Potentially modifiable factors associated with health-related quality of life among people with chronic kidney disease: baseline findings from the National Unified Renal Translational Research Enterprise CKD (NURTuRE-CKD) cohort.

Background: Many non-modifiable factors are associated with poorer health-related quality of life (HRQoL) experienced by people with chronic kidney disease (CKD). We hypothesize that potentially modifiable factors for poor HRQoL can be identified among CKD patients, providing potential targets for intervention. Method: The National Unified Renal Translational Research Enterprise Chronic Kidney Disease (NURTuRE-CKD) cohort study recruited 2996 participants from nephrology centres with all stages of non-dialysis-dependent CKD. Baseline data collection for sociodemographic, anthropometric, biochemical and clinical information, including Integrated Palliative care Outcome Scale renal, Hospital Anxiety and Depression score (HADS) and the 5-level EuroQol-5D (EQ-5D-5L) as HRQoL measure, took place between 2017 and 2019. EQ-5D-5L dimensions (mobility, self-care, usual activities, pain/discomfort, anxiety/depression) were mapped to an EQ-5D-3L value set to derive index value. Multivariable mixed effects regression models, adjusted for known factors affecting HRQoL with recruitment region as a random effect, were fit to assess potentially modifiable factors associated with index value (linear) and within each dimension (logistic). Results: Among the 2958/2996 (98.7%) participants with complete EQ-5D data, 2201 (74.4%) reported problems in at least one EQ-5D-5L dimension. Multivariable linear regression identified independent associations between poorer HRQoL (EQ-5D-3L index value) and obesity (body mass index ≥30.0 kg/m2, ß -0.037, 95% CI -0.058 to -0.016, P = .001), HADS depression score ≥8 (ß -0.159, -0.182 to -0.137, P < .001), anxiety score ≥8 (ß -0.090, -0.110 to -0.069, P < .001), taking ≥10 medications (ß -0.065, -0.085 to -0.046, P < .001), sarcopenia (ß -0.062, -0.080 to -0.043, P < .001) haemoglobin <100 g/L (ß -0.047, -0.085 to -0.010, P = .012) and pain (ß -0.134, -0.152 to -0.117, P < .001). Smoking and prescription of prednisolone independently associated with problems in self-care and usual activities respectively. Renin-angiotensin system inhibitor (RASi) prescription associated with fewer problems with mobility and usual activities. Conclusion: Potentially modifiable factors including obesity, pain, depression, anxiety, anaemia, polypharmacy, smoking, steroid use and sarcopenia associated with poorer HRQoL in this cohort, whilst RASi use was associated with better HRQoL in two dimensions.

Extracellular matrix stiffness activates mechanosensitive signals but limits breast cancer cell spheroid proliferation and invasion.

Breast cancer is characterized by physical changes that occur in the tumor microenvironment throughout growth and metastasis of tumors. Extracellular matrix stiffness increases as tumors develop and spread, with stiffer environments thought to correlate with poorer disease prognosis. Changes in extracellular stiffness and other physical characteristics are sensed by integrins which integrate these extracellular cues to intracellular signaling, resulting in modulation of proliferation and invasion. However, the co-ordination of mechano-sensitive signaling with functional changes to groups of tumor cells within 3-dimensional environments remains poorly understood. Here we provide evidence that increasing the stiffness of collagen scaffolds results in increased activation of ERK1/2 and YAP in human breast cancer cell spheroids. We also show that ERK1/2 acts upstream of YAP activation in this context. We further demonstrate that YAP, matrix metalloproteinases and actomyosin contractility are required for collagen remodeling, proliferation and invasion in lower stiffness scaffolds. However, the increased activation of these proteins in higher stiffness 3-dimensional collagen gels is correlated with reduced proliferation and reduced invasion of cancer cell spheroids. Our data collectively provide evidence that higher stiffness 3-dimensional environments induce mechano-signaling but contrary to evidence from 2-dimensional studies, this is not sufficient to promote pro-tumorigenic effects in breast cancer cell spheroids.

Occurrence of adverse events and change in disease activity after initiation of etanercept in paediatric patients with juvenile psoriatic arthritis in the CARRA Registry.

OBJECTIVE: Etanercept is commonly used to treat juvenile idiopathic arthritis, including juvenile psoriatic arthritis (JPsA); however, information on etanercept's safety and effectiveness in clinical practice is limited. We used data from the Childhood Arthritis and Rheumatology Research Alliance (CARRA) Registry to evaluate etanercept's safety and effectiveness in JPsA in clinical practice. METHODS: We analysed safety and effectiveness data for paediatric patients enrolled in the CARRA Registry who had a JPsA diagnosis and had used etanercept. Safety was assessed by calculating rates of prespecified adverse events of special interest (AESIs) and serious adverse events (SAEs). Effectiveness was assessed by a variety of disease activity measures. RESULTS: Overall, 226 patients had JPsA and received etanercept; 191 met criteria for safety analysis and 43 met criteria for effectiveness analysis. AESI and SAE incidence rates were low. There were five events: three uveitis, one new-onset neuropathy and one malignancy. Incidence rates were 0.55 (95% CI: 0.18, 1.69), 0.18 (95% CI: 0.03, 1.29) and 0.13 (95% CI: 0.02, 0.09) per 100 patient-years for uveitis, neuropathy and malignancy, respectively. Etanercept showed effectiveness for JPsA treatment; 7 of 15 (46.7%) had an American College of Rheumatology-Pediatric Response 90, 9 of 25 (36.0%) had a clinical Juvenile Arthritis Disease Activity Score 10-joint ≤1.1 and 14 of 27 (51.9%) had clinically inactive disease at the 6-month follow-up. CONCLUSION: Data in the CARRA Registry showed that etanercept treatment was safe in treating children with JPsA, with low AESIs and SAEs. Etanercept was also effective, even when assessed in a small sample size.

A method for reproducible high-resolution imaging of 3D cancer cell spheroids.

Multicellular tumour cell spheroids embedded within three-dimensional (3D) hydrogels or extracellular matrices (ECM) are widely used as models to study cancer growth and invasion. Standard methods to embed spheroids in 3D matrices result in random placement in space which limits the use of inverted fluorescence microscopy techniques, and thus the resolution that can be achieved to image molecular detail within the intact spheroid. Here, we leverage UV photolithography to microfabricate PDMS (polydimethylsiloxane) stamps that allow for generation of high-content, reproducible well-like structures in multiple different imaging chambers. Addition of multicellular tumour spheroids into stamped collagen structures allows for precise positioning of spheroids in 3D space for reproducible high-/super-resolution imaging. Embedded spheroids can be imaged live or fixed and are amenable to immunostaining, allowing for greater flexibility of experimental approaches. We describe the use of these spheroid imaging chambers to analyse cell invasion, cell-ECM interaction, ECM alignment, force-dependent intracellular protein dynamics and extension of fine actin-based protrusions with a variety of commonly used inverted microscope platforms. This method enables reproducible, high-/super-resolution live imaging of multiple tumour spheroids, that can be potentially extended to visualise organoids and other more complex 3D in vitro systems.

Dosing Variation at Initiation of Adalimumab and Etanercept and Clinical Outcomes in Juvenile Idiopathic Arthritis: A Childhood Arthritis and Rheumatology Research Alliance Registry Study.

OBJECTIVE: To determine the dose-response relationship of tumor necrosis factor (TNF) inhibition in the treatment of juvenile idiopathic arthritis (JIA). METHODS: Participants of the Childhood Arthritis and Rheumatology Research Alliance Registry were eligible for inclusion in the analyses if they started TNF inhibition treatment for JIA. The primary treatment response was determined 3 to 7 months after the start of treatment, based on the JIA American College of Rheumatology Pediatric criteria for improvement, clinical Juvenile Arthritis Disease Activity Score, and persistence of treatment after 6 months. Subsequently, pooled logistic regression models were performed to include long-term follow-up data. The models were adjusted for risk factors associated with poor treatment response. Dosing was expressed by body weight, body surface area, ideal body weight, fat free mass, and lean body mass. RESULTS: Participants treated with adalimumab (n = 328) and etanercept (n = 437) were included in the analyses (median dose 0.82 mg/kg body weight [interquartile range (IQR) 0.66-1.04] and 0.83 mg/kg body weight [IQR 0.75-0.95], respectively). The majority of analyses did not show a relationship between dose and outcome. Where associations were found, results were conflicting. Alternative dosing characteristics based on ideal body weight, fat free mass, and lean body mass did not result in stronger or more consistent associations. CONCLUSION: This study was not able to confirm our hypothesis that increased dosing of TNF inhibitors results in improved treatment outcomes. Although adjustment was performed for risk factors of impaired treatment response, residual confounding by indication likely explains the negative associations found in this study.

PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease-protective variant PLCγ2 R522.

Variants identified in genome-wide association studies have implicated immune pathways in the development of Alzheimer's disease (AD). Here, we investigated the mechanistic basis for protection from AD associated with PLCγ2 R522, a rare coding variant of the PLCG2 gene. We studied the variant's role in macrophages and microglia of newly generated PLCG2-R522-expressing human induced pluripotent cell lines (hiPSC) and knockin mice, which exhibit normal endogenous PLCG2 expression. In all models, cells expressing the R522 mutation show a consistent non-redundant hyperfunctionality in the context of normal expression of other PLC isoforms. This manifests as enhanced release of cellular calcium ion stores in response to physiologically relevant stimuli like Fc-receptor ligation or exposure to Aß oligomers. Expression of the PLCγ2-R522 variant resulted in increased stimulus-dependent PIP2 depletion and reduced basal PIP2 levels in vivo. Furthermore, it was associated with impaired phagocytosis and enhanced endocytosis. PLCγ2 acts downstream of other AD-related factors, such as TREM2 and CSF1R, and alterations in its activity directly impact cell function. The inherent druggability of enzymes such as PLCγ2 raises the prospect of PLCγ2 manipulation as a future therapeutic approach in AD.

Ethanedinitrile as a Fumigant for Lasioderma serricorne (Coleoptera: Anobiidae), and Rhyzopertha dominica (Coleoptera: Bostrichidae): Toxicity and Mode of Action.

This study evaluated the fumigant ethanedinitrile (EDN) against the cigarette beetle, Lasioderma serricorne, and phosphine-resistant and susceptible lesser grain borer, Rhyzopertha dominica, life stages under laboratory conditions. Eggs of both species were the most susceptible stage to EDN. EDN is, therefore, a promising alternative because eggs are generally tolerant to most common fumigants. Lasioderma serricorne eggs were the most susceptible with an LC50 estimated of 50.4 ppm, followed by adults, pupae and larvae with LC50 values of 160.2, 192.5, and 446.6 ppm, respectively, after 24-h exposure at 25°C. Eggs of phosphine-susceptible (LC50 = 11.2 ppm) and resistant (LC50 = 12.0 ppm) R. dominica strains were more susceptible to EDN than were adults of both strains, with LC50 values of 27.7 and 36.0 ppm, respectively. Lasioderma serricorne mixed life stage cultures were completely controlled at concentrations ≥2,000 ppm at 24 h. Fumigation with 600 ppm was enough to suppress adult emergence in the case of the phosphine-susceptible R. dominica strain (USDA), while an average of only 4.0 adults emerged from the phosphine-resistant R. dominica strain (Belle Glade) compared with 514.3 adults in the control. Lasioderma serricorne was more tolerant to EDN than both R. dominica strains. EDN caused 61.8 and 68.2 % inhibition of R. dominica (USDA) cytochrome c oxidase activity at concentrations of 0.0038 and 0.0076 mM in vitro, respectively, and it did not inhibit its activity in the case of an in vivo assay. These results suggest that cytochrome c oxidase may not be the main target for EDN toxicity.

Lightsheet fluorescence lifetime imaging microscopy with wide-field time-correlated single photon counting.

We report on wide-field time-correlated single photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single-photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide-field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.

Caffeine Exacerbates Postictal Hypoxia.

A stroke-like event follows seizures which may be responsible for the postictal state and a contributing factor to the development of seizure-induced brain abnormalities and behavioral dysfunction associated with epilepsy. Caffeine is the world's most popular drug with ∼85% of people in the USA consuming it daily. Thus, persons with epilepsy are likely to have caffeine in their body and brain during seizures. This preclinical study investigated the effects of acute caffeine on local hippocampal tissue oxygenation pre and post seizure. We continuously measured local oxygen levels in the CA1 region of the hippocampus and utilized the electrical kindling model in rats. Rats were acutely administered either caffeine, or one of its metabolites, or agonists and antagonists at adenosine sub-receptor types or ryanodine receptors prior to the elicitation of seizures. Acute caffeine administration caused a significant drop in pre-seizure hippocampal pO2. Following a seizure, caffeine, as well as two of its metabolites paraxanthine, and theophylline, increased the time below the severe hypoxic threshold (10 mmHg). Likewise, the specific A2A receptor antagonist, SCH-58261, mimicked caffeine by causing a significant drop in pre-seizure pO2 and the area and time below the severe hypoxic threshold. Moreover, the A2A receptor agonist, CGS-21680 was able to prevent the effect of both caffeine and SCH-58261 adding further evidence that caffeine is likely acting through the A2A receptor. Clinical tracking and investigations are needed to determine the effect of caffeine on postictal symptomology and blood flow in persons with epilepsy.

Pilot study comparing the childhood arthritis and rheumatology research alliance consensus treatment plans for induction therapy of juvenile proliferative lupus nephritis.

BACKGROUND: To reduce treatment variability and facilitate comparative effectiveness studies, the Childhood Arthritis and Rheumatology Research Alliance (CARRA) published consensus treatment plans (CTPs) including one for juvenile proliferative lupus nephritis (LN). Induction immunosuppression CTPs outline treatment with either monthly intravenous (IV) cyclophosphamide (CYC) or mycophenolate mofetil (MMF) in conjunction with one of three corticosteroid (steroid) CTPs: primarily oral, primarily IV or mixed oral/IV. The acceptability and in-practice use of these CTPs are unknown. Therefore, the primary aims of the pilot study were to demonstrate feasibility of adhering to the LN CTPs and delineate barriers to implementation in clinical care in the US. Further, we aimed to explore the safety and effectiveness of the treatments for induction therapy. METHODS: Forty-one patients were enrolled from 10 CARRA sites. Patients had new-onset biopsy proven ISN/RPS class III or IV proliferative LN, were starting induction therapy with MMF or IV CYC and high-dose steroids and were followed for up to 24 months. Routine clinical data were collected at each visit. Provider reasons for CTP selection were assessed at baseline. Adherence to the CTPs was evaluated by provider survey and medication logs. Complete and partial renal responses were reported at 6 months. RESULTS: The majority of patients were female (83%) with a mean age of 14.7 years, SD 2.8. CYC was used more commonly than MMF for patients with ISN/RPS class IV LN (vs. class III), those who had hematuria, and those with adherence concerns. Overall adherence to the immunosuppression induction CTPs was acceptable with a majority of patients receiving the target MMF (86%) or CYC (63%) dose. However, adherence to the steroid CTPs was poor (37%) with large variability in dosing. Renal response endpoints were exploratory and did not show a significant difference between CYC and MMF. CONCLUSIONS: Overall, the immunosuppression CTPs were followed as intended in the majority of patients however, adherence to the steroid CTPs was poor indicating revision is necessary. In addition, our pilot study revealed several sources of treatment selection bias that will need to be addressed in for future comparative effectiveness research.

Conducting electrospun fibres with polyanionic grafts as highly selective, label-free, electrochemical biosensor with a low detection limit for non-Hodgkin lymphoma gene.

A highly selective, label-free sensor for the non-Hodgkin lymphoma gene, with an aM detection limit, utilizing electrochemical impedance spectroscopy (EIS) is presented. The sensor consists of a conducting electrospun fibre mat, surface-grafted with poly(acrylic acid) (PAA) brushes and a conducting polymer sensing element with covalently attached oligonucleotide probes. The sensor was fabricated from electrospun NBR rubber, embedded with poly(3,4-ethylenedioxythiophene) (PEDOT), followed by grafting poly(acrylic acid) brushes and then electrochemically polymerizing a conducting polymer monomer with ssDNA probe sequence pre-attached. The resulting non-Hodgkin lymphoma gene sensor showed a detection limit of 1aM (1 × 10-18mol/L), more than 400 folds lower compared to a thin-film analogue. The sensor presented extraordinary selectivity, with only 1%, 2.7% and 4.6% of the signal recorded for the fully non-complimentary, T-A and G-C base mismatch oligonucleotide sequences, respectively. We suggest that such greatly enhanced selectivity is due to the presence of negatively charged carboxylic acid moieties from PAA grafts that electrostatically repel the non-complementary and mismatch DNA sequences, overcoming the non-specific binding.

Screening for At-Risk Alcohol Consumption in Primary Care: A Randomized Evaluation of Screening Approaches.

AIMS: The aim of the study was to explore the relative efficiency and effectiveness of targeted versus universal screening for at-risk alcohol use in a primary care population in the UK. METHODS: The study was a randomized evaluation of screening approach (targeted versus universal) for consecutive attendees at primary care aged 18 years or more. Targeted screening involved screening any patient attending with one of the targeted presentations, conditions associated with excessive alcohol consumption: mental health, gastrointestinal, hypertension, minor injuries or a new patient registration. In the universal arm of the study all presentations in the recruitment period were included. Universal screening included all patients presenting to allocated practices. RESULTS: A total of 3562 potential participants were approached. The odds ratio of being screen positive was higher for the targeted group versus the universal group. Yet the vast majority of those screening positive in the universal group of the study would have been missed by a targeted approach. A combination of age and gender was a more efficient approach than targeting by clinical condition or context. CONCLUSIONS: While screening targeted by age and gender is more efficient than universal screening, targeting by clinical condition or presentation is not. Further universal screening is more effective in identifying the full range of patients who could benefit from brief alcohol interventions, and would therefore have greater public health impact. TRIAL REGISTRATION: Current Controlled Trials ISRCTN06145674.

Alcohol Consumption, Early-Onset Drinking, and Health-Related Consequences in Adolescents Presenting at Emergency Departments in England.

PURPOSE: Globally, alcohol use is the leading cause of ill health and life years lost in adolescents, although its clinical impact is often overlooked, particularly in England where most research is based in schools. This study aims to examine the prevalence of alcohol consumption and the association between alcohol consumption and age of onset with health and social consequences among adolescents presenting to emergency departments (EDs). METHODS: Consecutive attenders (n = 5,576) aged 10-17 years at 10 EDs were included. Information was collected on general health and functioning, quality of life, alcohol use, and alcohol-related health and social consequences. RESULTS: Nearly 40% of adolescents reported the consumption of alcohol that was more than a sip in their lifetime. Age of the first alcohol consumption before the age of 15 years was associated with tobacco use (p < .001), lower quality of life (p = .003), and evidence of an alcohol use disorder (p = .002). It was also associated with general social functioning (problems with conduct p = .001 and hyperactivity p = .001) and alcohol-related health and social consequences (accident p = .046, problems with a parent p = .017, school p = .0117, or police p = .012). CONCLUSIONS: Rates of alcohol consumption in adolescents presenting to the ED were similar to those reported in schools in England and globally. Associations of alcohol consumption and earlier onset of drinking with poorer health and social functioning were observed. The ED can offer an opportunity for the identification of hazardous alcohol use in adolescents.

Resistance of Lasioderma serricorne (Coleoptera: Anobiidae) to Fumigation with Phosphine.

Lasioderma serricorne (F.) is a serious pest of stored products that is known to be resistant to the fumigant pesticide gas phosphine. This study investigated resistance in populations from the southeastern United States, and determined if a recommended treatment schedule could kill resistant insects. A laboratory assay for adult insects was developed that used a discriminating concentration of 50 ppm phosphine applied to insects for 20 h at 25°C followed by 7 d of recovery in air. Survivors were classified as resistant. L. serricorne from six different field populations associated with stored tobacco were surveyed with the assay and all had resistant individuals. Four populations had greater than 90% of their insects resistant. Two industry-recommended treatment schedules were evaluated in laboratory fumigations against mixed life stage cultures of the four most resistant populations: the first at 200 ppm for 4 d at 25°C for controlling phosphine-susceptible L. serricorne and the second at 600 ppm for 6 d at 25°C intended to control phosphine-resistant beetles. The four populations with the highest frequency of resistant individuals from the field sampling study were not controlled by the "normal" treatment intended for susceptible insects. The higher concentration treatment greatly reduced beetle progeny from mixed-stage colony jars, but there were substantial numbers of surviving adults from all four highly resistant populations that represented unacceptable levels of control.

Persistent liver biochemistry abnormalities are more common in older patients and those with cholestatic drug induced liver injury.

OBJECTIVES: The long-term outcomes of patients with drug induced liver injury (DILI) are not well described. The aim of this study was to determine the frequency and severity of persistent liver biochemistry abnormalities in DILI patients followed over 2 years. METHODS: Subjects with evidence of liver injury at 6 months after DILI onset were offered a month 12 and 24 study visit. RESULTS: Amongst the 99 patients with definite, probable, or very likely DILI and available laboratory data at 12 months after DILI onset, 74 (75%) had persistent liver injury (persisters) defined as a serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >1.5 × upper limit of normal (ULN) or an alkaline phosphatase >ULN, while 25 (25%) had resolved liver injury (resolvers). On multivariate analysis, month 12 persisters were significantly older (52.6 vs. 43.7 years, P=0.01) and more likely to have a cholestatic lab profile at DILI onset (54 vs. 20%, P<0.01) than resolvers. The month 12 persisters also had significantly poorer SF-36 physical summary scores at DILI onset and throughout follow-up compared with the resolvers (P<0.01). Amongst the 17 subjects with a liver biopsy obtained at a median of 387 days after DILI onset, 9 had chronic cholestasis, 3 had steatohepatitis, and 3 had chronic hepatitis. CONCLUSIONS: In all, 75% of subjects with liver injury at 6 months after DILI onset have laboratory evidence of persistent liver injury during prolonged follow-up. Higher serum alkaline phosphatase levels at presentation and older patient age were independent predictors of persistent liver injury. Subjects with persistent liver injury at 12 months after DILI onset should be carefully monitored and assessed for liver disease progression.

The effectiveness of electronic screening and brief intervention for reducing levels of alcohol consumption: a systematic review and meta-analysis.

BACKGROUND: Electronic screening and brief intervention (eSBI) has been shown to reduce alcohol consumption, but its effectiveness over time has not been subject to meta-analysis. OBJECTIVE: The current study aims to conduct a systematic review and meta-analysis of the available literature to determine the effectiveness of eSBI over time in nontreatment-seeking hazardous/harmful drinkers. METHODS: A systematic review and meta-analysis of relevant studies identified through searching the electronic databases PsychINFO, Medline, and EMBASE in May 2013. Two members of the study team independently screened studies for inclusion criteria and extracted data. Studies reporting data that could be transformed into grams of ethanol per week were included in the meta-analysis. The mean difference in grams of ethanol per week between eSBI and control groups was weighted using the random-effects method based on the inverse-variance approach to control for differences in sample size between studies. RESULTS: There was a statistically significant mean difference in grams of ethanol consumed per week between those receiving an eSBI versus controls at up to 3 months (mean difference -32.74, 95% CI -56.80 to -8.68, z=2.67, P=.01), 3 to less than 6 months (mean difference -17.33, 95% CI -31.82 to -2.84, z=2.34, P=.02), and from 6 months to less than 12 months follow-up (mean difference -14.91, 95% CI -25.56 to -4.26, z=2.74, P=.01). No statistically significant difference was found at a follow-up period of 12 months or greater (mean difference -7.46, 95% CI -25.34 to 10.43, z=0.82, P=.41). CONCLUSIONS: A significant reduction in weekly alcohol consumption between intervention and control conditions was demonstrated between 3 months and less than 12 months follow-up indicating eSBI is an effective intervention.

Alcohol screening and brief intervention for adolescents: the how, what and where of reducing alcohol consumption and related harm among young people.

AIM: The aim of the study was to explore the evidence base on alcohol screening and brief intervention for adolescents to determine age appropriate screening tools, effective brief interventions and appropriate locations to undertake these activities. METHODS: A review of existing reviews (2003-2013) and a systematic review of recent research not included in earlier reviews. RESULTS: The CRAFFT and AUDIT tools are recommended for identification of 'at risk' adolescents. Motivational interventions delivered over one or more sessions and based in health care or educational settings are effective at reducing levels of consumption and alcohol-related harm. CONCLUSION: Further research to develop age appropriate screening tools needs to be undertaken. Screening and brief intervention activity should be undertaken in settings where young people are likely to present; further assessment at such venues as paediatric emergency departments, sexual health clinics and youth offending teams should be evaluated. The use of electronic (web/smart-phone based) screening and intervention shows promise and should also be the focus of future research.

Combined targeting of MEK and PI3K/mTOR effector pathways is necessary to effectively inhibit NRAS mutant melanoma in vitro and in vivo.

Activating mutations in the neuroblastoma rat sarcoma viral oncogene homolog (NRAS) gene are common genetic events in malignant melanoma being found in 15-25% of cases. NRAS is thought to activate both mitogen activated protein kinase (MAPK) and PI3K signaling in melanoma cells. We studied the influence of different components on the MAP/extracellular signal-regulated (ERK) kinase (MEK) and PI3K/mammalian target of rapamycin (mTOR)-signaling cascade in NRAS mutant melanoma cells. In general, these cells were more sensitive to MEK inhibition compared with inhibition in the PI3K/mTOR cascade. Combined targeting of MEK and PI3K was superior to MEK and mTOR1,2 inhibition in all NRAS mutant melanoma cell lines tested, suggesting that PI3K signaling is more important for cell survival in NRAS mutant melanoma when MEK is inhibited. However, targeting of PI3K/mTOR1,2 in combination with MEK inhibitors is necessary to effectively abolish growth of NRAS mutant melanoma cells in vitro and regress xenografted NRAS mutant melanoma. Furthermore, we showed that MEK and PI3K/mTOR1,2 inhibition is synergistic. Expression analysis confirms that combined MEK and PI3K/mTOR1,2 inhibition predominantly influences genes in the rat sarcoma (RAS) pathway and growth factor receptor pathways, which signal through MEK/ERK and PI3K/mTOR, respectively. Our results suggest that combined targeting of the MEK/ERK and PI3K/mTOR pathways has antitumor activity and might serve as a therapeutic option in the treatment of NRAS mutant melanoma, for which there are currently no effective therapies.