VDR and MEK-ERK dependent induction of the antimicrobial peptide cathelicidin in keratinocytes by lithocholic acid

Cathelicidin is an antimicrobial peptide (AMP) and signaling molecule in innate immunity and a direct target of 1,25-dihydroxyvitamin D3 (1,25D3) in primary human keratinocytes (NHEK). The expression of cathelicidin is dysregulated in various skin diseases and its regulation differs depending on the epithelial cell type. The secondary bile acid lithocholic acid (LCA) is a ligand of the vitamin D receptor (VDR) and can carry out in vivo functions of vitamin D3. Therefore we analyzed cathelicidin mRNA- and peptide expression levels in NHEK and colonic epithelial cells (Caco-2) after stimulation with LCA. We found increased expression of cathelicidin mRNA and peptide in NHEK, in Caco-2 colon cells no effect was observed after LCA stimulation. The VDR as well as MEK-ERK signaled the upregulation of cathelicidin in NHEK induced by LCA. Collectively, our data indicate that cathelicidin induction upon LCA treatment differs in keratinocytes and colonic epithelial cells. Based on these observations LCA-like molecules targeting cathelicidin could be designed for the treatment of cutaneous diseases that are characterized by disturbed cathelicidin expression.

Overexpression of the microRNA miR-433 promotes resistance to paclitaxel through the induction of cellular senescence in ovarian cancer cells

Annually, ovarian cancer (OC) affects 240,000 women worldwide and is the most lethal gynecological malignancy. High-grade serous OC (HGSOC) is the most common and aggressive OC subtype, characterized by widespread genome changes and chromosomal instability and is consequently poorly responsive to chemotherapy treatment. The objective of this study was to investigate the role of the microRNA miR-433 in the cellular response of OC cells to paclitaxel treatment. We show that stable miR-433 expression in A2780 OC cells results in the induction of cellular senescence demonstrated by morphological changes, downregulation of phosphorylated retinoblastoma (p-Rb), and an increase in β-galactosidase activity. Furthermore, in silico analysis identified four possible miR-433 target genes associated with cellular senescence: cyclin-dependent kinase 6 (CDK6), MAPK14, E2F3, and CDKN2A. Mechanistically, we demonstrate that downregulation of p-Rb is attributable to a miR-433-dependent downregulation of CDK6, establishing it as a novel miR-433 associated gene. Interestingly, we show that high miR-433 expressing cells release miR-433 into the growth media via exosomes which in turn can induce a senescence bystander effect. Furthermore, in relation to a chemotherapeutic response, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that only PEO1 and PEO4 OC cells with the highest miR-433 expression survive paclitaxel treatment. Our data highlight how the aberrant expression of miR-433 can adversely affect intracellular signaling to mediate chemoresistance in OC cells by driving cellular senescence.

MIR-433 Predicts poor response to chemotherapy in ovarian cancer patients by the induction of cellular senescence

Annually, ovarian cancer (OC) affects 240,000 women worldwide and is the most lethal gynaecological malignancy. Such mortality is predominantly associated with the development of an intrinsic and acquired resistance to chemotherapy, the lack of targeted therapies and the lack of biomarkers predicting response to standard treatment.

Our clinical data demonstrates that increased miR-433 expression in primary high grade serous OC (HGSOCs) is significantly associated with poor PFS (n=46, p=0.024). Interestingly, the IHC analysis of two miR-433 targets: MAD2 [1] and HDAC6 shows that low IHC levels of both proteins is also significantly associated with worse outcome (p=0.002 and 0.002 respectively; n=43). Additionally, the analysis of miR 433 in the publicly available TCGA dataset corroborates that high miR-433 is significantly correlated with worse OS for patients presenting with OC (n=558 and p=0.027). In vito, in a panel of OC cell lines, higher miR-433 and lower MAD2 and HDAC6 levels were associated with resistance to paclitaxel.

To further investigate the role of miR-433 in the cellular response to chemotherapy, we generated an OC cell line stably expressing miR-433 or miR-control. MTT viability assays and Western Blot analyses established that miR-433 cells were more resistant to paclitaxel treatment (50nM) compared to miR-controls. Importantly, we have shown for the first time that miR 433 induced senescence resulting in a chracteristic flattened morphology and down-regulation of phosphorylated Retinoblastoma (p Rb), a molecular marker of senescence. Surprisingly, miR 433 induced senescence was independent from two well recognised senescent drivers: namely p53/p21 and p16. To explore this further we performed an in silico analysis of seven microRNA platforms which indicated that miR 433 potentially targets Cyclin-dependent kinase CDK6, which promotes sustained phosphorylation of Rb and thus cell cycle progression. In vitro, the overexpression of pre-miR-433 resulted in diminished CDK6 expression demonstrating a novel interaction between miR-433 and CDK6.

In conclusion, this study demonstrates that high miR-433 expression predicts poor outcome in OC patients by putatively rendering OC cells resistant to paclitaxel treatment through the induction of cellular senescence identifying this microRNA as a potential marker of chemoresponse.

Effect of feed space allowance and period of access to food on dairy cow performance

Two experiments were conducted to examine the ‘long-term’ effect of feed space allowance and period of access to feed on dairy cow performance. In Experiment 1, three horizontal feed space allowances (20, 40 and 60 cm cow−1) were examined over a 127-d period (14 cows per treatment). In Experiment 2, 48 dairy cows were used in a continuous design (10-week duration) 2 × 2 factorial design experiment comprising two horizontal feed space allowances (15 and 40 cm cow−1), and two periods of access to feed (unrestricted and restricted). With the former, uneaten feed was removed at 08·00 h, while feeding took place at 09·00 h. With the latter, uneaten feed was removed at 06·00 h, while feeding was delayed until 12·00 h. Mean total dry-matter (DM) intakes were 19·0, 18·7 and 19·3 kg cow−1 d−1 with the 20, 40 and 60 cm cow−1 treatments in Experiment 1, and 18·1 and 18·2 kg cow−1 d−1 with the ‘restricted feeding time’ treatments, and 17·8 and 18·1 kg d−1 with the ‘unrestricted feeding time’ treatments (15 and 40 cm respectively) in Experiment 2. None of milk yield, milk composition, or end-of-study live weight or condition score were significantly affected by treatment in either experiment (P > 0·05), while fat + protein yield was reduced with the 15-cm treatment in Experiment 2 (P < 0·05). When access to feed was restricted by space or time constraints, cows modified their time budgets and increased their rates of intake.

Psychotropic prescribing patterns among adolescents in Northern Ireland presenting with psychotic symptoms during a 5-year period

Objective: To report new prescriptions of psychotropic medications among adolescents presenting with new onset psychotic symptoms during a 5-year period.
Methods: The Northern Ireland Early Onset Psychosis Study is a naturalistic longitudinal observational study of patients with an early onset first psychotic episode. All patients aged <18 years presenting to specialist mental health services across Northern Ireland with new onset psychotic symptoms between 2001 and 2006 were recruited (n = 113). Clinical case notes were analysed retrospectively for details of subsequent treatment with psychotropic medications.
Results: A total of 100 patients (88.5%) were prescribed some form of psychotropic medication. Over three-quarters of patients received an antipsychotic as their first medication. Risperidone (45.8%), olanzapine (24.0%) and chlorpromazine (12.5%) were the most commonly prescribed first-line antipsychotic medications. Of a total of 160 antipsychotic prescriptions,81 (50.6%) were off-label. Prescriptions were most likely to have been deemed off-label owing to medications not being licensed in under-18s (71.6% of off-label prescriptions) but other reasons were medications being used outsidelicensed age ranges (23.5%) and outside licensed indications (4.9%).
Conclusions: This is the first study examining psychotropic prescribing patterns in a complete sample of all children and adolescents presenting with early onset psychotic episodes in a single geographical area. The observation of risperidoneas the most commonly prescribed antipsychotic was in keeping with previous studies in child and adolescent populations. Rates of off-label prescribing were lower than previously observed although our study was the first to investigateoff-label prescribing solely in children and adolescents presenting with psychotic symptoms.

A randomised comparison of daunorubicin 90mg/m2 vs 60mg/m2 in AML induction: results from the UK NCRI AML17 trial in 1206 patients

Modifying induction therapy in AML may improve the remission rate and reduce the risk of relapse thereby improving survival. Escalation of the daunorubicin dose to 90mg/m(2) has shown benefit for some patient subgroups when compared with a dose of 45mg/m(2) and has been recommended as a standard of care. However 60mg/m(2) is widely used and has never been directly compared to 90mg/m(2). As part of the UK NCRI AML17 trial 1206 adults with untreated AML or high risk MDS, mostly under 60 years of age, were randomised to a first induction course of chemotherapy which delivered either 90mg/m(2) or 60mg/m(2) on days 1,3 and 5 combined with cytosine arabinoside. All patients then received a second course which included daunorubicin 50mg/m(2) on days 1,3 and 5. There was no overall difference in complete remission rate (CR) (73% vs 75%, OR1.07 (0.83-1.39), p=0.6) or in any recognised subgroup. The 60 day mortality was increased in the 90mg/m2 arm (10% vs 5% (HR 1.98(1.30-3.02) p=0.001)), which resulted in no difference in overall 2 year survival (59% vs 60%, HR 1.16(0.95-1.43), p=0.15). In exploratory subgroup analysis there was no subgroup which showed significant benefit, although there was a significant interaction by FLT3 ITD mutation. The trial is registered to www.isrctn.com as ISRCTN55675535.

The Decaying Long-period Oscillation of a Stellar Megaflare

We analyze and interpret the oscillatory signal in the decay phase of the U-band light curve of a stellar megaflare observed on 2009 January 16 on the dM4.5e star YZ CMi. The oscillation is well approximated by an exponentially decaying harmonic function. The period of the oscillation is found to be 32 minutes, the decay time about 46 minutes, and the relative amplitude 15%. As this observational signature is typical of the longitudinal oscillations observed in solar flares at extreme ultraviolet and radio wavelengths, associated with standing slow magnetoacoustic waves, we suggest that this megaflare may be of a similar nature. In this scenario, macroscopic variations of the plasma parameters in the oscillations modulate the ejection of non-thermal electrons. The phase speed of the longitudinal (slow magnetoacoustic) waves in the flaring loop or arcade, the tube speed, of about 230 km s-1 would require a loop length of about 200 Mm. Other mechanisms, such as standing kink oscillations, are also considered.

Induction of the inflammatory regulator A20 by gibberellic acid in airway epithelial cells

BACKGROUND AND PURPOSE: NF-κB-driven inflammation is negatively regulated by the zinc finger protein A20. Gibberellic acid (GA3 ) is a plant-derived diterpenoid with documented anti-inflammatory activity, which is reported to induce A20-like zinc finger proteins in plants. Here, we sought to investigate the anti-inflammatory effect of GA3 in airway epithelial cells and determine if the anti-inflammatory action relates to A20 induction.

EXPERIMENTAL APPROACH: Primary nasal epithelial cells and a human bronchial epithelial cell line (16HBE14o-) were used. Cells were pre-incubated with GA3 , stimulated with Pseudomonas aeruginosa LPS; IL-6 and IL-8 release, A20, NF-κB and IκBα expression were then evaluated. To determine if any observed anti-inflammatory effect occurred via an A20-dependent mechanism, A20 was silenced using siRNA.

KEY RESULTS: Cells pre-incubated with GA3 had significantly increased levels of A20 mRNA (4 h) and protein (24 h), resulting in a significant reduction in IL-6 and IL-8 release. This effect was mediated via reduced IκBα degradation and reduced NF-κB (p65) expression. Furthermore, the anti-inflammatory action of GA3 was abolished in A20-silenced cells.

CONCLUSIONS AND IMPLICATIONS: We showed that A20 induction by GA3 attenuates inflammation in airway epithelial cells, at least in part through its effect on NF-κB and IκBα. GA3 or gibberellin-derived derivatives could potentially be developed into anti-inflammatory drugs for the treatment of chronic inflammatory diseases associated with A20 dysfunction.

The Impact of Colleague Peer Review on the Radiotherapy Treatment Planning Process in the Radical Treatment of Lung Cancer

AIMS: Modern radiotherapy uses techniques to reliably identify tumour and reduce target volume margins. However, this can potentially lead to an increased risk of geographic miss. One source of error is the accuracy of target volume delineation (TVD). Colleague peer review (CPR) of all curative-intent lung cancer plans has been mandatory in our institution since May 2013. At least two clinical oncologists review plans, checking treatment paradigm, TVD, prescription dose tumour and critical organ tolerances. We report the impact of CPR in our institution.

MATERIALS AND METHODS: Radiotherapy treatment plans of all patients receiving radical radiotherapy were presented at weekly CPR meetings after their target volumes were reviewed and signed off by the treating consultant. All cases and any resultant change to TVD (including organs at risk) or treatment intent were recorded in our prospective CPR database. The impact of CPR over a 13 month period from May 2013 to June 2014 is reported.

RESULTS: One hundred and twenty-two patients (63% non-small cell lung carcinoma, 17% small cell lung carcinoma and 20% 'clinical diagnosis') were analysed. On average, 3.2 cases were discussed per meeting (range 1-8). CPR resulted in a change in treatment paradigm in 3% (one patient proceeded to induction chemotherapy, two patients had high-dose palliative radiotherapy). Twenty-one (17%) had a change in TVD and one (1%) patient had a change in dose prescription. In total, 6% of patients had plan adjustment after review of dose volume histogram.

CONCLUSION: The introduction of CPR in our centre has resulted in a change in a component of the treatment plan for 27% of patients receiving curative-intent lung radiotherapy. We recommend CPR as a mandatory quality assurance step in the planning process of all radical lung plans.

Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1

The breast cancer susceptibility gene BRCA1 encodes a protein implicated in the cellular response to DNA damage, with postulated roles in homologous recombination as well as transcriptional regulation. To identify downstream target genes, we established cell lines with tightly regulated inducible expression of BRCA1. High-density oligonucleotide arrays were used to analyze gene expression profiles at various times following BRCA1 induction. A major BRCA1 target is the DNA damage-responsive gene GADD45. Induction of BRCA1 triggers apoptosis through activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), a signaling pathway potentially linked to GADD45 gene family members. The p53-independent induction of GADD45 by BRCA1 and its activation of JNK/SAPK suggest a pathway for BRCA1-induced apoptosis.