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Background The objective of this work to calculate prescribed quantity of an active pharmaceutical ingredient (API) in prescription medications for human use, to facilitate research on the prediction of amount of API released to the environment and create an open-data tool to facilitate spatiotemporal and long-term prescription trends for wider usage. Design We have developed an R package, PrAna to calculate the prescribed quantity (in kg) of an APIs by postcode using England’s national level prescription data provided by National Health Service, for the years 2015–2018. Datasets generated using PrAna can be visualized in a real-time interactive web-based tool, PrAnaViz to explore spatiotemporal and long-term trends. The visualisations can be customised by selecting month, year, API, and region. Results PrAnaViz’s targeted API approach is demonstrated with the visualisation of prescribed quantities of 14 APIs in the Bath and North East Somerset (BANES) region during 2018. Once the APIs list is loaded, the back end retrieves relevant data and populates the graphs based on user-defined data features in real-time. These plots include the prescribed quantity of APIs over a year, by month, and individual API by month, general practice, postcode, and medicinal form. The non-targeted API approach is demonstrated with the visualisation of clarithromycin prescribed quantities at different postcodes in the BANES region. Conclusion PrAna and PrAnaViz enables the analysis of spatio-temporal and long-term trends with prescribed quantities of different APIs by postcode. This can be used as a support tool for policymakers, academics and researchers in public healthcare, and environmental scientist to monitor different group of pharmaceuticals emitted to the environment and for prospective risk assessment of pharmaceuticals in the environment.

Lung cancer is a leading cause of death worldwide, with only a fraction of patients responding to immunotherapy. The correlation between increased T-cell infiltration and positive patient outcomes has motivated the search for therapeutics promoting T-cell infiltration. While transwell and spheroid platforms have been employed, these models lack flow and endothelial barriers, and cannot faithfully model T-cell adhesion, extravasation and migration through 3D tissue. Presented here is a 3D chemotaxis assay, in a lung tumor on chip model with 3D endothelium (LToC-Endo), to address this need. The described assay consists of a vascular tubule cultured under rocking flow, through which T-cells are added; a collagenous stromal barrier, through which T-cells infiltrate; and a chemoattractant/tumor compartment. Here, activated T-cells extravasate and infiltrate in response to gradients of rhCXCL11 and rhCXCL12. Adopting a T-cell activation protocol with a rest period enables proliferative burst prior to introducing T-cells into chips, increases T-cell expression of CXCR3 and CXCR4 receptors, and enhances assay sensitivity. In addition, incorporating this rest recovers endothelial activation in response to rhCXCL12. As a final control, we show that blocking ICAM-1 interferes with T-cell adhesion and chemotaxis. This microphysiological system, which mimics in vivo stromal and vascular barriers, can be used to evaluate potentiation of immune chemotaxis into tumors while probing for vascular responses to potential therapeutics. Finally, we propose a translational strategy by which this assay could be linked to preclinical and clinical models to support human dose prediction, personalized medicine, and the reduction, refinement, and replacement of animal models.

Inducible T cell Co-Stimulator (ICOS, CD278) is a receptor belonging to the CD28/CTLA-4 superfamily that is predominantly expressed on T cells following T cell receptor (TCR) activation and plays an important role in regulating adaptive immune response. ICOS represents possible node of therapeutic intervention for cancer, and as such, is under clinical evaluation. Although ICOS agonists have been shown to promote significant monotherapeutic efficacy in non-clinical studies, most patients do not respond to the single-agent approaches. Therefore, it is crucial to better understand the potential mechanisms of action as well as to develop novel combination strategies and biomarkers to best position therapeutic in the clinic. In an effort to identify novel combination partners and/or biomarkers for ICOS agonist, we utilized pooled in vivo genetic screening via CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 genome editing in the syngeneic CT26 colorectal mouse tumor model alongside agonist treatment for the target. We evaluated 509 druggable genes with four sgRNAs targeting each gene to identify genes where loss in CT26 tumor cells can increase sensitivity or resistance to this treatment. With a false discovery rate (FDR) Citation Format: Qila Sa, Hongyu Liang, Rumen Kostadinov, Jong Yu, Jeremy Waight, Stephanie Van Horn, Qing Xie, Sue Griffin, Chris Hopson, Ning Sun. In vivo CRISPR screening in syngeneic tumor models to identify novel combinations for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A18.

Only a subset of patients durable clinical responses to aPD-1 and/or aCTLA-4 immunotherapies, thus, developing new therapeutic agents to increase the proportion of responding patients is a priority. Combining aPD-1 with aLAG-3 has shown promising results; however, lack of mechanistic understanding of aPD-1/aLAG-3 synergy remains a barrier for its optimal clinical use. Here, we examined the mechanism of aPD-1/aLAG-3 synergy in multiple mouse models using flow cytometry and single cell RNA sequencing. Combined aPD-1/aLAG-3 immunotherapy significantly improved the survival of CT26 (BALB/c; colon carcinoma) and MCA-205 (C57BL/6; sarcoma) tumor-bearing mice compared to monotherapy. Regulatory T cells (Tregs) suppressed response to this therapy, as in the absence of CD4+ T cells, 100% of mice responded. To understand how responders overcome Treg suppression, we performed an in-depth analysis of tumor-infiltrating lymphocytes (TIL) comparing mice that responded to treatment (decreased tumor size post-treatment) to non-responders (same tumor growth trajectory as control). Responders had reduced Foxp3+ CD4+ Tregs in comparison to non-responders and, in addition, those Tregs had a ‘fragile’ phenotype, including a pro-inflammatory cytokine profile (TNF-a; IFN-g), increased LAG-3, and decreased NRP1 expression. Within responders, CD8+ TIL exhibited increased frequency, effector cytokine production (TNF-a; IFN-g), and LAG-3 expression as compared to non-responders. Together, these data suggest that aPD-1/aLAG-3 can reduce Treg frequency and function leading to expansion of active tumor-specific CD8+ T cells capable of supporting tumor regression and improved survival. Supported by the Providence Portland Medical Foundation and GlaxoSmithKline

To explore whether the variation in clinical response to immune checkpoint blockade (ICB) reflects intrinsic characteristics of tumor-infiltrating lymphocytes (TIL), TILs from multiple tumor types were analyzed by multiparameter flow cytometry. Recent work identified CD39+CD103+ double positive (DP) CD8 TIL as tumor-reactive, therefore we assessed this phenotype in primary NSCLC (n=28), BCa (n=23), HNSCC (n=23), and RCC (n=23) specimens. TILs from NSCLC and HNSCC had significantly higher frequencies of DP T cells than BCa and RCC (NSCLC: median=34.2, IQR=15.0–53.8%; HNSCC: median=28.6%, IQR=13.5–44.5%), while TILs from BCa and RCC had low frequencies of DP T cells (BCa: median=2.4%, IQR=1.1–3.9%; RCC: median=4.2%, IQR=1.5–16.6%). Additionally, DP cells in NSCLC and HNSCC were co-expressed immune checkpoint markers PD-1, TIM-3, and LAG-3 at a higher frequency than RCC or BCa (P Supported by GlaxoSmithKline and the Providence Portland Medical Foundation

DESPITE EMERGING EVIDENCE OF THE contributors to high-quality family day care, a comprehensive comparison of international family day care systems has not been undertaken. The aim of this paper is to compare regulated family day care (FDC) in Australia, Canada, England and Wales, Germany, Ireland, Japan, Norway, New Zealand, Sweden, and the USA, using standard information about FDC usage and each country's structural characteristics, regulatory approach, quality assurance indicators, supervision and support, and sector challenges. A template was developed by representatives of the International Family Day Care Organisation, including academics, FDC providers, and FDC educators and management staff. The paper demonstrates that, although the existing body of published research is sufficient to provide clear guidance for developing and maintaining quality FDC, this knowledge is not always being applied in current practices. Three practices, each pertaining to several countries, are of concern: (a) minimal requirements for being a regulated FDC provider, (b) minimal quality assurance guidelines, and (c) minimal provider supports. There is great potential to learn from countries that have a high level of success in providing high-quality FDC and strong support for providers and families.

Substantial evidence exists that social circumstances can affect children’s language development. As a result many children in socially deprived areas start school with delayed language, which may persist and adversely affect their attainment. We assessed the language of children in seven reception classes in a London (UK) borough and followed the progress of children with English as their first language (E1L) and with English as an additional language (EAL) during their first 2 years at school. Significant differences were found between schools. The effect of social factors on performance was reflected in a high correlation between the mean language score for each school and the percentage of children in the school receiving the pupil premium. Many of the children with EAL had very low scores reflecting their limited exposure to English prior to starting school. Most of these children attended schools where children with E1L also had low scores increasing the demands on the schools and their teachers. Children who had low initial scores made modest but significant progress during their reception year but failed to improve further during year 1 despite having non-verbal ability appropriate for their age. These results support previous findings that social deprivation can seriously delay language development, and that many children start school with weak communication skills. They add to previous findings by showing that the level of delay may differ substantially across schools in the same borough, by reporting data on children with EAL and by showing that children struggle to improve their abilities in the first 2 years of school.

Alzheimer's disease (AD) brain is characterized by excess deposition of amyloid beta-peptide (Abeta), particularly the 42-amino acid peptide [Abeta(1-42)] and by extensive oxidative stress. Several sources of the oxidative stress and inflammatory cascades are likely, including that induced by advanced glycation end products, microglial activation, and by Abeta(1-42) and its sequelae. This review briefly examines each of these sources of oxidative stress and inflammation in AD brain and discusses their potential roles in the clinical progression of AD dementia.

The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents an attractive target for therapeutic intervention. To investigate the primary phenotypes mediated by increased pathway signaling in a clean, patient-relevant context, an activating PIK3CA mutation (H1047R) was knocked-in to an endogenous allele of the MCF10A non-tumorigenic human breast epithelial cell line. Introduction of an endogenously mutated PIK3CA allele resulted in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype, compared to isogenic wild-type cells. The invasive phenotype was linked to enhanced PIP(3) production via a S6K-IRS positive feedback mechanism. Moreover, potent and selective inhibitors of PI3K were highly effective in reversing this phenotype, which is optimally revealed in 3-dimensional cell culture. In contrast, inhibition of Akt or mTOR exacerbated the invasive phenotype. Our results suggest that invasion is a core phenotype mediated by increased PTEN/PI3K pathway activity and that therapeutic agents targeting different nodes of the PI3K pathway may have dramatic differences in their ability to reverse or promote cancer metastasis.

Targeting tumour metabolism is becoming a major new area of pharmaceutical endeavour. Consequently, a systematic search to define whether there are specific energy source dependencies in tumours, and how these might be dictated by upstream driving genetic mutations, is required. The PI3K-AKT-mTOR signalling pathway has a seminal role in regulating diverse cellular processes including cell proliferation and survival, but has also been associated with metabolic dysregulation. In this study, we sought to define how mutations within PI3KCA may affect the metabolic dependency of a cancer cell, using precisely engineered isogenic cell lines. Studies revealed gene expression signatures in PIK3CA mutant cells indicative of a consistent up-regulation of glycolysis. Interestingly, the genes up- and down-regulated varied between isogenic models suggesting that the primary node of regulation is not the same between models. Additional gene expression changes were also observed, suggesting that metabolic pathways other than glycolysis, such as glutaminolysis, were also affected. Nutrient dependency studies revealed that growth of PIK3CA mutant cells is highly dependent on glucose, whereas glutamine dependency is independent of PIK3CA status. In addition, the glucose dependency exhibited by PIK3CA mutant cells could not be overridden by supplementation with other nutrients. This specific dependence on glucose for growth was further illustrated by studies evaluating the effects of targeted disruption of the glycolytic pathway using siRNA and was also found to be present across a wider panel of cancer cell lines harbouring endogenous PIK3CA mutations. In conclusion, we have found that PIK3CA mutations lead to a shift towards a highly glycolytic phenotype, and that despite suggestions that cancer cells are adept at utilising alternative nutrient sources, PIK3CA mutant cells are not able to compensate for glucose withdrawal. Understanding the metabolic dependencies of PIK3CA mutant cancers will provide critical information for the design of effective therapies and tumour visualisation strategies.

Continuing this series on going back to the principles behind inclusive practice to ensure it is implemented in a meaningful way, Sue Griffin considers why we should be conscious of the significance of the language we use.

Continuing this series reflecting on best practice for inclusion, equality and diversity, Sue Griffin considers planning around festivals, foods and cultural life and making it appropriate for your particular setting.

Your setting probably has dolls of all ethnicities and it may no longer seem necessary to consider why this is good practice. In this new series, Sue Griffin explores how going back to the reasons why we do things is hugely valuable

In the final article on equality and inclusion, Sue Griffin looks at ways of responding to discriminatory behaviour.

In the penultimate article on our series on inclusion and equality, Sue Griffin reviews some historical influences and the framework of the law today.

In the tenth article in the equality and inclusion series, Sue Griffin discusses ways of celebrating diversity.

Our series on inclusion and equality continues with Sue Griffin considering some of the implications of being an inclusive setting.

After earlier articles exploring different aspects of equality and inclusion, Sue Griffin moves on to consider what this means for resources in early years settings.

Disability is the theme of the sixth article by Sue Griffin in our series on inclusion and equality.

The fifth article in the series on equality and inclusion by Sue Griffin focuses on culture.

Sue Griffin continues our series on equality and inclusion by exploring issues concerning gender.

In the first article of a new series on the issues surrounding equality and inclusion, Sue Griffin outlines why these are so important in the early years sector.

In the penultimate article in our series about babies and under-threes, Sue Griffin reflects on the importance of practitioners’ relationships with parents

Our series on babies and under-threes moves on to consider the significance of play. Sue Griffin builds on previous articles in the series, reviewing practice in providing play opportunities for the youngest children.

In the ninth article in this year’s series on babies and under-threes, Sue Griffin turns attention to social development.

Child consultant Sue Griffin looks at good safeguarding practice.

When you work with babies and under-threes, you give them a lot of personal care. Sue Griffin continues our series on the youngest children by looking at some of the issues for good practice in this aspect of your work.

In the fourth of a series of articles about under-threes, Sue Griffin explores the development of motor skills.

In the second of our series on working with babies and under-threes, Sue Griffin looks at aspects of practice relating to nutrition.

In the first of our series of articles on babies and under-threes, Sue Griffin looks at the essential ingredients of working with the youngest children.

The Statutory Framework for the Early Years Foundation Stage says that providers must carry out a risk assessment, so it will be sensible for you to start this as soon as possible, to be ready for implementation of EYFS. Sue Griffin looks at what is involved in assessing risk and acting on what you find.

We often think about children’s development by dividing it up in a variety of ways. Sue Griffin reminds us that we must always keep the whole child in view.

When the Childcare Act 2006 became law in July this year, the Minister for Children, Beverley Hughes, called it ‘an historic and radical piece of legislation – the first solely dedicated to early years and childcare – that redraws the boundaries of the welfare state’. Sue Griffin explains the key elements of this important new law

You and the children should make the most of opportunities to be outdoors. But there are safety issues to keep in mind. Sue Griffin reviews the value of outdoor play and reminds you of the key safety factors

What goes on in the kitchen of your childminding setting? Children eating meals together – and helping to prepare food and clear up afterwards? Play activities, especially some of the messy ones? Great! But how do you make sure it’s a safe place for children to be? Sue Griffin offers a few reminders

You should try to take children outside regularly, whether on occasional special outings or just for a short walk. This can present challenges and needs some careful forethought. Sue Griffin looks at some of the safety aspects, and why it’s worth the effort!

One of the cornerstones of central government policy for children and families in England is now children’s centres. Sue Griffin explains the aims and aspirations of this policy, and raises some of the uncertainties surrounding it

Concluding this series on working in a team, Sue Griffin offers advice on taking part in meetings. Read on for tips on preparing, giving your opinions, expressing disagreement, and how to set an agenda and act as chair.