Oral Antiviral Defense: Saliva- and Beverage-like Hypotonicity Dynamically Regulate Formation of Membraneless Biomolecular Condensates of Antiviral Human MxA in Oral Epithelial Cells.

The oral mucosa represents a defensive barrier between the external environment and the rest of the body. Oral mucosal cells are constantly bathed in hypotonic saliva (normally one-third tonicity compared to plasma) and are repeatedly exposed to environmental stresses of tonicity, temperature, and pH by the drinks we imbibe (e.g., hypotonic: water, tea, and coffee; hypertonic: assorted fruit juices, and red wines). In the mouth, the broad-spectrum antiviral mediator MxA (a dynamin-family large GTPase) is constitutively expressed in healthy periodontal tissues and induced by Type III interferons (e.g., IFN-λ1/IL-29). Endogenously induced human MxA and exogenously expressed human GFP-MxA formed membraneless biomolecular condensates in the cytoplasm of oral carcinoma cells (OECM1 cell line). These condensates likely represent storage granules in equilibrium with antivirally active dispersed MxA. Remarkably, cytoplasmic MxA condensates were exquisitely sensitive sensors of hypotonicity-the condensates in oral epithelium disassembled within 1-2 min of exposure of cells to saliva-like one-third hypotonicity, and spontaneously reassembled in the next 4-7 min. Water, tea, and coffee enhanced this disassembly. Fluorescence changes in OECM1 cells preloaded with calcein-AM (a reporter of cytosolic "macromolecular crowding") confirmed that this process involved macromolecular uncrowding and subsequent recrowding secondary to changes in cell volume. However, hypertonicity had little effect on MxA condensates. The spontaneous reassembly of GFP-MxA condensates in oral epithelial cells, even under continuous saliva-like hypotonicity, was slowed by the protein-phosphatase-inhibitor cyclosporin A (CsA) and by the K-channel-blocker tetraethylammonium chloride (TEA); this is suggestive of the involvement of the volume-sensitive WNK kinase-protein phosphatase (PTP)-K-Cl cotransporter (KCC) pathway in the regulated volume decrease (RVD) during condensate reassembly in oral cells. The present study identifies a novel subcellular consequence of hypotonic stress in oral epithelial cells, in terms of the rapid and dynamic changes in the structure of one class of phase-separated biomolecular condensates in the cytoplasm-the antiviral MxA condensates. More generally, the data raise the possibility that hypotonicity-driven stresses likely affect other intracellular functions involving liquid-liquid phase separation (LLPS) in cells of the oral mucosa.

Evaluation of the Healthy Start voucher scheme on maternal vitamin use and child breastfeeding: a natural experiment using data linkage.

Background: Having a good start in life during pregnancy and infancy has been shown to be important for living both a healthy life and a longer life. Despite the introduction of many policies for the early-years age group, including voucher schemes, with the aim of improving nutrition, there is limited evidence of their impact on health. Objectives: To assess the effectiveness of the Healthy Start voucher scheme on infant, child and maternal outcomes, and to capture the lived experiences of the Healthy Start voucher scheme for low-income women. Design: This was a natural experiment study using existing data sets, linked to routinely collected health data sets, with a nested qualitative study of low-income women and an assessment of the health economics. Setting: Representative sample of Scottish children and UK children. Participants: Growing Up in Scotland cohort 2 (n = 2240), respondents to the 2015 Infant Feeding Study (n = 8067) and a sample of 40 participants in the qualitative study. Interventions: The Health Start voucher, a means-tested scheme that provides vouchers worth £3.10 per week to spend on liquid milk, formula milk, fruit and vegetables. Main outcome measures: Infant and child outcomes - breastfeeding initiation and duration; maternal outcomes - vitamin use pre and during pregnancy. Results: The exposed group were women receiving the Healthy Start voucher (R), with two control groups: eligible and not claiming the Healthy Start voucher (E) and nearly eligible. There was no difference in vitamin use during pregnancy for either comparison (receiving the Healthy Start voucher, 82%; eligible and not claiming the Healthy Start voucher, 86%; p = 0.10 vs. receiving the Healthy Start voucher, 87%; nearly eligible, 88%; p = 0.43) in the Growing Up in Scotland cohort. Proportions were similar for the Infant Feeding Study cohort (receiving the Healthy Start voucher, 89%; eligible and not claiming the Healthy Start voucher, 86%; p = 0.01 vs. receiving the Healthy Start voucher, 89%; nearly eligible, 87%; p = 0.01); although results were statistically significantly different, these were small effect sizes. There was no difference for either comparison in breastfeeding initiation or breastfeeding duration in months in Growing Up in Scotland, but there was a negative effect of the Healthy Start voucher in the Infant Feeding Survey. This contrast between data sets indicates that results are inconclusive for breastfeeding. The qualitative study found that despite the low monetary value the women valued the Healthy Start voucher scheme. However, the broader lives of low-income women are crucial to understand the constraints to offer a healthy diet. Limitations: Owing to the policy being in place, it was difficult to identify appropriate control groups using existing data sources, especially in the Infant Feeding Study. Conclusions: As the Healthy Start voucher scheme attempts to influence health behaviour, this evaluation can inform other policies aiming to change behaviour and use voucher incentives. The null effect of Healthy Start vouchers on the primary outcomes may be due to the value of the vouchers being insufficient to change the broader lives of low-income women to offer a healthy diet. Future work: The methods developed to undertake an economic evaluation alongside a natural experiment using existing data can be used to explore the cost-effectiveness of the Healthy Start voucher scheme. Funding: This project was funded by the National Institute for Health and Care Research (NIHR) Public Health Research programme and will be published in full in Public Health Research; Vol. 11, No. 11. See the NIHR Journals Library website for further project information.

United Kingdom governments have introduced many policies to support infants and their families. Most of these policies have not been evaluated in terms of health outcomes. Therefore, there is limited evidence for policy-makers about whether or not the right policies are in place to make a difference to the health of young children and their families. We investigated the impact of the Healthy Start voucher scheme (worth £3.10 per week to spend on milk, fruit and vegetables) on the health of low-income mothers, and their infants and young children, in particular vitamin use of mothers and breastfeeding of infants. Using survey data, there were high rates of vitamin use during pregnancy, but fewer women taking vitamins before pregnancy. There was no effect of Healthy Start vouchers on taking vitamins before or during pregnancy. There was inconclusive evidence of the effect of Healthy Start vouchers on breastfeeding, indicating that use of the vouchers does not discourage breastfeeding in women with low incomes. From interviews with mothers, we found that they valued the Healthy Start vouchers and understood the aims of the policy. Healthy Start vouchers were not mentioned in decision-making around breastfeeding. Women's choice to breast or formula feed was based on a range of other factors, such as support to breastfeed. They wanted to provide a healthy diet for their families, but owing to living on low incomes did not always manage it. Policy-makers still need more evidence about the effects of voucher schemes to improve the health of low-income mothers, and their infants and young children. The decision-makers require evidence to determine where to allocate limited resources. There is a need to improve support for low-income families to provide their families with a healthy diet.

True prevalence of long-COVID in a nationwide, population cohort study.

Long-COVID prevalence estimates vary widely and should take account of symptoms that would have occurred anyway. Here we determine the prevalence of symptoms attributable to SARS-CoV-2 infection, taking account of background rates and confounding, in a nationwide population cohort study of 198,096 Scottish adults. 98,666 (49.8%) had symptomatic laboratory-confirmed SARS-CoV-2 infections and 99,430 (50.2%) were age-, sex-, and socioeconomically-matched and never-infected. While 41,775 (64.5%) reported at least one symptom 6 months following SARS-CoV-2 infection, this was also true of 34,600 (50.8%) of those never-infected. The crude prevalence of one or more symptom attributable to SARS-CoV-2 infection was 13.8% (13.2%,14.3%), 12.8% (11.9%,13.6%), and 16.3% (14.4%,18.2%) at 6, 12, and 18 months respectively. Following adjustment for potential confounders, these figures were 6.6% (6.3%, 6.9%), 6.5% (6.0%, 6.9%) and 10.4% (9.1%, 11.6%) respectively. Long-COVID is characterised by a wide range of symptoms that, apart from altered taste and smell, are non-specific. Care should be taken in attributing symptoms to previous SARS-CoV-2 infection.

Natural history of long-COVID in a nationwide, population cohort study.

Previous studies on the natural history of long-COVID have been few and selective. Without comparison groups, disease progression cannot be differentiated from symptoms originating from other causes. The Long-COVID in Scotland Study (Long-CISS) is a Scotland-wide, general population cohort of adults who had laboratory-confirmed SARS-CoV-2 infection matched to PCR-negative adults. Serial, self-completed, online questionnaires collected information on pre-existing health conditions and current health six, 12 and 18 months after index test. Of those with previous symptomatic infection, 35% reported persistent incomplete/no recovery, 12% improvement and 12% deterioration. At six and 12 months, one or more symptom was reported by 71.5% and 70.7% respectively of those previously infected, compared with 53.5% and 56.5% of those never infected. Altered taste, smell and confusion improved over time compared to the never infected group and adjusted for confounders. Conversely, late onset dry and productive cough, and hearing problems were more likely following SARS-CoV-2 infection.

Outcomes among confirmed cases and a matched comparison group in the Long-COVID in Scotland study.

With increasing numbers infected by SARS-CoV-2, understanding long-COVID is essential to inform health and social care support. A Scottish population cohort of 33,281 laboratory-confirmed SARS-CoV-2 infections and 62,957 never-infected individuals were followed-up via 6, 12 and 18-month questionnaires and linkage to hospitalization and death records. Of the 31,486 symptomatic infections,1,856 (6%) had not recovered and 13,350 (42%) only partially. No recovery was associated with hospitalized infection, age, female sex, deprivation, respiratory disease, depression and multimorbidity. Previous symptomatic infection was associated with poorer quality of life, impairment across all daily activities and 24 persistent symptoms including breathlessness (OR 3.43, 95% CI 3.29-3.58), palpitations (OR 2.51, OR 2.36-2.66), chest pain (OR 2.09, 95% CI 1.96-2.23), and confusion (OR 2.92, 95% CI 2.78-3.07). Asymptomatic infection was not associated with adverse outcomes. Vaccination was associated with reduced risk of seven symptoms. Here we describe the nature of long-COVID and the factors associated with it.

Widening or narrowing inequalities? The equity implications of digital tools to support COVID-19 contact tracing: A qualitative study.

BACKGROUND: As digital tools are increasingly used to support COVID-19 contact tracing, the equity implications must be considered. As part of a study to understand the public's views of digital contact tracing tools developed for the national 'Test and Protect' programme in Scotland, we aimed to explore the views of groups often excluded from such discussions. This paper reports on their views about the potential for contact tracing to exacerbate inequalities. METHODS: A qualitative study was carried out; interviews were conducted with key informants from organizations supporting people in marginalized situations, followed by interviews and focus groups with people recruited from these groups. Participants included, or represented, minority ethnic groups, asylum seekers and refugees and those experiencing multiple disadvantage including severe and enduring poverty. RESULTS: A total of 42 people participated: 13 key informants and 29 members of the public. While public participants were supportive of contact tracing, key informants raised concerns. Both sets of participants spoke about how contact tracing, and its associated digital tools, might increase inequalities. Barriers included finances (inability to afford smartphones or the data to ensure access to the internet); language (digital tools were available only in English and required a degree of literacy, even for English speakers); and trust (many marginalized groups distrusted statutory organizations and there were concerns that data may be passed to other organizations). One strength was that NHS Scotland, the data guardian, is seen as a generally trustworthy organization. Poverty was recognized as a barrier to people's ability to self-isolate. Some participants were concerned about giving contact details of individuals who might struggle to self-isolate for financial reasons. CONCLUSIONS: The impact of contact tracing and associated digital tools on marginalized populations needs careful monitoring. This should include the contact tracing process and the ability of people to self-isolate. Regular clear messaging from trusted groups and community members could help maintain trust and participation in the programme. PATIENT AND PUBLIC CONTRIBUTION: Our patient and public involvement coapplicant, L. L., was involved in all aspects of the study including coauthorship. Interim results were presented to our local Public and Patient Involvement and Engagement Group, who commented on interpretation and made suggestions about further recruitment.

Patterns of multimorbidity and their effects on adverse outcomes in rheumatoid arthritis: a study of 5658 UK Biobank participants.

OBJECTIVE: To investigate how the type and number of long-term conditions (LTCs) impact on all-cause mortality and major adverse cardiovascular events (MACE) in people with rheumatoid arthritis (RA). DESIGN: Population-based longitudinal cohort study. SETTING: UK Biobank. PARTICIPANTS: UK Biobank participants (n=502 533) aged between 37 and 73 years old. PRIMARY OUTCOME MEASURES: Primary outcome measures were risk of all-cause mortality and MACE. METHODS: We examined the relationship between LTC count and individual comorbid LTCs (n=42) on adverse clinical outcomes in participants with self-reported RA (n=5658). Risk of all-cause mortality and MACE were compared using Cox's proportional hazard models adjusted for lifestyle factors (smoking, alcohol intake, physical activity), demographic factors (sex, age, socioeconomic status) and rheumatoid factor. RESULTS: 75.7% of participants with RA had multimorbidity and these individuals were at increased risk of all-cause mortality and MACE. RA and >4 LTCs showed a threefold increased risk of all-cause mortality (HR 3.30, 95% CI 2.61 to 4.16), and MACE (HR 3.45, 95% CI 2.66 to 4.49) compared with those without LTCs. Of the comorbid LTCs studied, osteoporosis was most strongly associated with adverse outcomes in participants with RA compared with those without RA or LTCs: twofold increased risk of all-cause mortality (HR 2.20, 95% CI 1.55 to 3.12) and threefold increased risk of MACE (HR 3.17, 95% CI 2.27 to 4.64). These findings remained in a subset (n=3683) with RA diagnosis validated from clinical records or medication reports. CONCLUSION: Those with RA and other LTCs, particularly comorbid osteoporosis, are at increased risk of adverse outcomes, although the role of corticosteroids could not be evaluated in this study. These results are clinically relevant for the monitoring and management of RA across the healthcare system, and future clinical guidelines for RA should acknowledge the importance of multimorbidity.

Biomolecular condensates in cell biology and virology: Phase-separated membraneless organelles (MLOs).

Membraneless organelles (MLOs) in the cytoplasm and nucleus in the form of 2D and 3D phase-separated biomolecular condensates are increasingly viewed as critical in regulating diverse cellular functions. These functions include cell signaling, immune synapse function, nuclear transcription, RNA splicing and processing, mRNA storage and translation, virus replication and maturation, antiviral mechanisms, DNA sensing, synaptic transmission, protein turnover and mitosis. Components comprising MLOs often associate with low affinity; thus cell integrity can be critical to the maintenance of the full complement of respective MLO components. Phase-separated condensates are typically metastable (shape-changing) and can undergo dramatic, rapid and reversible assembly and disassembly in response to cell signaling events, cell stress, during mitosis, and after changes in cytoplasmic "crowding" (as observed with condensates of the human myxovirus resistance protein MxA). Increasing evidence suggests that neuron-specific aberrations in phase-separation properties of RNA-binding proteins (e.g. FUS and TDP-43) and others (such as the microtubule-binding protein tau) contribute to the development of degenerative neurological diseases (e.g. amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer's disease). Thus, studies of liquid-like phase separation (LLPS) and the formation, structure and function of MLOs are of considerable importance in understanding basic cell biology and the pathogenesis of human diseases.

Discovery, Optimization, and Biological Characterization of 2,3,6-Trisubstituted Pyridine-Containing M4 Positive Allosteric Modulators.

Herein we describe the discovery and optimization of a new series of 2,3-disubstituted and 2,3,6-trisubstituted muscarinic acetylcholine receptor 4 (M4 ) positive allosteric modulators (PAMs). Iterative libraries enabled rapid exploration of one-dimensional structure-activity relationships (SAR) and identification of potency-enhancing heterocycle and N-alkyl pyrazole substituents. Further optimization led to identification of the potent, receptor-subtype-selective, brain-penetrant tool compound 24 (7-[3-[1-[(1-fluorocyclopentyl)methyl]pyrazol-4-yl]-6-methyl-2-pyridyl]-3-methoxycinnoline). It is efficacious in preclinical assays that are predictive of antipsychotic effects, producing dose-dependent reversal of amphetamine-induced hyperlocomotion in rats and mice, but not in M4 knockout mice. Cholinergic-related adverse effects observed in rats treated with 24 at unbound plasma concentrations more than 3-fold higher than an efficacious dose in the hyperlocomotion assay were fewer and less severe than those observed in rats treated with the nonselective M4 agonist xanomeline, suggesting a receptor-subtype-selective PAM has the potential for an improved safety profile.

Making sense of bodily sensations: Do shared cancer narratives influence symptom appraisal?

Though new or altered bodily sensations are a common occurrence they rarely transition to biomedically defined symptoms. When they do, sensations are subject to an appraisal process that can culminate in help-seeking. The transition has particular relevance for cancer diagnoses. Studies of 'symptom appraisal' in cancer patients typically conclude that failure to regard sensations as serious or 'symptom misattribution' results in lengthier help-seeking intervals. Though multiple influences on appraisal processes are acknowledged, including the socio-cultural context, detailed description and analyses of how socio-cultural factors shape appraisal is lacking. In this paper we explore one substantial component of the sociocultural context, namely, publicly recognised shared cancer narratives, and their impact on appraisal. We undertook a secondary analysis of 24 interviews with Scottish colorectal cancer patients originally completed in 2006-2007. Our analysis showed that fear, death and severity dominated cancer narratives and were frequently restated throughout interviews. Yet, early bodily changes were often mild and vague, were commonly experienced in the context of 'feeling well' and failed to match preconceived ideas of what cancer 'feels like'. Moreover, few perceived themselves to be 'at risk' of cancer and diagnoses were characterised as 'shocking' events. Participants engaged in self-monitoring strategies and severe or painful changes prompted help-seeking. Far from misattributing symptoms, responses to bodily changes were sensible and measured; responses are particularly apt in relation to current policy rhetoric, which urges measured use of services. Our findings have resonance across healthcare settings as patients are required to negotiate a narrow and challenging space when making decisions to seek help. There is a pressing need for a more realistic approach to symptom appraisal in order to reduce help-seeking intervals. Future awareness campaigns should emphasise the importance of vague/minor bodily changes although this will necessitate discussions with health professionals on referral thresholds to achieve earlier detection.

Physiological alterations of GS-CHO cells in response to adenosine monophosphate treatment.

Growth-arrested strategies (e.g. hypothermia and hyperosmolarity) have been widely employed to enhance cell-specific productivity (qP) in mammalian cell culture bioprocess. In addition to enhanced qP, alterations in cell physiology, such as cell size and cell cycle phase, have also attracted extensive attention under growth-arrested conditions. However, to date, very few reports on associations between physiological changes in growth-inhibiting approaches have been published. In this study, we explored associations between the physiological changes of GS-CHO cells in response to adenosine monophosphate (AMP) treatment. In dose response studies, AMP treatment resulted in suppressed proliferation, accumulated S-phase cells, increased cell size and enhanced qP. Subsequently, six GS-CHO clones exhibited the physiological alterations in varying degrees when treated with 7 mM AMP. But more importantly, a significant positive correlation between total intracellular protein content and mean electronic volume, an indicator of cell size (P < 0.01) was found, indicating that total intracellular protein was the determining factor in increasing cell size in this growth-arrested strategy. Besides, our results provide additional evidence that treatment with growth-arrested agents may increase cell size; the agent per se did not cause the increased productivity.

Illness identity as an important component of candidacy: Contrasting experiences of help-seeking and access to care in cancer and heart disease.

How and when we use health services or healthcare provision has dominated exploration of and debates around healthcare access. Levels of utilisation are assumed as a proxy for access. Yet, focusing on utilisation conceals an important aspect of the access conundrum: the relationships that patients and potential patients have with the healthcare system and the professionals within those systems. Candidacy has been proposed as an antidote to traditional utilisation models. The Candidacy construct offers the ability to include patient-professional aspects alongside utilisation and thus promotes a deeper understanding of access. Originally applied to healthcare access for vulnerable populations, additional socio-demographic factors, including age and ethnicity, have also been shown to influence the Candidacy process. Here we propose a further extension of the Candidacy construct and illustrate the importance of illness identities when accessing healthcare. Drawing on a secondary data analysis of three data sets of qualitative interviews from colorectal cancer and heart failure patients we found that though similar access issues are apparent pre-diagnosis, diagnosis marks a critical juncture in the experience of access. Cancer patients describe a person-centred responsive healthcare system where their patienthood requires only modest assertion. Cancer speaks for itself. In marked contrast heart failure patients, describe struggling within a seemingly impermeable system to understand their illness, its implications and their own legitimacy as patients. Our work highlights the pressing need for healthcare professionals, systems and policies to promote a person centred approach, which is responsive and timely, regardless of illness category. To achieve this, attitudes regarding the importance or priority afforded to different categories of illness need to be tackled as they directly influence ideas of Candidacy and consequently access and experiences of care.

Metabolic profiling of hematopoietic stem and progenitor cells during proliferation and differentiation into red blood cells.

An understanding of the metabolic profile of cell proliferation and differentiation should support the optimization of culture conditions for hematopoietic stem and progenitor cell (HSPC) proliferation, differentiation, and maturation into red blood cells. We have evaluated the key metabolic parameters during each phase of HSPC culture for red blood cell production in serum-supplemented (SS) and serum-free (SF) conditions. A simultaneous decrease in growth rate, total protein content, cell size, and the percentage of cells in the S/G2 phase of cell cycle, as well as an increase in the percentage of cells with a CD71(-)/GpA(+) surface marker profile, indicates HSPC differentiation into red blood cells. Compared with proliferating HSPCs, differentiating HSPCs showed significantly lower glucose and glutamine consumption rates, lactate and ammonia production rates, and amino acid consumption and production rates in both SS and SF conditions. Furthermore, extracellular acidification was associated with late proliferation phase, suggesting a reduced cellular metabolic rate during the transition from proliferation to differentiation. Under both SS and SF conditions, cells demonstrated a high metabolic rate with a mixed metabolism of both glycolysis and oxidative phosphorylation (OXPHOS) in early and late proliferation, an increased dependence on OXPHOS activity during differentiation, and a shift to glycolytic metabolism only during maturation phase. These changes indicate that cell metabolism may have an important impact on the ability of HSPCs to proliferate and differentiate into red blood cells.

Delivering digital health and well-being at scale: lessons learned during the implementation of the dallas program in the United Kingdom.

OBJECTIVE: To identify implementation lessons from the United Kingdom Delivering Assisted Living Lifestyles at Scale (dallas) program-a large-scale, national technology program that aims to deliver a broad range of digital services and products to the public to promote health and well-being. MATERIALS AND METHODS: Prospective, longitudinal qualitative research study investigating implementation processes. Qualitative data collected includes semi-structured e-Health Implementation Toolkit-led interviews at baseline/mid-point (n = 38), quarterly evaluation, quarterly technical and barrier and solutions reports, observational logs, quarterly evaluation alignment interviews with project leads, observational data collected during meetings, and ethnographic data from dallas events (n > 200 distinct pieces of qualitative data). Data analysis was guided by Normalization Process Theory, a sociological theory that aids conceptualization of implementation issues in complex healthcare settings. RESULTS: Five key challenges were identified: 1) The challenge of establishing and maintaining large heterogeneous, multi-agency partnerships to deliver new models of healthcare; 2) The need for resilience in the face of barriers and set-backs including the backdrop of continually changing external environments; 3) The inherent tension between embracing innovative co-design and achieving delivery at pace and at scale; 4) The effects of branding and marketing issues in consumer healthcare settings; and 5) The challenge of interoperability and information governance, when commercial proprietary models are dominant. CONCLUSIONS: The magnitude and ambition of the dallas program provides a unique opportunity to investigate the macro level implementation challenges faced when designing and delivering digital health and wellness services at scale. Flexibility, adaptability, and resilience are key implementation facilitators when shifting to new digitally enabled models of care.

Online Continuing Education for Expanding Clinicians' Roles in Breastfeeding Support.

Lack of health professional support is an important variable affecting mothers' achievement of breastfeeding goals. Online continuing education is a recognized pathway for disseminating content for improving clinicians' knowledge and supporting efforts to change practices. At the time we developed our project, free, accredited continuing education for physicians related to breastfeeding management that could be easily accessed using portable devices (via tablets/smartphones) was not available. Such resources were in demand, especially for facilities pursuing designation through the Baby-Friendly Hospital Initiative. We assembled a government, academic, health care provider, and professional society partnership to create such a tutorial that would address the diverse content needed for supporting breastfeeding mothers postdischarge in the United States. Our 1.5-hour-long continuing medical and nursing education was completed by 1606 clinicians (1172 nurses [73%] and 434 physicians [27%]) within 1 year. More than 90% of nurses and over 98% of physicians said the tutorial achieved its 7 learning objectives related to breastfeeding physiology, broader factors in infant feeding decisions and practices, the American Academy of Pediatrics' policy statement, and breastfeeding management/troubleshooting. Feedback received from the tutorial led to the creation of a second tutorial consisting of another 1.5 hours of continuing medical and nursing education related to breast examination and assessment prior to delivery, provision of anticipatory guidance to pregnant women interested in breastfeeding, maternity care practices that influence breastfeeding outcomes, breastfeeding preterm infants, breastfeeding's role in helping address disparities, and dispelling common myths. The tutorials contribute to achievement of 8 Healthy People 2020 Maternal, Infant and Child Health objectives.

Reducing dementia risk by targeting modifiable risk factors in mid-life: study protocol for the Innovative Midlife Intervention for Dementia Deterrence (In-MINDD) randomised controlled feasibility trial.

BACKGROUND: Dementia prevalence is increasing as populations live longer, with no cure and the costs of caring exceeding many other conditions. There is increasing evidence for modifiable risk factors which, if addressed in mid-life, can reduce the risk of developing dementia in later life. These include physical inactivity, low cognitive activity, mid-life obesity, high blood pressure, and high cholesterol. This study aims to assess the acceptability and feasibility and impact of giving those in mid-life, aged between 40 and 60 years, an individualised dementia risk modification score and profile and access to personalised on-line health information and goal setting in order to support the behaviour change required to reduce such dementia risk. A secondary aim is to understand participants' and practitioners' views of dementia prevention and explore the acceptability and integration of the Innovative Midlife Intervention for Dementia Deterrence (In-MINDD) intervention into daily life and routine practice. METHODS/DESIGN: In-MINDD is a multi-centre, primary care-based, single-blinded randomised controlled feasibility trial currently being conducted in four European countries (France, Ireland, the Netherlands and the UK). Participants are being recruited from participating general practices. Inclusion criteria will include age between 40 and 60 years; at least one modifiable risk factor for dementia risk (including diabetes, hypertension, obesity, renal dysfunction, current smoker, raised cholesterol, coronary heart disease, current or previous history of depression, self-reported sedentary lifestyle, and self-reported low cognitive activity) access to the Internet. Primary outcome measure will be a change in dementia risk modification score over the timescale of the trial (6 months). A qualitative process evaluation will interview a sample of participants and practitioners about their views on the acceptability and feasibility of the trial and the links between modifiable risk factors and dementia prevention. This work will be underpinned by Normalisation Process Theory. DISCUSSION: This study will explore the feasibility and acceptability of a risk profiler and on-line support environment to help individuals in mid-life assess their risk of developing dementia in later life and to take steps to alleviate that risk by tackling health-related behaviour change. Testing the intervention in a robust and theoretically informed manner will inform the development of a future, full-scale randomised controlled trial. TRIAL REGISTRATION: ISRCTN Registry: ISRCTN 98553005 (DOI: 10.1186/ISRCTN98553005).

Measuring dissolved oxygen to track erythroid differentiation of hematopoietic progenitor cells in culture.

As stem cell technologies move from the developmental to the commercial stage strategies must be developed to monitor culture operations. These will ensure consistency of differentiation programs and maintenance of optimum cell viability during production runs. Due to the sensitivity of stem cells to their environment, and their variability in response to external stimuli, accurate monitoring of in vitro conditions will be crucial for effective large-scale culturing of therapeutic stem cells. Here we describe a simple method to monitor the expansion and maturation of adult human haematopoietic stem/progenitor cells into red blood cells in vitro by measuring the oxygen consumption rate of cultures. Cell cultures followed a characteristic pattern of oxygen consumption that is reflective of in vivo erythroid maturation. This method could be easily developed as an online system to map erythroid differentiation and maturation of cultured cells as effectively as the more time consuming process of flow cytometric analysis of surface marker expression patterns.

Characterization of the novel positive allosteric modulator of the metabotropic glutamate receptor 4 ADX88178 in rodent models of neuropsychiatric disorders.

There is growing evidence that activation of metabotropic glutamate receptor 4 (mGlu4) leads to anxiolytic- and antipsychotic-like efficacy in rodent models, yet its relevance to depression-like reactivity remains unclear. Here, we present the pharmacological evaluation of ADX88178 [5-methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine], a novel potent, selective, and brain-penetrant positive allosteric modulator of the mGlu4 receptor in rodent models of anxiety, obsessive compulsive disorder (OCD), fear, depression, and psychosis. ADX88178 dose-dependently reduced the number of buried marbles in the marble burying test and increased open-arm exploration in the elevated plus maze (EPM) test, indicative of anxiolytic-like efficacy. Target specificity of the effect in the EPM test was confirmed using male and female mGlu4 receptor knockout mice. In mice, ADX88178 reduced the likelihood of conditioned freezing in the acquisition phase of the fear conditioning test, yet had no carryover effect in the expression phase. Also, ADX88178 dose-dependently reduced duration of immobility in the forced swim test, indicative of antidepressant-like efficacy. ADX88178 reduced DOI (2,5-dimethoxy-4-iodoamphetamine)-mediated head twitches (albeit with no dose-dependency), and MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]-induced locomotor hyperactivity in mice, but was inactive in the conditioned avoidance response test in rats. The compound showed good specificity as it had no effect on locomotor activity in mice and rats at efficacious doses. Thus, allosteric activation of mGlu4 receptors can be a promising new therapeutic approach for treatment of anxiety, OCD, fear-related disorders, and psychosis.