The impact of COVID-19 on mental health and well-being in critical care nurses - a longitudinal, qualitative study.

BACKGROUND: The COVID-19 pandemic has had both a psychological and physiological effect on the human race. For those working in health care, particularly in critical care, the pandemic has put unprecedented strain on staff. Witnessing suffering during crisis in an organizational setting can be a traumatic experience and critical care nurses often risked, not only their own lives, but their psychological well-being, so that those infected with the virus might have a better chance at survival. AIM: The aim of this study was to explore the challenges to mental health and psychological well-being experienced by Critical Care Nurses during the COVID-19 pandemic. STUDY DESIGN: A longitudinal, qualitative study involving semi-structured interviews with 54 critical care nurses across 38 hospitals in the United Kingdom and Ireland. Interviews were transcribed verbatim and analysed using thematic analysis. RESULTS: Four key themes were identified which represent the challenges faces by critical care nurses during the COVID-19 pandemic: Lack of control, Psychological trauma, Unexpected leadership, Public-political betrayal. CONCLUSIONS: While public-political praise may lead to a short-term lift in morale for front line workers; where it is not accompanied by practical support in terms of appropriate equipment, leadership, emotional support and renumeration it is likely to be damaging in the longer term. RELEVANCE TO CLINICAL PRACTICE: This study has provided a greater understanding of the factors which affected the well-being and mental health of critical care nurses during a global pandemic.

What can the uptake of online pharmacy education in Scotland, during the COVID-19 pandemic, tells us about the future of CPD?

Pharmacy CPD in Scotland has been evolving towards online, asynchronous delivery since 2014. The COVID-19 pandemic accelerated this movement by restricting face-to-face education (Zlotos & Stewart, 2021). This study utilised Google analytics to describe web traffic, and electronic e-learning completion records to describe learner activity on the Virtual Learning Environment of a national CPD provider in Scotland. The aim was to describe patterns of learning activity in the years spanning the COVID-19 pandemic, to help predict what future education practice may look like. This study identified that there was an increase in estimated time spent on learning from 8085.5 vs 16,061.5 hours of learning in 2018-19 and 2020-21, respectively. Completion of non-mandatory clinical modules and mandatory service modules increased each year. Mandatory, service focussed modules were most popular each year and the number of completions peaked to coincide with new services or updated content. The findings suggest asynchronous, online pharmacy education continues to grow in popularity. CPD providers should prioritise Mandatory, Service focussed education for pharmacy staff; although, they cannot neglect non-mandatory and Clinical education too. Future education for CPD should be designed to reflect the growing and diverse learner population.

A network of phosphatidylinositol (4,5)-bisphosphate (PIP2) binding sites on the dopamine transporter regulates amphetamine behavior in Drosophila Melanogaster.

Reward modulates the saliency of a specific drug exposure and is essential for the transition to addiction. Numerous human PET-fMRI studies establish a link between midbrain dopamine (DA) release, DA transporter (DAT) availability, and reward responses. However, how and whether DAT function and regulation directly participate in reward processes remains elusive. Here, we developed a novel experimental paradigm in Drosophila melanogaster to study the mechanisms underlying the psychomotor and rewarding properties of amphetamine (AMPH). AMPH principally mediates its pharmacological and behavioral effects by increasing DA availability through the reversal of DAT function (DA efflux). We have previously shown that the phospholipid, phosphatidylinositol (4, 5)-bisphosphate (PIP2), directly interacts with the DAT N-terminus to support DA efflux in response to AMPH. In this study, we demonstrate that the interaction of PIP2 with the DAT N-terminus is critical for AMPH-induced DAT phosphorylation, a process required for DA efflux. We showed that PIP2 also interacts with intracellular loop 4 at R443. Further, we identified that R443 electrostatically regulates DA efflux as part of a coordinated interaction with the phosphorylated N-terminus. In Drosophila, we determined that a neutralizing substitution at R443 inhibited the psychomotor actions of AMPH. We associated this inhibition with a decrease in AMPH-induced DA efflux in isolated fly brains. Notably, we showed that the electrostatic interactions of R443 specifically regulate the rewarding properties of AMPH without affecting AMPH aversion. We present the first evidence linking PIP2, DAT, DA efflux, and phosphorylation processes with AMPH reward.

Evaluation of Inter-Professional Education (IPE) with medical, nursing and pharmacy students through a simulated IPL Educational Intervention.

INTRODUCTION: Inter-Professional Education (IPE) is becoming an integral part of many professional programmes throughout the United Kingdom, ensuring health professionals are competent to work as part of an inter-professional team upon entry into their profession. IPE has become a fundamental component of curriculum content in health and social care degrees. AIMS: Research aim - to evaluate a simulated IPE intervention. METHODS: A one day IPE intervention, "Evening On-Call" was run involving nursing and medical students and pre-registration pharmacists (student pharmacists in year 5 of training) in an on-call setting. This IPE incorporated manikin and actor patients in a simulated ward. During the intervention, the 3 groups of students are assessed under observation on their clinical, prioritisation and communication skills. Participants perceptions of this intervention were evaluated by completion of a questionnaire to capture their perceptions regarding the experience, the pre-IPL briefing and post-IPL feedback and perceived relevance of this training. Free text sections collected additional comments and a follow-up questionnaire was sent 6 months later. RESULTS: Initial questionnaire feedback was predominantly positive for each professional group. The majority perceived the simulated IPL had given them a greater understanding of other professionals' roles, had enhanced their professional confidence and would help them prioritise workload once qualified. The 6-months follow-up questionnaire supported the initial questionnaire findings. Some responses highlighted that participants believed the simulated IPL had helped them work more effectively with other healthcare professionals, communicate more effectively and better prioritise their workload. There may be some evidence of sustained self-reported effectiveness in teaching certain professional and clinical skills to participants using this type of simulated intervention.

Neuroendocrine drivers of risk and resilience: The influence of metabolism & mitochondria.

The manifestation of risk versus resilience has been considered from varying perspectives including genetics, epigenetics, early life experiences, and type and intensity of the challenge with which the organism is faced. Although all of these factors are central to determining risk and resilience, the current review focuses on what may be a final common pathway: metabolism. When an organism is faced with a perturbation to the environment, whether internal or external, appropriate energy allocation is essential to resolving the divergence from equilibrium. This review examines the potential role of metabolism in the manifestation of stress-induced neural compromise. In addition, this review details the current state of knowledge on neuroendocrine factors which are poised to set the tone of the metabolic response to a systemic challenge. The goal is to provide an essential framework for understanding stress in a metabolic context and appreciation for key neuroendocrine signals.

Using the Analytical Target Profile to Drive the Analytical Method Lifecycle.

Quality by design (ICH-Topic Q8) requires a prospective summary of the desired quality characteristics of a drug product. This is known as the Quality Target Product Profile (QTPP), which forms the basis for the design and development of the product. An analogous term has been established for analytical procedures called the Analytical Target Profile (ATP). The ATP, in a similar fashion to the QTPP, prospectively summarizes the requirements associated with a measurement on a quality attribute which needs to be met by an analytical procedure. Criteria defined in the ATP relate to the maximum uncertainty associated with the reportable result that is required to maintain acceptable confidence in the quality decision made from the result. The ATP is used to define and assess the fitness of an analytical procedure in the development phase and during all changes across the analytical lifecycle. One or more analytical procedures can meet the requirements of an ATP. The ATP can be applied to any quality attribute across any pharmaceutical modality where an analytical procedure is used to generate a reportable result, and this paper provides examples from three of these modalities: small molecules, oligonucleotides, and vaccines. Some key performance characteristics will be discussed for each ATP, namely specificity, accuracy, and precision, taking into account the expected range of the analyte. The combination of accuracy and precision into a combined uncertainty characteristic is also discussed as a more holistic approach. The use of the ATP concept will help focus attention on the properties of a method which impact quality decisions rather than method descriptions and may enable greater regulatory flexibility across the lifecycle using established conditions based on method performance criteria as proposed in the Step 2 version of ICHQ12. The revision of ICHQ2(R1) and development of the new ICHQ14 guideline (Analytical Procedure Development) will provide a golden opportunity to harmonize the definition of new QbD concepts such as the ATP.

Embracing an integromic approach to tissue biomarker research in cancer: Perspectives and lessons learned.

Modern approaches to biomedical research and diagnostics targeted towards precision medicine are generating 'big data' across a range of high-throughput experimental and analytical platforms. Integrative analysis of this rich clinical, pathological, molecular and imaging data represents one of the greatest bottlenecks in biomarker discovery research in cancer and other diseases. Following on from the publication of our successful framework for multimodal data amalgamation and integrative analysis, Pathology Integromics in Cancer (PICan), this article will explore the essential elements of assembling an integromics framework from a more detailed perspective. PICan, built around a relational database storing curated multimodal data, is the research tool sitting at the heart of our interdisciplinary efforts to streamline biomarker discovery and validation. While recognizing that every institution has a unique set of priorities and challenges, we will use our experiences with PICan as a case study and starting point, rationalizing the design choices we made within the context of our local infrastructure and specific needs, but also highlighting alternative approaches that may better suit other programmes of research and discovery. Along the way, we stress that integromics is not just a set of tools, but rather a cohesive paradigm for how modern bioinformatics can be enhanced. Successful implementation of an integromics framework is a collaborative team effort that is built with an eye to the future and greatly accelerates the processes of biomarker discovery, validation and translation into clinical practice.

Integrated tumor identification and automated scoring minimizes pathologist involvement and provides new insights to key biomarkers in breast cancer.

Digital image analysis (DIA) is becoming central to the quantitative evaluation of tissue biomarkers for discovery, diagnosis and therapeutic selection for the delivery of precision medicine. In this study, automated DIA using a new purpose-built software platform (QuPath) is applied to a cohort of 293 breast cancer patients to score five biomarkers in tissue microarrays (TMAs): ER, PR, HER2, Ki67 and p53. This software is able to measure IHC expression following fully automated tumor recognition in the same immunohistochemical (IHC)-stained tissue section, as part of a rapid workflow to ensure objectivity and accelerate biomarker analysis. The digital scores produced by QuPath were compared with manual scores by a pathologist and shown to have a good level of concordance in all cases (Cohen's κ>0.6), and almost perfect agreement for the clinically relevant biomarkers ER, PR and HER2 (κ>0.86). To assess prognostic value, cutoff thresholds could be applied to both manual and automated scores using the QuPath software, and survival analysis performed for 5-year overall survival. DIA was shown to be capable of replicating the statistically significant stratification of patients achieved using manual scoring across all biomarkers (P<0.01, log-rank test). Furthermore, the image analysis scores were shown to consistently lead to statistical significance across a wide range of potential cutoff thresholds, indicating the robustness of the method, and identify sub-populations of cases exhibiting different expression patterns within the p53 and Ki67 data sets that warrant further investigation. These findings have demonstrated QuPath's suitability for fast, reproducible, high-throughput TMA analysis across a range of important biomarkers. This was achieved using our tumor recognition algorithms for IHC-stained sections, trained interactively without the need for any additional tumor recognition markers, for example, cytokeratin, to obtain greater insight into the relationship between biomarker expression and clinical outcome applicable to a range of cancer types.

Withdrawal from repeated morphine administration augments expression of the RhoA network in the nucleus accumbens to control synaptic structure.

The nucleus accumbens (NAc) is a critical brain reward region that mediates the rewarding effects of drugs of abuse, including those of morphine and other opiates. Drugs of abuse induce widespread alterations in gene transcription and dendritic spine morphology in medium spiny neurons (MSNs) of the NAc that ultimately influence NAc excitability and hence reward-related behavioral responses. Growing evidence indicates that within the NAc small GTPases are common intracellular targets of drugs of abuse where these molecules regulate drug-mediated transcriptional and spine morphogenic effects. The RhoA small GTPase is among the most well-characterized members of the Ras superfamily of small GTPases, and recent work highlights an important role for hippocampal RhoA in morphine-facilitated reward behavior. Despite this, it remains unclear how RhoA pathway signaling in the NAc is affected by withdrawal from morphine. To investigate this question, using subcellular fractionation and subsequent protein profiling we examined the expression of key components of the RhoA pathway in NAc nuclear, cytoplasmic, and synaptosomal compartments during multiple withdrawal periods from repeated morphine administration. Furthermore, using in vivo viral-mediated gene transfer, we determined the consequences of revealed RhoA pathway alterations on NAc MSN dendritic spine morphology. Our findings reveal an important role for RhoA signaling cascades in mediating the effects of long-term morphine withdrawal on NAc MSN dendritic spine elimination. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Validation of the systematic scoring of immunohistochemically stained tumour tissue microarrays using QuPath digital image analysis.

AIMS: Output from biomarker studies involving immunohistochemistry applied to tissue microarrays (TMA) is limited by the lack of an efficient and reproducible scoring methodology. In this study, we examine the functionality and reproducibility of biomarker scoring using the new, open-source, digital image analysis software, QuPath. METHODS AND RESULTS: Three different reviewers, with varying experience of digital pathology and image analysis, applied an agreed QuPath scoring methodology to CD3 and p53 immunohistochemically stained TMAs from a colon cancer cohort (n = 661). Manual assessment was conducted by one reviewer for CD3. Survival analyses were conducted and intra- and interobserver reproducibility assessed. Median raw scores differed significantly between reviewers, but this had little impact on subsequent analyses. Lower CD3 scores were detected in cases who died from colorectal cancer compared to control cases, and this finding was significant for all three reviewers (P-value range = 0.002-0.02). Higher median p53 scores were generated among cases who died from colorectal cancer compared with controls (P-value range = 0.04-0.12). The ability to dichomotise cases into high versus low expression of CD3 and p53 showed excellent agreement between all three reviewers (kappa score range = 0.82-0.93). All three reviewers produced dichotomised expression scores that resulted in very similar hazard ratios for colorectal cancer-specific survival for each biomarker. Results from manual and QuPath methods of CD3 scoring were comparable, but QuPath scoring revealed stronger prognostic stratification. CONCLUSIONS: Scoring of immunohistochemically stained tumour TMAs using QuPath is functional and reproducible, even among users of limited experience of digital pathology images, and more accurate than manual scoring.

Targeted Epigenetic Remodeling of the Cdk5 Gene in Nucleus Accumbens Regulates Cocaine- and Stress-Evoked Behavior.

UNLABELLED: Recent studies have implicated epigenetic remodeling in brain reward regions following psychostimulant or stress exposure. It has only recently become possible to target a given type of epigenetic remodeling to a single gene of interest, and to probe the functional relevance of such regulation to neuropsychiatric disease. We sought to examine the role of histone modifications at the murine Cdk5 (cyclin-dependent kinase 5) locus, given growing evidence of Cdk5 expression in nucleus accumbens (NAc) influencing reward-related behaviors. Viral-mediated delivery of engineered zinc finger proteins (ZFP) targeted histone H3 lysine 9/14 acetylation (H3K9/14ac), a transcriptionally active mark, or histone H3 lysine 9 dimethylation (H3K9me2), which is associated with transcriptional repression, specifically to the Cdk5 locus in NAc in vivo We found that Cdk5-ZFP transcription factors are sufficient to bidirectionally regulate Cdk5 gene expression via enrichment of their respective histone modifications. We examined the behavioral consequences of this epigenetic remodeling and found that Cdk5-targeted H3K9/14ac increased cocaine-induced locomotor behavior, as well as resilience to social stress. Conversely, Cdk5-targeted H3K9me2 attenuated both cocaine-induced locomotor behavior and conditioned place preference, but had no effect on stress-induced social avoidance behavior. The current study provides evidence for the causal role of Cdk5 epigenetic remodeling in NAc in Cdk5 gene expression and in the control of reward and stress responses. Moreover, these data are especially compelling given that previous work demonstrated opposite behavioral phenotypes compared with those reported here upon Cdk5 overexpression or knockdown, demonstrating the importance of targeted epigenetic remodeling tools for studying more subtle molecular changes that contribute to neuropsychiatric disease. SIGNIFICANCE STATEMENT: Addiction and depression are highly heritable diseases, yet it has been difficult to identify gene sequence variations that underlie this heritability. Gene regulation via epigenetic remodeling is an additional mechanism contributing to the neurobiological basis of drug and stress exposure. In particular, epigenetic regulation of the Cdk5 gene alters responses to cocaine and stress in mouse and rat models. In this study, we used a novel technology, zinc-finger engineered transcription factors, to remodel histone proteins specifically at the Cdk5 gene. We found that this is sufficient to regulate the expression of Cdk5 and results in altered behavioral responses to cocaine and social stress. These data provide compelling evidence of the significance of epigenetic regulation in the neurobiological basis of reward- and stress-related neuropsychiatric disease.

Statin use, candidate mevalonate pathway biomarkers, and colon cancer survival in a population-based cohort study.

BACKGROUND: Statin use after colorectal cancer diagnosis may improve survival but evidence from observational studies is conflicting. The anti-cancer effect of statins may be restricted to certain molecular subgroups. In this population-based cohort study, the interaction between p53 and 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGCR) expression, KRAS mutations, and the association between statin use and colon cancer survival was assessed. METHODS: The cohort consisted of 740 stage II and III colon cancer patients diagnosed between 2004 and 2008. Statin use was determined through clinical note review. Tissue blocks were retrieved to determine immunohistochemical expression of p53 and HMGCR in tissue microarrays and the presence of KRAS mutations in extracted DNA. Cox proportional hazards models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for colorectal cancer-specific and overall survival. RESULTS: Statin use was not associated with improved cancer-specific survival in this cohort (HR=0.91, 95% CI 0.64-1.28). Statin use was also not associated with improved survival when the analyses were stratified by tumour p53 (wild-type HR=1.31, 95% CI 0.67-2.56 vs aberrant HR=0.80, 95% CI 0.52-1.24), HMGCR (HMGCR-high HR=0.69, 95% CI 0.40-1.18 vs HMGCR-low HR=1.10, 95% CI 0.66-1.84), and KRAS (wild-type HR=0.73, 95% CI 0.44-1.19 vs mutant HR=1.21, 95% CI 0.70-2.21) status. CONCLUSIONS: Statin use was not associated with improved survival either independently or when stratified by potential mevalonate pathway biomarkers in this population-based cohort of colon cancer patients.

A gene-signature progression approach to identifying candidate small-molecule cancer therapeutics with connectivity mapping.

BACKGROUND: Gene expression connectivity mapping has gained much popularity recently with a number of successful applications in biomedical research testifying its utility and promise. Previously methodological research in connectivity mapping mainly focused on two of the key components in the framework, namely, the reference gene expression profiles and the connectivity mapping algorithms. The other key component in this framework, the query gene signature, has been left to users to construct without much consensus on how this should be done, albeit it has been an issue most relevant to end users. As a key input to the connectivity mapping process, gene signature is crucially important in returning biologically meaningful and relevant results. This paper intends to formulate a standardized procedure for constructing high quality gene signatures from a user's perspective. RESULTS: We describe a two-stage process for making quality gene signatures using gene expression data as initial inputs. First, a differential gene expression analysis comparing two distinct biological states; only the genes that have passed stringent statistical criteria are considered in the second stage of the process, which involves ranking genes based on statistical as well as biological significance. We introduce a "gene signature progression" method as a standard procedure in connectivity mapping. Starting from the highest ranked gene, we progressively determine the minimum length of the gene signature that allows connections to the reference profiles (drugs) being established with a preset target false discovery rate. We use a lung cancer dataset and a breast cancer dataset as two case studies to demonstrate how this standardized procedure works, and we show that highly relevant and interesting biological connections are returned. Of particular note is gefitinib, identified as among the candidate therapeutics in our lung cancer case study. Our gene signature was based on gene expression data from Taiwan female non-smoker lung cancer patients, while there is evidence from independent studies that gefitinib is highly effective in treating women, non-smoker or former light smoker, advanced non-small cell lung cancer patients of Asian origin. CONCLUSIONS: In summary, we introduced a gene signature progression method into connectivity mapping, which enables a standardized procedure for constructing high quality gene signatures. This progression method is particularly useful when the number of differentially expressed genes identified is large, and when there is a need to prioritize them to be included in the query signature. The results from two case studies demonstrate that the approach we have developed is capable of obtaining pertinent candidate drugs with high precision.

QUADrATiC: scalable gene expression connectivity mapping for repurposing FDA-approved therapeutics.

BACKGROUND: Gene expression connectivity mapping has proven to be a powerful and flexible tool for research. Its application has been shown in a broad range of research topics, most commonly as a means of identifying potential small molecule compounds, which may be further investigated as candidates for repurposing to treat diseases. The public release of voluminous data from the Library of Integrated Cellular Signatures (LINCS) programme further enhanced the utilities and potentials of gene expression connectivity mapping in biomedicine. RESULTS: We describe QUADrATiC ( http://go.qub.ac.uk/QUADrATiC ), a user-friendly tool for the exploration of gene expression connectivity on the subset of the LINCS data set corresponding to FDA-approved small molecule compounds. It enables the identification of compounds for repurposing therapeutic potentials. The software is designed to cope with the increased volume of data over existing tools, by taking advantage of multicore computing architectures to provide a scalable solution, which may be installed and operated on a range of computers, from laptops to servers. This scalability is provided by the use of the modern concurrent programming paradigm provided by the Akka framework. The QUADrATiC Graphical User Interface (GUI) has been developed using advanced Javascript frameworks, providing novel visualization capabilities for further analysis of connections. There is also a web services interface, allowing integration with other programs or scripts. CONCLUSIONS: QUADrATiC has been shown to provide an improvement over existing connectivity map software, in terms of scope (based on the LINCS data set), applicability (using FDA-approved compounds), usability and speed. It offers potential to biological researchers to analyze transcriptional data and generate potential therapeutics for focussed study in the lab. QUADrATiC represents a step change in the process of investigating gene expression connectivity and provides more biologically-relevant results than previous alternative solutions.

Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion.

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions.

PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein.

Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux without altering the hDAT physiological function of DA uptake. We evaluated the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present what is to our knowledge the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms.

Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate.

Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP2) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.

Whole slide image cytometry: a novel method to detect abnormal DNA content in Barrett's esophagus.

Barrett's esophagus (BE) is a precursor of esophageal adenocarcinoma (EAC). Both low-grade dysplasia (LGD) and high-grade dysplasia (HGD) are associated with an increased risk of progression to EAC. However, histological interpretation and grading of dysplasia (particularly LGD) is subjective and poorly reproducible. This study has combined whole slide imaging with DNA image cytometry to provide a novel method for the detection of abnormal DNA content through image analysis of tissue sections. A total of 20 cases were evaluated, including 8 negative for dysplasia (NFD), 6 LGD, and 6 HGD. Feulgen-stained esophageal sections were scanned in their entirety. Barrett's mucosa was interactively chosen for automatic nuclei segmentation where irrelevant cell types were ignored. The combined DNA content histogram for all nuclei within selected image regions was then obtained. In addition, three histogram measurements were computed, including xER-5C, 2cDI, and DNA-MG. Visual evaluation suggested the shape of DNA content histograms from NFD, LGD, and HGD cases exhibiting identifiable differences. The histogram measurements, xER-5C, 2cDI, and DNA-MG, were shown to be effective in differentiating metaplastic from dysplastic cases with statistical significance. Moreover, they also successfully separated NFD, LGD, and HGD patients with statistical significance. Whole slide image cytometry is a novel and effective method for the detection of abnormal DNA content in BE. Compared with histological review, it is more objective. Compared with flow cytometry and cytology-preparation image cytometry, it is low cost, simple to use, only requires a single 1 µm section, and facilitates selection of tissue and topographical correlation. Whole slide image cytometry can detect differences in DNA content between NFD, LGD, and HGD patients in this cross-sectional study. Abnormal DNA content detection by whole slide image cytometry is a promising biomarker of progression that could affect future diagnostics in BE.

Epidermal growth factor receptor immunohistochemistry: new opportunities in metastatic colorectal cancer.

The treatment of cancer is becoming more precise, targeting specific oncogenic drivers with targeted molecular therapies. The epidermal growth factor receptor has been found to be over-expressed in a multitude of solid tumours. Immunohistochemistry is widely used in the fields of diagnostic and personalised medicine to localise and visualise disease specific proteins. To date the clinical utility of epidermal growth factor receptor immunohistochemistry in determining monoclonal antibody efficacy has remained somewhat inconclusive. The lack of an agreed reproducible scoring criteria for epidermal growth factor receptor immunohistochemistry has, in various clinical trials yielded conflicting results as to the use of epidermal growth factor receptor immunohistochemistry assay as a companion diagnostic. This has resulted in this test being removed from the licence for the drug panitumumab and not performed in clinical practice for cetuximab. In this review we explore the reasons behind this with a particular emphasis on colorectal cancer, and to suggest a way of resolving the situation through improving the precision of epidermal growth factor receptor immunohistochemistry with quantitative image analysis of digitised images complemented with companion molecular morphological techniques such as in situ hybridisation and section based gene mutation analysis.