Nuclear targeting of dystroglycan promotes the expression of androgen regulated transcription factors in prostate cancer.

Dystroglycan is frequently lost in adenocarcinoma, but the mechanisms and consequences are poorly understood. We report an analysis of ß-dystroglycan in prostate cancer in human tissue samples and in LNCaP cells in vitro. There is progressive loss of ß-dystroglycan immunoreactivity from basal and lateral surfaces of prostate epithelia which correlates significantly with increasing Gleason grade. In about half of matched bone metastases there is significant dystroglycan re-expression. In tumour tissue and in LNCaP cells there is also a tyrosine phosphorylation-dependent translocation of ß-dystroglycan to the nucleus. Analysis of gene expression data by microarray, reveals that nuclear targeting of ß-dystroglycan in LNCaP cells alters the transcription of relatively few genes, the most unregulated being the transcription factor ETV1. These data suggest that proteolysis, tyrosine phosphorylation and translocation of dystroglycan to the nucleus resulting in altered gene transcription could be important mechanisms in the progression of prostate cancer.

Dystroglycan function is a novel determinant of tumor growth and behavior in prostate cancer.

BACKGROUND: Dystroglycan is a ubiquitously expressed cell adhesion molecule frequently found to be altered or reduced in adenocarcinomas, however the mechanisms or consequences of dystroglycan loss have not been studied extensively. METHODS: We examined the consequence of overexpression or RNAi depletion of dystroglycan on properties of in vitro growth migration and invasion of LNCaP, PC3, and DU145 prostate cancer cell lines. RESULTS: Using LNCaP cells we observed cell density-dependent changes in ß-dystroglycan with the appearance of several lower molecular weight species ranging in size from 43 to 26 kDa. The bands of 31 and 26 kDa were attributed to proteolysis, whereas bands between 43 and 38 kDa were a consequence of mis-glycosylation. The localization of ß-dystroglycan in LNCaP colonies in culture also varied, cells with a mesenchymal appearance at the periphery of the colony had more pronounced membrane localization of dystroglycan. Whereas some cells demonstrated nuclear dystroglycan. Increased dystroglycan levels were inhibitory to growth in soft agar but promoted Matrigel invasion, whereas reduced dystroglycan levels promoted growth in soft agar but inhibited invasion. Similar results were also obtained for PC3 and DU145 cells. CONCLUSIONS: This study suggests that changes in ß-dystroglycan distribution within the cell and/or the loss of dystroglycan during tumorigenesis, through a combination of proteolysis and altered glycosylation, leads to an increased ability to grow in an anchorage independent manner, however dystroglycan may need to be re-expressed for cell invasion and metastasis to occur.

Imaging patients with renal colic: a comparative analysis of the impact of non-contrast helical computed tomography versus intravenous pyelography on the speed of patient processing in the Emergency Department.

INTRODUCTION: Non-contrast helical CT (NHCT) became the procedure of choice for investigating Emergency Department (ED) patients presenting with suspected renal colic at Beaumont Hospital, Dublin, in 2008. The impact of NHCT on waiting times and patient management was compared with intravenous pyelography (IVP). METHODS: A retrospective, comparative cohort analysis of 95 patients who had IVP and 109 patients who had NHCT was performed. Length of ED stay from time of scan ordering to referral or discharge was analysed relative to time of day and scan result. RESULTS: Patients having NHCT who attended between 00:00-08:00 h, had a twofold longer length of stay than those who had IVP between the same hours (median 7.07 h vs 3.03 h, p=0.0294). The length of ED stay for patients attending between 08:00 and 24:00 h was similar in both groups. The presence of urolithiasis did not impact on length of stay. A significant alternate/incidental diagnosis was reported in 28 patients having NHCT, of which 12 were cancerous growths. CONCLUSION: NHCT allows for the detection of incidental/alternate diagnoses that may not be otherwise detected in patients with renal colic. Compared to IVP, NHCT has not impacted positively on the speed of patient processing in the ED under study. For patients presenting after midnight, it is associated with over a twofold longer length of stay from the time of scan ordering to referral or discharge. This leads to prolonged patient stays in the ED, and as such contributes to overcrowding.

Impact of admission screening for methicillin-resistant Staphylococcus aureus on the length of stay in an emergency department.

Preventing and controlling methicillin-resistant Staphylococcus aureus (MRSA) includes early detection and isolation. In the emergency department (ED), such measures have to be balanced with the requirement to treat patients urgently and transfer quickly to an acute hospital bed. We assessed, in a busy and overcrowded ED, the contribution made to a patient's stay by previous MRSA risk group identification and by selective rescreening of those patients who were previously documented in the research hospital as being MRSA positive. Patients with a previous diagnosis of MRSA colonisation were flagged automatically as 'risk group' (RG) on their arrival in the ED and were compared with 'non-risk group' (NRG), i.e. not previously demonstrated in the research hospital to be infected or colonised with MRSA. Over an 18 month period, there were 16 456 admissions via the ED, of which 985 (6%) were RG patients. The expected median times to be admitted following a request for a ward bed for NRG and RG patients were 10.4 and 12.9h, respectively. Female sex, age >65 years, and RG status all independently predicted a statistically significantly longer stay in the ED following a request for a hospital bed. We consider that national and local policies for MRSA need to balance the welfare of patients in the ED with the need to comply with best practice, when there are inadequate ED and inpatient isolation facilities. Patients with MRSA requiring emergency admission must have a bed available for them.

DNW--"did not wait" or "demographic needing work": a study of the profile of patients who did not wait to be seen in an Irish emergency department.

BACKGROUND: Patients who fail to wait for medical assessment in the emergency department (ED) have been referred to in the international literature as "did not wait" (DNW) or "left without being seen" (LWBS) patients or, indeed, simply as "walkouts". This is taken as a performance indicator internationally. In common with many countries, Ireland has very considerable problems in the delivery of ED care due largely to inadequate resources and the inappropriate use of EDs as holding bays for admitted patients. This is the first study of this size to profile the DNW phenomenon in Ireland. METHODS: The charts of DNW patients were identified and the DNW status was entered into the ED computer record. Data concerning age, sex, time of arrival, date of arrival, triage category and presenting complaint were recorded. RESULTS: In the study period there were 11 662 patient attendances, of whom 871 patients (7.47%) did not wait for assessment. Triage category was highly statistically significant, with those in the lowest triage category having the highest numbers not waiting to be seen (p<0.001). Those attending at night (p<0.001) and at the weekend (p = 0.03) were most likely to leave without being assessed. CONCLUSION: Failure to provide the service in a timely manner gives rise to patients leaving without receiving the medical assessment they came to obtain. This is a serious clinical problem and puts both those requiring care and those providing it at risk of adverse outcomes.

The Transit/Admission Lounge study.

In response to persistent overcrowding of Emergency Departments in Ireland, the Department of Health and Health Service Executive provided funding for "Transit Lounge" areas to be built. These lounges were to provide a location for patients to wait in beds pending the availability of a ward bed. This research was performed to assess the impact of such a lounge on the overcrowding of the Emergency Department and on patient outcomes. The time period from the opening of the Transit Lounge was compared with the same period a year earlier. The Transit Lounge delivers a comfortable place for patients to wait. It does not reduce Emergency Department overcrowding and has been associated with an increased time waiting for a ward bed. The solution to overcrowding is the creation of real capacity in the system so that ward beds are available in acute hospitals for the "unscheduled unwell".

Expression of beta-dystroglycan is reduced or absent in many human carcinomas.

AIMS: Dystroglycan is an important structural and signalling protein that is expressed in most human cells. alpha-Dystroglycan has been investigated and found to be reduced in human cancers, but there is only one published study on the expression of beta-dystroglycan in human cancer and that was only on small numbers of breast and prostatic cancers. The aim was to conduct a comprehensive immunohistochemical survey of the expression of beta-dystroglycan in normal human tissues and common cancers. METHODS AND RESULTS: Triplicate tissue microarrays of 681 samples of normal human tissues and common cancers were stained using an antibody directed against the cytoplasmic component of beta-dystroglycan. beta-Dystroglycan was strongly expressed at the intercellular junctions and basement membranes of all normal human epithelia. Expression of beta-dystroglycan was absent or markedly reduced in 100% of oesophageal adenocarcinomas, 97% of colonic cancers, 100% of transitional cell carcinomas of the urothelium and 94% of breast cancers. In the breast cancers, the only tumours that showed any retention of beta-dystroglycan expression were small low-grade oestrogen receptor-positive tumours. The only cancers that showed retention of beta-dystroglycan expression were cutaneous basal cell carcinomas. CONCLUSIONS: There is loss or marked reduction of beta-dystroglycan expression (by immunohistochemistry) in the vast majority of human cancers surveyed. Since beta-dystroglycan is postulated to have a tumour suppressor effect, this loss may have important functional significance.

Evaluation of formal feedback on endoscopic competence among trainees: the EFFECT trial.

INTRODUCTION: The medical literature describes disparity in colonoscopy performance. This randomised, controlled study aimed to characterise the impact of feedback on colonoscopy performance among gastroenterology (GI) trainees. METHODS: Gastroenterology trainees of similar experience levels who independently performed 581 colonoscopies over the study period were randomised to receive feedback/no feedback on their colonoscopy performance. RESULTS: Baseline colonoscopy performance was similar in both groups. Following feedback, caecal intubation improved by 10.5% (from 72.9 to 83.4%, p = 0.04) in the feedback group and declined by 6.1% (from 78 to 71.9%, p = 0.2) in the control group; polyp detection improved by 5.1% (from 12.9 to 18.0%, p = 0.2) in the feedback group and by 2.9% (from 16.7 to 19.6%, p = 0.5) in the control group. CONCLUSIONS: Systematic feedback appears to enhance colonoscopy performance among GI trainees.

The Boarders in the Emergency Department (BED) study.

BACKGROUND AND OBJECTIVES: In the Boarders in the Emergency Department (BED) study the impact of overcrowding due to boarders on patients' mortality and the likelihood of being diagnosed with methicillin resistant Staphylococcus aureus (MRSA) during admission was examined. With regard to efficiency, the impact of overcrowding on the time to first medical assessment for admitted patients, the number of patients leaving without being seen, and the rate of admission as a percentage of total emergency department attendances was explored. METHODS: The retrospective cohort analysis study of all emergency department admissions was performed using information accessed via the Diver Solution. The software integrated information from several databases. RESULTS: The average number of patients awaiting hospital admission in the emergency department at 09:00 was 20.4 (range 0-45). The average duration of stay in the emergency department following the decision to admit was 16.1 h (range 0-161 h). The number who did not wait (DNW) to be seen was strongly correlated with the time waiting for medical assessment, which in turn was correlated with the total number of attendances to the emergency department (p<0.001). The elderly waited longer for admission and had the highest mortality and the highest chance of being diagnosed with MRSA during their overall admission. CONCLUSION: It is wrong for patients who are sick enough to require admission to hospital to be kept in the emergency department, and the entire health system must respond to their plight.

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.

Targeting of dystroglycan to the cleavage furrow and midbody in cytokinesis.

Dystroglycan is a cell adhesion molecule that interacts with ezrin family proteins and also components of the extracellular signal-regulated kinase pathway. Ezrin and extracellular signal-regulated kinase are both involved in aspects of the cell division cycle. We therefore examined the role of dystroglycan during cytokinesis. Endogenous dystroglycan colocalised with ezrin at the cleavage furrow and midbody during cytokinesis in REF52 cells. Live cell imaging of green fluorescent protein-tagged dystroglycan in Swiss 3T3 and Hela cells revealed a similar localisation. Live cell imaging of a dystroglycan lacking its cytoplasmic domain revealed an even membrane localisation but no cleavage furrow or midbody localisation. Deletion of a previously identified ezrin-binding site in the dystroglycan cytoplasmic domain however only resulted in a slight reduction in cleavage furrow localisation but loss of midbody staining. There was no apparent cytokinetic defect in cells depleted for dystroglycan, however apoptosis levels were considerably higher in dystroglycan knockdown cells. Cell cycle analysis showed a delay in G2/M transition, possibly caused by a more than 50% reduction in extracellular signal-regulated kinase levels in the knockdown cells. Dystroglycan may therefore not only have a role in organising the contractile ring through direct or indirect associations with actin, but can also modulate the cell cycle by affecting extracellular signal-regulated kinase levels.

Why are we waiting? A study of the patients' perspectives about their protracted stays in an emergency department.

The overcrowding of Emergency Departments compromises their critical function and the safety of patients and staff. This study asked the patients how the wait in overcrowded conditions impacted on them and the care they received and what they believed the reasons for the overcrowding were. A prospective questionnaire based structured interview study was performed. Over half (57.7%) of patients felt that the lack of inpatient beds and wards was the main reason that they experienced delays. An overwhelming 85.9% felt that the Health Authorities were not doing enough to address the overcrowding issue. Overcrowding of Emergency Departments has been identified as a major problem the solution is to be found in increasing the capacity of the acute hospital system according to the majority of our study population.

Dystroglycan: a multifunctional adaptor protein.

Dystroglycan, a ubiquitous membrane-spanning cell adhesion molecule, is a crucial link between the actin cytoskeleton and the extracellular matrix. With a wide expression pattern and multiple interacting proteins, not only is dystroglycan now thought to be important as a structural molecule but also new research has suggested that it has a role in cell signalling, cytoskeleton reorganization and as a potential tumour suppressor.

Spectrin, alpha-actinin, and dystrophin.

Spectrin family proteins represent an important group of actin-bundling and membrane-anchoring proteins found in diverse structures from yeast to man. Arising from a common ancestral alpha-actinin gene through duplications and rearrangements, the family has increased to include the spectrins and dystrophin/utrophin. The spectrin family is characterized by the presence of spectrin repeats, actin binding domains, and EF hands. With increasing divergence, new domains and functions have been added such that spectrin and dystrophin also contain specialized protein-protein interaction motifs and regions for interaction with membranes and phospholipids. The acquisition of new domains also increased the functional complexity of the family such that the proteins perform a range of tasks way beyond the simple bundling of actin filaments by alpha-actinin in S. pombe. We discuss the evolutionary, structural, functional, and regulatory roles of the spectrin family of proteins and describe some of the disease traits associated with loss of spectrin family protein function.

Ezrin-dependent regulation of the actin cytoskeleton by beta-dystroglycan.

Dystroglycan is part of an adhesion receptor complex linking the extracellular matrix to the actin cytoskeleton. Previous studies have implicated dystroglycan in basement membrane formation and as a crucial link between dystrophin and laminin in muscle. We report here a further novel function for dystroglycan which appears to be in addition to its role as an adhesion molecule. beta-dystroglycan has been localized to microvilli structures in a number of cell types where it associates with the cytoskeletal adaptor ezrin, through which it is able to modulate the actin cytoskeleton and induce peripheral filopodia and microvilli. Ezrin is able to interact with dystroglycan through a cluster of basic residues in the juxtamembrane region of dystroglycan, and mutation of these residues both prevents ezrin binding and the induction of actin-rich surface protrusions. These studies reveal novel functions and additional signalling roles for dystroglycan, raising the possibility of new avenues for therapeutic intervention in diseases such as Duchenne muscular dystrophy.

A prospective, case controlled study of the natural history of diabetic retinopathy and maculopathy after uncomplicated phacoemulsification cataract surgery in patients with type 2 diabetes.

AIM: To determine if uncomplicated phacoemulsification cataract surgery is associated with an accelerated rate of progression of diabetic retinopathy or maculopathy postoperatively. METHODS: A prospective trial of 50 type 2 diabetics undergoing monocular phacoemulsification cataract surgery by a single consultant surgeon. The grade of diabetic retinopathy and diabetic maculopathy in the operated and non-operated fellow eye was assessed preoperatively and for 12 months postoperatively. RESULTS: Overall, retinopathy progression was observed in 11 patients. In seven the retinopathy progressed in both eyes, in three it progressed in the operated eye alone, and in one it progressed in the fellow eye alone. Macular oedema was observed in 13 eyes postoperatively. Four had transient pseudophakic cystoid macular oedema and nine true diabetic maculopathy. Where maculopathy progressed it did so symmetrically in five patients, it progressed in the operated eye alone in four patients, and the fellow eye alone in two patients. There was no significant difference in the number of operated and fellow eyes whose retinopathy or maculopathy progressed postoperatively. In both the operated (OE) and non-operated (NoE) eyes retinopathy progression was associated with a higher mean HbA(1)C (OE p=0.003; NoE p=0.001) and insulin treatment (OE p=0.008, NoE p=0.04). CONCLUSION: Uncomplicated phacoemulsification cataract surgery does not cause acceleration of diabetic retinopathy postoperatively and any progression that is observed probably represents the natural history of the disease. Although macular oedema is common after cataract surgery it may follow a benign course and in many patients the development of clinically significant macular oedema postoperatively probably represents natural disease progression rather than being a direct effect of surgery.

The interaction of dystrophin with beta-dystroglycan is regulated by tyrosine phosphorylation.

Dystrophin and the dystrophin-associated protein complex (DAPC) have recently been implicated in cell signalling events. These proteins are ideally placed to transduce signals from the extracellular matrix (ECM) to the cytoskeleton. Here we show that beta-dystroglycan is tyrosine-phosphorylated in C2/C4 mouse myotubes. Tyrosine phosphorylation was detected by mobility shifts on SDS-polyacrylamide gels (SDS-PAGE) and confirmed by immunoprecipitation and two-dimensional gel electrophoresis. The potential functional significance of this tyrosine phosphorylation was investigated using peptide 'SPOTs' assays. Phosphorylation of tyrosine in the 15 most C-terminal amino acids of beta-dystroglycan disrupts its interaction with dystrophin. The tyrosine residue in beta-dystroglycan's WW-binding motif PPPY appears to be the most crucial in disrupting the beta-dystroglycan-dystrophin interaction. beta-dystroglycan forms the essential link between dystrophin and the rest of the DAPC. This regulation by tyrosine phosphorylation may have implications in the pathogenesis and treatment of Duchenne's muscular dystrophy (DMD).