A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer.

BACKGROUND: miR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent. METHODS: RNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq). RESULTS: miR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival. INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes. NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy. CONCLUSIONS: A balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.

The tumor microenvironment and immune responses in prostate cancer patients.

The landscape of cancer treatment has been transformed over the past decade by the success of immune-targeting therapies. However, despite sipuleucel-T being the first-ever approved vaccine for cancer and the first immunotherapy licensed for prostate cancer in 2010, immunotherapy has since seen limited success in the treatment of prostate cancer. The tumour microenvironment of prostate cancer presents particular barriers for immunotherapy. Moreover, prostate cancer is distinguished by being one of only two solid tumours where increased T cell-infiltration correlates with a poorer, rather than improved, outlook. Here, we discuss the specific aspects of the prostate cancer microenvironment that converge to create a challenging microenvironment, including myeloid-derived immune cells and cancer-associated fibroblasts. By exploring the immune microenvironment of defined molecular subgroups of prostate cancer, we propose an immunogenomic subtyping approach to single-agent and combination immune-targeting strategies that could lead to improved outcomes in prostate cancer treatment.

The multiple effects of aspirin in prostate cancer patients.

Aspirin is a commonly used medication with anti-inflammatory and analgesic properties, and it is widely used to reduce the risk of ischaemic heart disease-related events and/or cerebrovascular accidents. However, there is also evidence from epidemiological and interventional studies to suggest that regular aspirin use can reduce the risk of prostate cancer development and progression, and can reduce the risk of disease recurrence following anti-prostate cancer therapy. Aspirin use in African-American men is associated with a reduced incidence of advanced PCa and reduced disease recurrence, and there is evidence from other studies of an association between regular aspirin use and decreased PCa-related mortality. The cyclooxygenase-2 enzyme inhibited by Aspirin and other NSAIDs, and which catalyses prostaglandin synthesis and mediates inflammation, is overexpressed in prostate cancer, therefore inhibition of cyclooxygenase-2 may have direct, and indirect, therapeutic effects. This review explores the evidence suggesting that aspirin use can modify prostate cancer biology and disease characteristics, and explores the potential mechanisms underpinning the observed associations between aspirin use and modification of prostate cancer risk. It also summarises the potential for adjuvant aspirin use to combine with other therapeutic approaches such as radical surgery and radiotherapy.

The drebrin/EB3 pathway drives invasive activity in prostate cancer.

Prostate cancer is the most common cancer in men and the metastatic form of the disease is incurable. We show here that the drebrin/EB3 pathway, which co-ordinates dynamic microtubule/actin filament interactions underlying cell shape changes in response to guidance cues, plays a role in prostate cancer cell invasion. Drebrin expression is restricted to basal epithelial cells in benign human prostate but is upregulated in luminal epithelial cells in foci of prostatic malignancy. Drebrin is also upregulated in human prostate cancer cell lines and co-localizes with actin filaments and dynamic microtubules in filopodia of pseudopods of invading cells under a chemotactic gradient of the chemokine CXCL12. Disruption of the drebrin/EB3 pathway using BTP2, a small molecule inhibitor of drebrin binding to actin filaments, reduced the invasion of prostate cancer cell lines in 3D in vitro assays. Furthermore, gain- or loss-of-function of drebrin or EB3 by over-expression or siRNA-mediated knockdown increases or decreases invasion of prostate cancer cell lines in 3D in vitro assays, respectively. Finally, expression of a dominant-negative construct that competes with EB3 binding to drebrin, also inhibited invasion of prostate cancer cell lines in 3D in vitro assays. Our findings show that co-ordination of dynamic microtubules and actin filaments by the drebrin/EB3 pathway drives prostate cancer cell invasion and is therefore implicated in disease progression.

Nuclear iASPP may facilitate prostate cancer progression.

One of the major challenges in prostate cancer (PCa) research is the identification of key players that control the progression of primary cancers to invasive and metastatic disease. The majority of metastatic PCa express wild-type p53, whereas loss of p63 expression, a p53 family member, is a common event. Here we identify inhibitor of apoptosis-stimulating protein of p53 (iASPP), a common cellular regulator of p53 and p63, as an important player of PCa progression. Detailed analysis of the prostate epithelium of iASPP transgenic mice, iASPP(Δ8/Δ8) mice, revealed that iASPP deficiency resulted in a reduction in the number of p63 expressing basal epithelial cells compared with that seen in wild-type mice. Nuclear and cytoplasmic iASPP expression was greater in PCa samples compared with benign epithelium. Importantly nuclear iASPP associated with p53 accumulation in vitro and in vivo. A pair of isogenic primary and metastatic PCa cell lines revealed that nuclear iASPP is enriched in the highly metastatic PCa cells. Nuclear iASPP is often detected in PCa cells located at the invasive leading edge in vivo. Increased iASPP expression associated with metastatic disease and PCa-specific death in a clinical cohort with long-term follow-up. These results suggest that iASPP function is required to maintain the expression of p63 in normal basal prostate epithelium, and nuclear iASPP may inactivate p53 function and facilitate PCa progression. Thus iASPP expression may act as a predictive marker of PCa progression.

Changes in circulating microRNA levels associated with prostate cancer.

BACKGROUND: The aim of this study was to investigate the hypothesis that changes in circulating microRNAs (miRs) represent potentially useful biomarkers for the diagnosis, staging and prediction of outcome in prostate cancer. METHODS: Real-time polymerase chain reaction analysis of 742 miRs was performed using plasma-derived circulating microvesicles of 78 prostate cancer patients and 28 normal control individuals to identify differentially quantified miRs. RESULTS: A total of 12 miRs were differentially quantified in prostate cancer patients compared with controls, including 9 in patients without metastases. In all, 11 miRs were present in significantly greater amounts in prostate cancer patients with metastases compared with those without metastases. The association of miR-141 and miR-375 with metastatic prostate cancer was confirmed using serum-derived exosomes and microvesicles in a separate cohort of patients with recurrent or non-recurrent disease following radical prostatectomy. An analysis of five selected miRs in urine samples found that miR-107 and miR-574-3p were quantified at significantly higher concentrations in the urine of men with prostate cancer compared with controls. CONCLUSION: These observations suggest that changes in miR concentration in prostate cancer patients may be identified by analysing various body fluids. Moreover, circulating miRs may be used to diagnose and stage prostate cancer.

Attractants for the green June beetle (Coleoptera: Scarabaeidae).

The purpose of this study was to develop and evaluate lures for adult green June beetles, Cotinis nitida (L.) (Coleoptera: Scarabaeidae), for future use in a mass trapping program. Volatile organic compounds collected from headspace of green June beetles feeding on fermenting ripe apple (Malus spp.), the natural lure that elicits feeding aggregations, were identified and confirmed by gas chromatography and mass spectrometry. Yellow funnel traps baited with 91% isopropanol or the five component blend were equally effective in eliciting aggregation behavior and often more attractive to green June beetles than the natural lure. In 2008, three trap lines adjacent and parallel to the perimeter of two vineyards, each with 12 Xpando yellow funnel traps baited with either 91% isopropanol or the five component blend, differed in catch of green June beetles across sample dates, and sample date by bait interaction but there were no differences among these two baits. A season total of 324,007 green June beetle were captured by these 36 baited traps. A brief review is included of fermentation volatiles attractive to insects. We conclude with the potential cost to use mass trapping against adult green June beetles.

Laparoscopic pyeloplasty: the retroperitoneal approach is suitable for establishing a de novo practice.

BACKGROUND: Laparoscopic dismembered pyeloplasty has become the "gold-standard" procedure for pelviureteric junction (PUJ) obstruction but consists of a steep learning curve especially via the retroperitoneal route. AIMS: To examine the feasibility and safety of introducing this technique via the retroperitoneal approach to a laparoscopic naïve center. SETTINGS AND DESIGN: A retrospective data analysis of a single surgeon's (NEO) series from a large UK teaching hospital. MATERIALS AND METHODS: The notes and imaging of all patients who underwent pyeloplasty for PUJ obstruction by NEO during a five-year period were reviewed. STATISTICAL ANALYSIS: Parametric and nonparametric data are presented analyzed with Excel XP (Microsoft, Redmond, WA, USA). RESULTS: Our series consists of 67 patients. Three ports were used in 47/57 (82%), and the antegrade technique for stent insertion was utilized in 41/67 (61%). Median time to drink, eat, and mobilize was one day (range one to two), and to discharge three days (range three to four). Two patients required conversion to an open procedure, and two developed intraoperative complications. Postoperative complications at 30 (three major, seven minor) and 90 days (three major, three minor) are presented. With median follow-up of 15 months 61/65 (94%) patients were unobstructed, and 57/63 (90%) of patients were pain-free. Two patients re-obstructed requiring further surgery. CONCLUSIONS: Analysis of our series of patients illustrates that adopting a policy of retroperitoneal laparoscopic pyeloplasty for primary PUJ obstruction is feasible without compromising patient safety or functional results. There is no need to breach the peritoneum to facilitate the learning curve.

Effects of milling on functional properties of rice flour.

A commercial long-grain rice flour (CRF) and the flours made by using a pin mill and the Udy mill from the same batch of broken second-head white long-grain rice were evaluated for their particle size and functional properties. The purpose of this study was to compare the commercial rice flour milling method to the pin and Udy milling methods used in our laboratory and pilot plant. The results showed that pin milled flour had more uniform particle size than the other 2 milled flours. The chalky kernels found in broken white milled rice were pulverized more into fines in both Udy milled flour and CRF than in the pin milled flour. The excessive amount of fines in flours affected their functional properties, for example, WSI and their potential usage in the novel foods such as rice breads (RB). The RB made from CRF collapsed more than loaves made from pin milled Cypress long-grain flours.

Ownership and uses of human tissue: what are the opinions of surgical in-patients?

AIMS: To investigate whether patient opinion about the uses of tissue removed at therapeutic operations has changed since the adverse publicity surrounding the Alder Hey and Bristol Royal Infirmary Inquiries, and to see whether it aligns with the Human Tissue Act 2004. METHODS: A questionnaire was given to 220 postoperative patients in a teaching hospital during an 11 week period. Aggregated responses to each question were ranked in frequency order. Unweighted centroid linkage hierarchical clustering analysis was performed with dendrogram display for the main data on tissue usage. RESULTS: 203 completed questionnaires were collected (compliance rate 92.3%). 96.3% of patients indicated that they would not object to their tissue being used in research, significantly higher than in the 1996 study (89.1%) with no overlap of the 95% CIs. 29.1% of patients believed that the hospital had ownership of tissue once it has been removed during surgery, 23.2% believed they had ownership, 19.7% believed that the pathology laboratory had ownership, and 15.3% believed that nobody had ownership rights in the case of tissue samples. CONCLUSIONS: This new survey indicates that despite a turbulent decade for those involved in human tissue retention in the UK, public support for a wide range of human tissue based activities, especially biomedical research, has not diminished and that patient opinion aligns well with the Human Tissue Act 2004.

The Polycomb Group protein EZH2 regulates actin polymerization in human prostate cancer cells.

BACKGROUND: The Polycomb Group protein EZH2 is implicated in prostate cancer progression. EZH2 promotes prostate cancer cell proliferation and invasiveness. We describe a link between EZH2 function and actin polymerization in prostate cancer cells. METHODS: Nuclear and cytoplasmic EZH2 expression in benign and malignant prostate tissue samples was assessed. An association between EZH2 function and actin polymerization in prostate cancer cells was investigated using siRNA-mediated knock-down of EZH2. Effects of EZH2 knock-down on actin polymerization dynamics were analyzed biochemically using immunoblot analysis of cell lysate fractions, and morphologically using immunocytochemistry. RESULTS: Cytoplasmic EZH2 is expressed at low levels in benign prostate epithelial cells and over-expressed in prostate cancer cells. Cytoplasmic EZH2 expression levels correlate with nuclear EZH2 expression in prostate cancer samples. Knock-down of EZH2 in PC3 prostate cancer cells increases the amount of F-actin polymerization, cell size, and formation of actin-rich filaments. CONCLUSIONS: Cytoplasmic EZH2 is over-expressed in prostate cancer cells. EZH2 function promotes a reduction in the pool of insoluble F-actin in invasive prostate cancer cells. EZH2 may regulate actin polymerization dynamics and thereby promote prostate cancer cell motility and invasiveness.

EZH2 promotes proliferation and invasiveness of prostate cancer cells.

BACKGROUND: The transcriptional repressor EZH2 is implicated in control of cell proliferation in embryonic, immortalized and transformed cells. EZH2 expression in prostate cancer correlates with progression to hormone-refractory and metastatic disease, but it is unknown whether EZH2 plays a specific role in the acquisition of an advanced prostate cancer phenotype. METHODS: Using siRNA knockdown, we investigated the role of EZH2 in maintenance of prostate cancer cell proliferation and invasiveness. Using LNCaP cells with inducible EZH2 overexpression, we investigated whether EZH2 upregulation promotes an aggressive phenotype. RESULTS: Knockdown of endogenous EZH2 reduced proliferation of androgen-responsive and androgen-independent prostate cancer cells. EZH2 knockdown also inhibited prostate cancer cell invasion. However, overexpression of EZH2 in androgen-responsive cancer cells did not appreciably affect either proliferation or invasiveness. CONCLUSIONS: EZH2 promotes proliferation and invasion of prostate cancer cells, which can account for the correlation between EZH2 expression levels and an adverse prostate cancer prognosis.

Ki67 staining pattern as a diagnostic tool in the evaluation of lymphoproliferative disorders.

AIMS: To evaluate a group of lymphoid proliferations using only Ki67-stained slides to determine the value of the pattern of proliferating cells in diagnosis. Ki67 immunohistochemistry allows evaluation of the distribution of proliferating cells in addition to simply determining the proliferation rate of cells. METHODS AND RESULTS: Three observers, using a Ki67-stained slide only, studied 149 cases from five diagnostic groupings: follicular hyperplasia, mixed pattern hyperplasia, localized Castleman's disease, follicular lymphoma and marginal zone lymphoma. The sensitivity for benign lesions varied from 94% to 97% among the three observers. Follicular lymphomas were recognized as neoplastic with a sensitivity of 96% and 100% by two of the observers. Marginal zone lymphoma was recognized as neoplastic in 67-73% of cases. CONCLUSIONS: The Ki67 stain alone is a powerful tool for distinguishing benign from malignant proliferations within the selected groups. Nuances and pitfalls in the interpretation of Ki67 staining pattern are discussed.

Racial differences in bone turnover and calcium metabolism in adolescent females.

Blacks develop a higher peak bone mass than whites which is associated with a reduced risk for bone fracture. The physiological basis for the difference in bone mass was investigated by metabolic balance and calcium kinetic studies in adolescent black and white girls. The hypothesis that the greater peak bone mass in blacks compared with whites is due to suppressed bone resorption was tested. Subjects were housed in a supervised environment for 3 wk during which time they consumed a controlled diet and collected all excreta. Subjects were given stable calcium isotopes orally and intravenously after 1 wk adaptation. Blacks have greater calcium retention (mean +/- SD, 11.5 +/- 6.1 vs. 7.3 +/- 4.1 mmol/d, P < 0.05) consistent with greater bone formation rates (49.4 +/- 13.5 vs. 36.5 +/- 13.6 mmol/d, P < 0.05) relative to bone resorption rates (37.4 +/- 13.2 vs. 29.4 +/- 10.9 mmol/d, P = 0.07), increased calcium absorption efficiency (54 +/- 19 vs. 38 +/- 18%, P < 0.05) and decreased urinary calcium (1.15 +/- 0.95 vs. 2.50 +/- 1.35 mmol/d, P < 0.001), compared with whites. The racial differences in calcium retention in adolescence can account for the racial differences in bone mass of adults.

Alpha and beta-galactosidase activities and oligosaccharide content in peanuts.

Thirty-three peanut cultivars were examined for their alpha-1,6 and beta-1,4 galactosidase activities and oligosaccharide contents along with proximate compositions. The average moisture, protein, fat, ash, and carbohydrate contents were: 4.9%, 26.6%, 43.1%, 2.3% and 23.1%, respectively. The corresponding coefficients of variation were: 5.2%, 10.1%, 7.2%, 7.8% and 15.7%, respectively. Raffinose and stachyose contents (%) ranged from 0.05 to 0.12 and 0.31 to 0.61, respectively. The specific activity (micromol product/min/mg protein) of crude preparation of alpha-galactosidase for the 33 cultivars ranged from 1.096 to 2.784 for the non-germinated seeds and from not being detected in some samples up to 2.432 for the germinated seeds; the mean values for non-germinated and germinated seeds were: 1.781 and 1.410, respectively. The specific activity of beta-galactosidase ranged from 0.101 to 1.727 in the non-germinated seeds and from not being detected in some samples up to 0.898 in the germinated seeds. Germination decreased the activity of both galactosidases significantly (p < or = 0.05).

The new dietary reference intakes for calcium: implications for osteoporosis.

The new Dietary Reference Intakes (DRI) for calcium were released August 13, 1997 by the Food and Nutrition Board of the National Academy of Science. Unlike the RDA's which established the minimal amounts of nutrients needed to be protective against possible deficiency, the new values are designed to reflect the latest understanding about nutrient requirements directed at optimizing health in individuals and groups. Adequate calcium intake recommendations were set at levels associated with desirable retention of body calcium since high bone density is known to be less susceptible to fractures. Recommended intake for adults is 1000 to 1200 mg/day. The majority of people do not consume these levels of calcium. The upper tolerable limit for calcium was set at 2.5 g/day.

Mouse chromosome classification by radial basis function network with fast orthogonal search.

This paper provides the results of our study on automatic classification of mouse chromosomes. A radial basis function neural network was compared with a multi-layer perceptron and a probabilistic neural network. The networks were trained and tested with 3723 chromosomes presented to each network as 30-point banding profiles. The radial basis function classifier trained with the fast orthogonal search learning rule provided the best unconstrained classification error rate of 12.7% which was obtained with a training set of 2250 chromosomes.

Ownership and uses of human tissue: does the Nuffield bioethics report accord with opinion of surgical inpatients?

OBJECTIVE: To compare opinion of surgical inpatients with the conclusions of the report of the Nuffield Council on Bioethics regarding the ownership and uses of human tissue. DESIGN: Survey of results of questionnaires completed by patients. SETTING: Large teaching hospital. SUBJECTS: 384 postoperative adult surgical patients. RESULTS: There was strong support among patients for the use of tissues in medical education, research, and science with the exception of those tissues which may transmit disease to others. Few patients (39; 10%) believed that they retained ownership of tissue removed at surgery. Most believed that the tissue belonged to the hospital (103; 27%), to nobody (103; 27%), or to the laboratory (77; 20%). Most patients had not been given any information about the possible uses of their tissues after removal. CONCLUSIONS: Surgical inpatients seem to endorse the conclusions of the Nuffield report regarding the ownership and uses of human tissue. The recommendations regarding patient information and consent procedures should be implemented at the earliest opportunity.

Interactions of bacterial and amoebal populations in soil microcosms with fluctuating moisture content.

Sterilized soil samples (20 g of soil per 50-ml flask), amended with 600 mug of glucose-carbon and 60 mug of NH(4)-N . g of dry soil, were inoculated with bacteria (Pseudomonas paucimobilis) alone or with bacteria and amoebae (Acanthamoeba polyphaga). We used wet-dry treatments, which involved air drying the samples to a moisture content of approximately 2% and remoistening the samples three times during the 83-day experiment. Control treatments were kept moist. In the absence of amoebae, bacterial populations were reduced by the first drying to about 60% of the moist control populations, but the third drying had no such effect. With amoebae present, bacterial numbers were not significantly affected by the dryings. Amoebal grazing reduced bacterial populations to 20 to 25% of the ungrazed bacterial populations in both moisture treatments. Encystment was an efficient survival mechanism for amoebae subjected to wet-dry cycles. The amoebal population was entirely encysted in dry soil, but the total number of amoebae was not affected by the three dryings. Growth efficiencies for amoebae feeding on bacteria were 0.33 and 0.39 for wet-dry and constantly moist treatments, respectively, results that compared well with those previously reported for Acanthamoeba spp.