunteer use pre-COVID-19. These were across a number of roles (from 458): direct patient/family facing support (58.7%), indirect support (e.g. driving) (52.0%), back office (48.5%) and fundraising (45.6%). 11% had volunteers with COVID-19. Of those responding to a question on change in volunteer deployment (328 of 458) most (256/328, 78%) indicated less or much less use of volunteers. Less use of volunteers was associated with being an in-patient hospice, (OR=0.15, 95%CI = 0.07-0.3 pDiscussion and conclusionVolunteers were mostly prevented from supporting many forms of palliative care, particularly in in-patient hospices, which may have quality and safety implications given their previously central roles. Volunteer re-deployment plans are needed that take a more considered approach, using volunteers more flexibly to enhance care while ensuring safe working practices. Consideration needs to be given to widening the volunteer base away from those who may be considered to be most vulnerable to COVID-19.

BackgroundPalliative rehabilitation involves multi-professional processes and interventions aimed at optimising patients’ symptom self-management, independence, and social participation throughout advanced illness. Rehabilitation services were highly disrupted during the Covid-19 pandemic.AimTo understand rehabilitation provision in palliative care services during the Covid-19 pandemic, identifying and reflecting on adaptative and innovative practice to inform ongoing provision.DesignCross-sectional national online survey.Setting/participantsRehabilitation leads for specialist palliative care services across hospice, hospital, or community settings, conducted from 30/07/20 to 21/09/2020.Findings61 completed responses (England, n=55; Scotland, n=4; Wales, n=1; and Northern Ireland, n=1) most frequently from services based in hospices (56/61, 92%) providing adult rehabilitation. Most services (55/61, 90%) reported rehabilitation provision becoming remote during Covid-19 and half reported reduced caseloads. Rehabilitation teams frequently had staff members on sick-leave with suspected/confirmed Covid-19 (27/61, 44%), redeployed to other services/organisations (25/61, 41%) or furloughed (15/61, 26%). Free text responses were constructed into four themes: (i) fluctuating shared spaces; (ii) remote and digitised rehabilitation offer; (iii) capacity to provide and participate in rehabilitation; (iv) Covid-19 as a springboard for positive change. These represent how rehabilitation services contracted, reconfigured, and were redirected to more remote modes of delivery, and how this affected the capacity of clinicians and patients to participate in rehabilitation.ConclusionThis study demonstrates how changes in provision of rehabilitation during the pandemic could act as a springboard for positive changes. Hybrid models of rehabilitation have the potential to expand the equity of access and reach of rehabilitation within specialist palliative care.Key StatementsWhat is already known about the topic?Guidelines recommend that rehabilitation targeting function, well-being, and social participation is provided by specialist palliative care services.Prior to Covid-19, there was variable provision of palliative rehabilitation in the UK. This variation was related to local service priorities, funding, and commissioning constraints.What this paper addsOver time, Covid-19 related disruptions forced services to reconfigure and adapt which caused fluctuations in the shared spaces in which health professionals, patients and family care givers met to participate in rehabilitation.These fluctuations resulted in the adoption of digital and remote forms of care which altered health professionals’ and patients’ capacity to participate in, and the equity of access to and reach of, rehabilitation.Covid-19 has acted as a springboard for learning, with many rehabilitation services hoping to move into the future by (re)gaining losses and integrating these with lessons learned during the pandemic.Implications for practice, theory or policyRecommendations are made to support extended reach and more equitable access to rehabilitation in palliative care services.We recommend mixed methods evaluations of hybrid models of in-person and online rehabilitation across palliative care settings.

Epigenetic therapies describe drug molecules such as DNA methyltransferase, histone methyltransferase and histone acetylase/deacetylase inhibitors, which target epigenetic mechanisms such as DNA methylation and histone modifications. Many DNA damage response (DDR) genes are epigenetically regulated in cancer leading to transcriptional silencing and the loss of DNA repair capacity. Epigenetic marks at DDR genes, such as DNA methylation at gene promoters, have the potential to be used as stratification biomarkers, identifying which patients may benefit from particular chemotherapy treatments. For genes such as MGMT and BRCA1, promoter DNA methylation is associated with chemosensitivity to alkylating agents and platinum coordination complexes, respectively, and they have use as biomarkers directing patient treatment options. In contrast to epigenetic change leading to chemosensitivity, DNA methylation of DDR genes involved in engaging cell death responses, such as MLH1, are associated with chemoresistance. This contrasting functional effect of epigenetic modification on chemosensitivity raises challenges in using DNA-demethylating agents, and other epigenetic approaches, to sensitise tumours to DNA-damaging chemotherapies and molecularly targeted agents. Demethylation of MGMT/BRCA1 could lead to drug resistance whereas demethylation of MLH1 could sensitise cells to chemotherapy. Patient selection based on a solid understanding of the disease pathway will be one means to tackle these challenges. The role of epigenetic modification of DDR genes during tumour development, such as causing a mutator phenotype, has different selective pressures and outcomes compared to epigenetic adaptation during treatment. The prevention of epigenetic adaptation during the acquisition of drug resistance will be a potential strategy to improve the treatment of patients using epigenetic therapies.

Introduction Methods for measuring frailty over-emphasise physical health, and consensus for a more holistic approach is increasing. However, holistic tools have had mixed success in meeting the validation criteria required of a frailty index. We report on the further development and validation of a Frailty Tool designed for use in the community with a greater emphasis on psychological markers, Holland et al’s Community-Oriented Frailty Index (COM-FI). Method A total of 351 participants aged 58–96 were recruited from Retirement Villages and local communities across the West Midlands of the UK. Participants completed a series of measures designed to assess frailty and outcomes associated with frailty over a 2-year period. Results All three candidate items (‘polypharmacy’, ‘exercise frequency’, and the Coronary Heart Disease and Diabetes ‘joint effect’) were incorporated into the tool, and one variable, ‘falls’ was removed from the index. The revised COM-FI was shown to be valid and met Rockwood's validation criteria (Rockwood et al., 2006), with the exception that in this specific sample there was no significant gender difference and the index did not predict mortality. Discussion Overall, the COM-FI is a valid and reliable tool, although the capacity for the COM-FI to predict mortality over a 2-year period remains inconclusive given the small numbers of people at the higher ends of the frailty range. Prediction of need for social care was good, showing the utility of this community based tool.

Background according to the World Health Organisation, the role of the environment for older adults is to maintain and facilitate independence and promote quality of life. However, measures that examine the environment in terms of its potential impact on older people are either oriented towards specific aspects of the environment, specifically designed for community-level assessment rather than individually oriented, or are unwieldy for everyday use. Objectives this article describes the development and validation of the Age-Friendly Environment Assessment Tool (AFEAT), assessing whether individual function and frailty impact on perceptions of environmental age-friendliness. The extent to which such perceptions may have moderate impacts of frailty on outcomes such as need for care support, quality of life and loneliness is examined. Methods a total of 132 participants aged 58–96 were recruited from retirement villages and local communities in the Midlands of the UK. Participants completed the AFEAT, and a series of measures designed to assess frailty and assessments of quality of life, loneliness and perceptions of functional limitations. Results internal reliability assessment indicated that the AFEAT possesses a Cronbach’s Alpha score of 0.745. The AFEAT significantly predicted quality of life and loneliness, accounting for 17.1% and 5.8% of variance respectively, indicating high concurrent and predictive validity. Furthermore, the AFEAT moderated the predictive strength of frailty in predicting the amount of formal care an individual receives, but not quality of life or loneliness. Discussion the AFEAT is a valid and reliable tool, and analyses highlight the need for an individual-oriented Age-Friendly environment tool.

Frailty is often seen as the absence of resilience. There is evidence to support prevention and intervention strategies for frailty across the range of robust to frail older adults. The role of health psychology in the design and implementation of interventions and the development of understanding that frailty can be addressed even amongst the very old is central to progress in improving quality of life for people at all stages of frailty and also to improving resilience. This chapter reviews the concept of frailty, currently based largely on a physically described syndrome, and examines the role of psychological and social variables in the interaction between frailty and outcomes for the individual. The phenomenon of resilience despite frailty is examined in the context of outcomes of research that suggest that support for developing resilience is needed alongside interventions for frailty.

SummaryIn high-risk and complicated coronary intervention, the risk of acute closure is unpredictable. Thrombus and platelet deposition at the intervention site may also have further effects on subsequent restenosis. In vivo infusion of activated protein C has previously been shown to achieve potent anticoagulation without any haemostatic side effects. We now evaluated the in vitro and in vivo efficacy of polymer-coated coronary stents loaded with purified rabbit Activated Protein C (APC). By measuring 125I-fibrinogen/fibrin deposition APC-loaded stent-wires were antithrombotic compared to albumin-loaded, inhibited-APCloaded, plain polymer-coated and stainless steel stent-wires. In a balloon injury rabbit iliac artery model, APC-loaded stents did not occlude (0/14) compared to plain stents (9/15) and BSA-loaded stents (2/4). Relative 111In-labelled platelet deposition showed a similarly significant degree of inhibition. In conclusion, APC-loading could render stents significantly less thrombotic. Whether an effective antithrombogenic stent like this effectively reduces restenosis rates warrants further evaluation.

Salmon calcitonin (sCT) is an example of one of the many bioactive peptides that require amidation of the carboxy terminus for full potency. We describe a method for the production of amidated sCT in the mammary gland of transgenic rabbits. Expression of a fusion protein comprising human alpha lactalbumin joined by an enterokinase cleavable linker to sCT was directed to the mammary gland under the control of the ovine beta lactoglobulin promoter. C-terminal amidation in vivo was achieved by extending the sCT by a single glycine residue that provides a substrate for endogenous amidating activity in the mammary gland. Full characterization of the released sCT demonstrated it to be equivalent to synthetic standard in terms of structure, purity, and potency.

Recombinant human proteins are generally recovered in low yields from mammalian tissue culture following transfection with commercially available vectors. We have constructed a novel vector containing both the neomycin-resistance-encoding gene (neo) as a dominant selectable marker, and the dihydrofolate reductase-encoding gene (DHFR) to enable amplification of transfected DNA followed by stable expression in mammalian cell lines. Levels of 5 micrograms/ml of the coagulation proteins, factor VII (FVII) and factor XI (FXI), have been achieved in serum-free media. N-terminal sequencing of the purified proteins, and of their separated chains after proteolytic activation, demonstrated correct processing of the recombinant products. In addition, the ratios of clotting activity to antigen for each are close to unity, and the recombinant and plasma-derived proteins had identical mobilities upon electrophoresis in the presence of SDS. The vector described will be of use for the synthesis of recombinant proteins, both wild-type and variants produced by site-directed mutagenesis, especially where complex post-translational modification of the protein makes it essential to use mammalian cells.

The two sarcomeric actin genes, encoding alpha-cardiac and alpha-skeletal actins, are co-expressed in striated muscle, but in the adult the respective isoform predominates in cardiac or skeletal muscle of the normal mouse. We have investigated the interaction between this gene pair in different genetic contexts. Northern blot analysis of alpha-actin mRNA levels in different inbred mice (129/SJ, C3H, C57BL/6) demonstrates variation of as much as threefold in skeletal muscle and eightfold in cardiac muscle. High or low-level expression is seen for both skeletal and cardiac muscle in a given line, suggesting common regulatory phenomena affecting the abundant alpha-skeletal or alpha-cardiac transcript. In the BALB/c mouse, which has a mutant cardiac actin locus, skeletal as well as cardiac actin mRNA and protein accumulate in the adult heart. We have analysed the role of the two alpha-actin genes in this phenomenon in seven recombinant inbred mouse lines (BALB/c x C57BL/6) and in a cross (BALB/c x C3H). The results demonstrate that neither alpha-actin gene alone is sufficient, and implicate other regulatory loci. DNA sequencing of the C3H and BALB/c alpha-skeletal actin gene promoters shows that they are virtually identical over 830 nucleotides. The relative levels of alpha-skeletal and alpha-cardiac actin proteins have been measured by N-terminal peptide analysis in the different mouse lines. The results point to regulatory loci affecting mRNA utilization and protein stability.

This report seeks to surface any possible environmental benefits from a locally based watershed coalition and public funding in California's Santa Ana Watershed. Chartered as a "Special District" with a focus on clean water, the Santa Ana Watershed Project Authority is a regional coalition that works with water planners and lawmakers. The regional coalition is important as it ensures the implementation of regulations based on science and the distribution of funding needed for water projects. Santa Ana River water quality data archived by the United States Geological Survey presents an opportunity to evaluate the accomplishments of this watershed coalition from 1975 to 1985., The second part of the thesis focuses on the benefit of public funding - both federal and state - to the watershed from 1971 to 1999. This is an important topic as infrastructure investment becomes central in the 111th Congress and different programs are audited to ensure they are not wasteful. There are several federal programs that have been used in the Santa Ana Watershed: the Drinking Water State Revolving Fund and environmental education grants program. On the state level, California's programs include: the Nonpoint Source Pollution (NPS) Control and Watershed Protection grant program, Southern California Integrated Watershed Program (SCIWP) and Proposition 50 Integrated Regional Water Management Implementation Grant Program.

Fibrinogen is a complex plasma protein composed of two each of three different polypeptide chains. We have targeted expression of r-human fibrinogen to the mammary gland of transgenic mice. Three expression cassettes, each containing the genomic sequence for one of the three human fibrinogen chains controlled by sheep whey protein beta-lactoglobulin promoter sequences, were coinjected into fertile mouse eggs. Southern blot analysis demonstrated that more than 80% of the transgenic founders contained all three fibrinogen genes. Reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis of milk from the highest producing founder animal demonstrated the presence of human fibrinogen subunits at concentrations of 2000 micrograms/ml. In several animals with a balanced ratio of the individual fibrinogen subunits, up to 100% of the protein was incorporated into fully assembled fibrinogen hexamers. Incubation of the transgenic milk with thrombin and factor XIII resulted in a cross-linked fibrin clot, indicating that a major portion of the secreted fibrinogen was functional. These studies represent the first report of high-level biosynthesis and secretion of a functional, complex, hexameric protein in the milk of a transgenic animal.

We describe the structure and transcriptional activity of the 5' portion of the alpha-cardiac actin gene of BALB/c mice. Southern blotting and DNA sequencing reveal that the promoter and first three exons of the gene are present as perfect repeats in a direct duplication of 9.5 kbp situated immediately upstream of the gene. Both promoters are active in adult cardiac tissue. Transcripts from the partial gene duplication give rise to novel RNAs that are spliced correctly in the actin region and polyadenylated. The level of mature alpha-cardiac actin mRNA is only 16.5% that found in mice that do not possess the duplication. This is due, at least in part, to interference at the transcriptional level. Transcripts from the alpha-skeletal actin gene accumulate to abnormally high levels in the hearts of such mutant mice. This result suggests tight regulatory coupling for this actin gene pair.

There are three principal myosin alkali light chain (MLC) genes expressed in mouse striated muscle. The skeletal muscle gene MLC1F/MLC3F, the ventricular muscle/slow skeletal muscle gene MLC1V(MLC1S), and the atrial muscle/foetal striated muscle gene MLC1A(MLC1emb). MLC1V and MLC1A are expressed in both cardiac and skeletal muscle, and we show here that these genes use a single site of initiation of transcription, and therefore the same proximal promoter region, in both muscle types, and in myogenic cell lines in culture. We have previously shown that for the MLC1F/MLC3F gene, 1200bp of upstream sequence from the MLC1F promoter is sufficient to allow tissue specific and developmentally regulated expression. We have therefore isolated, characterised, and sequenced over 1200bp upstream of each of the three MLC genes in order to look for elements which may be involved in their regulation. Detailed comparison of their promoter sequences, as well as those of the cardiac and skeletal muscle alpha-actin genes, reveals a number of common elements. Among these is an "MLC-sequence" (CCTTTTATAG) common to all MLC genes, including those of chick and rat, and a "cardiac sequence" common to the mouse MLC1A, MLC1V and alpha-cardiac actin genes expressed in the heart.

Mammalian cardiac muscle contains two myosin alkali light chains which are the major isoforms present in either atrial (MLC1A) or ventricular (MLC1V) muscle, and which are different from the fast skeletal muscle isoforms (MLC1F and MLC3F). The atrial isoform is also expressed in fetal skeletal and fetal ventricular muscle, where this isoform is also described as the fetal isoform MLC1emb. We have previously isolated a cDNA clone encoding part of the mouse MLC1A/MLC1emb isoform and have used this clone to demonstrate the identity of MLC1A and MLC1emb in the mouse. To date no information on the amino acid sequence of this mammalian atrial/fetal isoform has been available. Here we present the complete structure and sequence of the mouse MLC1A/MLC1emb gene, together with the predicted amino acid sequence of this isoform. Comparison of the MLC1A/MLC1emb gene and polypeptide with those of MLC1F and MLC1V suggests that MLC1A/MLC1emb and MLC1V were generated from a common ancestral gene. The NH2-terminal region of MLC1A/MLC1emb, thought to be involved in the actomyosin interaction, shows conservation with MLC1V but not with MLC1F suggesting a shared functional domain in these cardiac isoforms. Comparison with the chicken embryonic MLC (L23) suggests that although MLC1A/MLC1emb and L23 show very different patterns of expression, both during development and in the adult, they probably represent the homologous gene in these two species.

The genes encoding the beta and beta' subunits of RNA polymerase in E.coli, rpoB and rpoC, lie downstream of at least two ribosomal protein genes, rplJ (encoding L10) and rplL (L7/12), in a common operon. All four genes are served by promoter PL10, and an attenuator (partial terminator) of transcription, t1, lies between rplJL and rpoBC. Treatment of E.coli with rifampicin, under conditions producing partial inhibition of general RNA synthesis, can stimulate transcription of rpoBC. We have investigated the locus of this effect by fusing PL10 and t1 separately to galK, in suitable plasmids. Our studies of these fusions, and similar fusions involving transcriptional terminators derived from coliphage T7, indicate that low concentrations of rifampicin cause increased readthrough of several different transcriptional terminators in E.coli in vivo, including rpo t1. We discuss whether or not this unspecific mechanism is solely responsible for the observed stimulatory effects of the drug on rpoBC transcription.

We report the effects in vivo of four segments of coliphage T7 DNA upon expression, from an upstream promoter, of galK in plasmids of the pKO family. Three of the segments carry the known major or putative distal terminators of host-dependent T7 early transcription. The fourth carries a novel terminator and maps in the late region of T7. We report the efficiencies of termination in these regions: evidence, based on studies with the E. coli nusA1 mutation, for an involvement of the transcription factor NusA in events at the major early and novel terminators: and the nature of the latter transcription signal.

Proximal upstream flanking sequences of the mouse myosin alkali light chain gene encoding MLC1F and MLC3F, the mouse alpha-cardiac actin gene and the chicken gene for the alpha-subunit of the acetylcholine receptor were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into primary cultures derived from mouse skeletal muscle or into myogenic cell lines. We demonstrate that the mouse MLC1F/MLC3F gene has two functional promoters. In primary muscle cultures, a 1200 bp sequence flanking exon 1 (MLC1F) and a 438 bp sequence flanking exon 2 (MLC3F) direct CAT activity in myotubes, but not in myoblasts or in non myogenic 3T6 and CV1 cells. Developmentally regulated expression is also seen with the alpha-cardiac actin (320 bp) and acetylcholine receptor alpha-subunit (850 bp) upstream sequences in the primary culture system. Transfection experiments with myogenic cell lines show different results with a given promoter construct, reflecting possible differences in the levels of regulatory factors between lines. Different muscle gene promoters behave differently in a given cell line, suggesting different regulatory factor requirements between these promoters.

A spontaneous rifampicin-resistant mutant of E. coli, RpoB26, which inhibits the growth of bacteriophage T7, has been described in the accompanying paper (Schwarz et al.). The rifr mutation appears to increase the rate of transcriptional termination in a rho-deficient strain. T7 mutants with the ability to grow (Gor+) on the Rifr mutant were isolated, and some of their properties were investigated. One of these Gor+ mutants has a small deletion, located between nucleotides 9694 and 9820 of the T7 DNA sequence (Dunn and Studier 1980), which affects the size of the T7 single-stranded DNA binding protein (ssDBP), the product of gene 2.5 (p 2.5) (Dunn and Studier 1980). The Gor+ phenotype was also mapped to this region by genetic methods. Gor+ is recessive to the wild-type (Gor-) phenotype. This suggests that the T7 ssDBP may normally increase the frequency of transcriptional termination in the early region by binding to single-stranded nucleic acid configurations, and so affecting molecular conformations involved in the termination process. Excessive termination in RpoB26 could therefore be compensated for by alterations of the ssDBP.

International audience; A genomic expression library of P.falciparum has been differentially screened with a number of immune sera. The response of 9 clones to the various sera is presented, together with the DNA sequence encoding the epitopes. All but one clone are extremely A+T rich and unlike the other P.falciparum epitopes described, are not composed of amino acid repeats. One clone, which responds specifically with a protective serum, has been analysed in detail. The epitope is carried on a 160kd antigen which is transcribed from a single gene to give a protein expressed in all of the erythrocytic forms. DNA sequence of this clone reveals it to have more than one open reading frame, only one of which is transcribed in the blood stages. The possible significance of the other open readings frames is discussed

The initial formation of skeletal muscle fibers is accompanied by the expression of muscle-type actin and myosin genes. During subsequent maturation of muscle fibers in vivo, developmental changes in the fetal/adult isoforms of these proteins occur. Skeletal muscle-specific transcripts coding for different myosin heavy chains accumulate sequentially both in vivo and in vitro. A genetic analysis demonstrates that these genes are clustered, implicating cis-acting regulatory factors. In contrast, actin and myosin light chain genes are dispersed in the mouse genome. These gene families show a different developmental "strategy": Genes expressed in adult cardiac tissue are coexpressed with the corresponding skeletal muscle sequence during fetal development. This phenomenon also occurs in adult tissue. Under conditions of cardiac overload, adult rat hearts accumulate skeletal actin mRNA and cardiac actin transcripts. In some mouse lines, a mutant cardiac actin gene locus is present. The presence of a second active upstream promoter at this locus depresses transcription of the bone fide gene, resulting in low levels of mature cardiac actin mRNA. In this situation skeletal actin gene transcripts accumulate. Genes expressed in the same fetal or adult muscle phenotype are not linked, suggesting that their coexpression is regulated by transacting factors. The promoter regions of such genes in the mouse have no common characteristics of primary structure with the exception of an E1A-type enhancer core sequence, which has a conserved 5' flanking element, seen for actin and myosin light chain genes. Reintroduction of these promoter regions into muscle cells provides a functional test for such potential regulatory sequences.