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.

Multiplex PCR allows simultaneous detection of pathogens in ships' ballast water.

There is enormous potential for global transfer of microorganisms, including pathogens, in ships' ballast water. We contend that a major advancement in the study of ballast-water microorganisms in particular, and of aquatic pathogens in general, will be expedited sample analysis, such as provided by the elegant technology of DNA microarrays. In order to use DNA microarrays, however, one must establish the appropriate conditions to bind target sequences in samples to multiple probes on the microarrays. We conducted proof-of-concept experiments to optimize simultaneous detection of multiple microorganisms using polymerase chain reaction (PCR) and Southern hybridization. We chose three target organisms, all potentially found in ballast water: a calicivirus, the bacterium Vibrio cholerae, and the photosynthetic protist Aureococcus anophagefferens. Here, we show simultaneous detection of multiple pathogens is possible, a result supporting the promising future use of microarrays for simultaneous detection of pathogens in ballast water.

A time for change and a time for equality--infertility services and the NHS.

The provision of infertility services has always provoked controversy. On the one hand, patients' groups and clinicians involved with infertile couples have long campaigned against the 'postcode lottery'. On the other hand, commissioners have been reluctant to commit resources to what they see as a low priority in health care. The issue has been brought back into the news with the government's decision to ask the National Institute of Clinical Excellence to review the inequalities in provision of fertility services. This article sets out the evidence for viewing infertility as an illness that deserves public funding, and argues that assisted conception should be viewed in the same light as other chronic non-life-threatening conditions that are currently funded by the NHS.

The effect of introduction of a guideline on the management of vaginal discharge and in particular bacterial vaginosis in primary care.

BACKGROUND: Bacterial vaginosis (BV) is the commonest cause of vaginal discharge, and its association with obstetric and gynaecological complications is being recognized increasingly. It was our impression that BV was poorly understood and underdiagnosed in family practice. OBJECTIVE: The aim of this study was to explore the management of patients with vaginal symptoms by family practitioners and to see if the management changed after the assimilation of best practice guidelines. METHOD: Family practitioners were invited to complete a baseline questionnaire of their perceived practice, and to record actual practice when consulted about vaginal symptoms, for a minimum of 4 weeks. Consensus best practice guidelines were then provided and practice recorded for a similar period. RESULTS: Baseline data was received from 34 practitioners and suggested that the symptoms and signs of different vaginal infections were not well known. Most symptomatic patients were only investigated at re-presentation with unresolved symptoms or at recurrence, and 43% of respondents treated with empirical antifungals as a first line approach. Pregnant patients were only occasionally asked about symptoms and only occasionally examined if symptomatic. Pre-guideline practice data from 30 practitioners showed 1.2 patient consultations/week, of which 60% were examined and 55% had a high vaginal swab (HVS) sent. Only 2% had near-patient tests done. Post-guideline data from 23 family practitioners showed a lower recorded consultation rate at 0.7/week, but 90% of these were examined, 77% had an HVS sent and 69% had near-patient tests done. Of the 36 HVS examined by Gram stain, 19 (53%) showed Lactobacillus predominant flora and 10 (28%) suggested BV. Seven (19%) were borderline or ungradable. Only three (8%) showed yeasts, one of which also showed BV. CONCLUSIONS: Baseline data supported our impression that BV was under-recognized. Guidelines appeared to improve the rate of investigation of women consulting with vaginal symptoms.

Reduction of fine airborne particulates (PM3) in a small city centre office, by altering electrostatic forces.

A two stage intervention study was carried out to establish the degree to which a newly developed, electrostatic air cleaning (EAC) system can improve indoor air quality (IAQ) by reducing the number of airborne fine particles. The IAQ and how employees in a city centre office (49 m2) perceived it, was monitored from May until November 1998. The number of fine particles, PM3 (0.3-3.0 microm); number of coarse particles, PM7 (3.0-7.0 microm); number of small positive and negative air ions; relative humidity and temperature were recorded in and out of doors. To assess the employees' perception of any changes in their work environment, a questionnaire was completed. Number of particles, relative humidity and temperature were also recorded in a nearby office, equipped with an identical air processor, where no interventions were made. The results from the first intervention (Stage 1), comparing number of airborne particles outdoors to indoors, gave a 19% reduction for PM3 and a 67% reduction for PM7 (P < 0.001). The reduction in PM3 was inconsistent and not statistically significant (P = 0.3). The reduction in PM7 from outdoors and the removal of PM7 created indoors was achieved by optimizing the existing air moving equipment. The results from the second intervention (Stage 2--with EAC units installed) comparing indoor to outdoor values, gave a further reduction in PM3 of 21% (P < 0.001) and a further 3% reduction for PM7 (P > 0.05). Therefore, at the end of Stage 2, the total reductions in particles from outdoors to indoors were 40% for PM3 and 70% for PM7 (P < 0.001). The Stage 2 results strongly suggest that electrostatic forces, created by the EAC unit(s) improved the removal of PM3, with no further significant improvement in the reduction of PM7. The questionnaire indicated an improvement in the IAQ, as perceived by the employees. The results suggest that the EAC system is effective in reducing PM3 and thereby improving IAQ in an urban office.

Randomized controlled trial of nettle sting for treatment of base-of-thumb pain.

There are numerous published references to use of nettle sting for arthritis pain but no randomized controlled trials have been reported. We conducted a randomized controlled double-blind crossover study in 27 patients with osteoarthritic pain at the base of the thumb or index finger. Patients applied stinging nettle leaf (Urtica dioica) daily for one week to the painful area. The effect of this treatment was compared with that of placebo, white deadnettle leaf (Lamium album), for one week after a five-week washout period. Observations of pain and disability were recorded for the twelve weeks of the study. After one week's treatment with nettle sting, score reductions on both visual analogue scale (pain) and health assessment questionnaire (disability) were significantly greater than with placebo (P = 0.026 and P = 0.0027).

Nettle sting of Urtica dioica for joint pain--an exploratory study of this complementary therapy.

This exploratory study aims to explore the present use of the common stinging nettle to treat joint pain. Eighteen self-selected patients using the nettle sting of Urtica dioica were interviewed. Information regarding patients' use of nettle therapy was elicited, in particular mode of application, dosage and effects. All except one respondent were sure that netles had been very helpful and several considered themselves cured. No observed side effects were reported, except a transient urticarial rash. This exploratory study suggests nettle sting is a useful, safe and cheap therapy which needs further study. A randomized controlled trial is planned in collaboration with a rheumatology specialist.

A retrospective study of the relationship between childhood asthma and respiratory infection during gestation.

BACKGROUND: Wheeze in children has been found to be associated with prior antepartum haemorrhage and raised levels of IgE in cord blood, and acute wheezing episodes are intimately linked with respiratory viral infections. OBJECTIVE: To assess the relationship between maternal presentation with respiratory tract infections in pregnancy and childhood asthma, taking into account factors which could affect presentation. METHODS: This was a case-control study of 200 asthmatic children, 5-16-year-old, age-matched with one control, having no recorded history of wheeze. Data on respiratory tract infections, maternal wheeze, atopy and smoking was collected from primary care records. Deprivation score was assessed according to small residential areas and subjects were equally distributed between four general practices in Plymouth, UK. RESULTS: Presentation with respiratory tract infections during pregnancy was significantly associated with childhood asthma (OR 1.69, 95% confidence interval 1.05-2.77, P = 0.03). The association was marginally stronger for infections in the first trimester (OR 2.30, 95% CI 1.05-5.41, P = 0.04) and for those with cough during pregnancy (OR 2.24, 95% CI 1.23-4.22, P = 0.007). The associations remained significant after allowing for the effect of the independent variables (gender, maternal smoking, maternal wheeze, allergic rhinitis, eczema, asthma treatment in pregnancy and deprivation [Townsend] score), using multiple logistic regression analysis (ORs and 95% CIs 1.91, 1.14-3.22; 2.32, 1.01-5.34 and 2.29, 1.17-4.48, respectively). There was also an association between numbers of presentations with respiratory infections and childhood asthma (test for trend, P = 0.02). CONCLUSIONS: This study has shown an association between presentation with respiratory infection during gestation and childhood asthma. The results were not affected by the other independent variable factors studied and therefore provide some evidence to support the theory that respiratory viruses may be implicated in the aetiology of asthma.

Comparison of two kinds of Biolog microplates (GN and ECO) in their ability to distinguish among aquatic microbial communities.

We compared the abilities of Biolog's GN and ECO plates to distinguish among aerobic and heterotrophic bacterial communities in samples from six aquatic environments. The Biolog system is based on interpreting patterns of sole-carbon substrate utilization indicated by color development in a 96-well microtiter plate. Whether of fresh or saltwater origin, bacterial communities utilized > 95% of substrates in both types of plates. Samples from any one environment exhibited similar time courses of average well color development (AWCD) in both GN and ECO plates. Principal component analysis was performed on data sets resulting from combinations of algorithms (AWCD and curve-integration methods) and levels of color development (end-point and set-point approaches). In all cases, the two types of plates demonstrated an equal capacity to discriminate among the heterotrophic expressions of the six microbial communities. Substantial deviation from an anticipated 1:1 correspondence occurred when color development of 25 substrates common to both types of plates was compared. The discrepancies likely are related to the different formulations of low-nutrient media in GN and ECO plates.

Mobility screening in the elderly and resulting referral.

The aims of this study were to test the usefulness of the Royal College of General Practitioners' (RCGP) mobility screening questionnaire in Irish general practice and to use it to assess the level of mobility problems in the over-75 year old population. Questionnaire screening survey in six faculty areas of the Irish College of General Practitioners(ICGP) well-spread geographically to be representative of the Irish population. Participants were a random sample of 627 people aged 75 years or over, from the General Medical Services list of 92 volunteer general practitioners. The Royal College of General Practitioners' screening questionnaire for mobility was administered by each person's doctor. Suggested interventions were also noted. 92% felt able to get around the house independently, with 78% able to manage stairs independently. The commonest aid to mobility was a stick, used by 27%. Mental impairment was closely associated with inability to get around the house (86% of those not independent around the house scored less than 8 on the Abbreviated Mental Test Score). Analysis of referral patterns indicated possible deficiencies in the availability of physiotherapy in certain areas. The RCGP mobility screening questionnaire was easy to use, and helpful in detecting problems which might require attention.

Case-finding incontinence in the over-75s.

BACKGROUND: The under-reporting of incontinence in older persons is well known. However, the general practitioner's (GP's) knowledge of incontinence in this population is less wall documented. AIM: To examine the knowledge of Irish GPs regarding incontinence in patients aged over 75, and to examine the relationship between incontinence and cognitive function in this age group. METHOD: Sixty-four GPs from six faculties of the Irish College of General Practitioners (ICGP), spread geographically over Ireland, administered a questionnaire to 10 of their patients, selected from the General Medical Services list, as part of the ICGP 1993 Care of the Elderly Study. A short test of cognitive function, the AMTS, was administered followed by RCGP (Royal College of General Practitioners) screening questions for incontinence, plus questions with regard to patients' prior reporting of incontinence. GPs were also asked to state their prior knowledge of the incontinence status of the patient and to choose from a list of management options including referral, assigning to a public health nurse, and hospital surgical service. RESULTS: Data were analysed on 527 patients aged over 75 years. Forty-four per cent of persons over 75 years reported having experienced urinary incontinence; 9% reported having experienced faecal incontinence. GPs reported full knowledge of the incontinence status in only 33% of their patients. The effects of low cognitive function, sex, and age on the probability of wetting were analysed using logistic regression modelling. Female sex and low cognitive score increased the likelihood of ever wetting; increasing age increased the likelihood of daily wetting. Poor sensitivities for these models limit their usefulness in clinical practice. CONCLUSION: GPs should have a high index of suspicion for incontinence in persons aged over 75. Thorough history-taking, physical examination, and examinations of therapeutic options for individual cases are recommended.

Epibiotic microorganisms on copepods and other marine crustaceans.

Although the occurrence of microbial (algal, protozoan, bacterial, and fungal) epibionts on marine crustaceans and other invertebrates has been documented repeatedly, the ecological context and significance of these relationships generally are not well understood. Recently, several studies have examined the population and community ecology of algal and protozoan epibionts on freshwater crustaceans. Even so, the study of microbial epibionts in aquatic environments is still in its infancy. In this review, we summarize associations of microalgae, protozoans, and bacteria with marine crustaceans, especially copepods. We note differences and commonalities across epibiont taxa, consider host-epibiont cycling of nutrients, generate hypotheses relevant to the ecology of the host and the epibiont, and suggest future research opportunities.

A scoring system for predicting group A streptococcal throat infection.

BACKGROUND: Sore throat is very common in general practice and is usually caused by viral infection. Nevertheless, up to 95% of patients may be treated with antibiotics. Previous diagnostic systems have not transferred well from one area to another because of an inability to allow for changing prevalence of streptococcus. AIM: To measure the occurrence rates of symptoms and signs in sore throat patients with and without streptococcal infection, and to develop a Bayesian scoring system which is easily adapted for prevalence to predict if patients have bacterial infection. METHOD: Occurrence rates of symptoms and signs were measured for 206 patients with sore throat symptoms over a 3-year period. Bayesian probability scores (B-scores) for each data item were calculated from the ocurrence rates in the patients with positive throat cultures for group A streptococci and the rates in patients with negative throat cultures. The B-score values were then used to predict the probability of positive culture for each patient. RESULTS: The streptococcal throat B-score system predicted positive culture with a sensitivity of 71% and a specificity of 71%. In comparison, the unaided general practitioners predicted infection with a sensitivity of 61% and a specificity of 65%. If the B-score prediction had been used to decide on treatment, more patients with streptococci present on culture would have been treated with antibiotic (71% instead of 68%) and appreciably fewer patients with negative streptococcal cultures would have been treated (29% instead of 59%). CONCLUSION: Use of the B-score system could result in significant savings in unnecessary antibiotic prescription, and unnecessary throat swab cultures, while achieving better levels of treatment.

A computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rRNA genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature.

An assessment of 10 tetrameric restriction enzymes (TREs) was conducted by using a computer-simulated restriction fragment length polymorphism (RFLP) analysis for over 100 proximally and distally related bacterial small-subunit (SSU) rRNA gene sequences. Screening SSU rDNA clone libraries with TREs has become an effective strategy because of logistic simplicity, commercial availability, and economy. However, the rationale for selecting the type and number of TREs has not been systematically evaluated. Our objective was to identify the optimal combination of TREs for RFLP screening of cloned SSU rRNA genes from undefined bacterial clone libraries. After computer-simulated TRE digestion, the resultant fragments were categorized on the basis of the frequency of different restriction fragment size classes. Three groups of distribution patterns for the TREs were determined and further examined via graphical exploratory data analysis. The RFLP size-frequency distribution data for each group of enzymes were then used to infer phylogenetic relationships via the neighbor-joining method. The resulting bootstrap values and the correct placement of node bifurcations were used as additional criteria to evaluate the efficacy of the selected TREs. These RFLP data were compared with known phylogenetic relationships based on SSU rRNA sequence analysis as defined by the Ribosomal Database Project. A heuristic approach testing random combinations of TREs showed that three or more TRE combinations detected > 99% of the operational taxonomic units (OTUs) within the model data set. OTUs that remained undetected after three TRE treatments had a median sequence similarity of 96.1%. Of the 10 restriction enzymes examined, HhaI, RsaI, and BstUI (group 3) were the most efficacious at detecting and differentiating bacterial SSU rRNA genes on the basis of their ability to correctly classify OTUs. Group 3 TREs are therefore recommended for screening in studies using bacterial SSU rRNA genes as descriptors of in situ microbial diversity.

Changes in myocardial perfusion abnormalities by positron emission tomography after long-term, intense risk factor modification.

OBJECTIVE: To quantify changes in size and severity of myocardial perfusion abnormalities by positron emission tomography (PET) in patients with coronary artery disease after 5 years of risk factor modification. DESIGN: Randomized controlled trial. SETTING: Outpatient community setting. INTERVENTION: Randomization of patients to risk factor modification consisting of very low-fat vegetarian diet, mild to moderate exercise, stress management, and group support (experimental group, n = 20) or to usual care by their own physicians, consisting principally of antianginal therapy (control group, n = 15). MAIN OUTCOME MEASURES: Quantitative coronary arteriography and PET at baseline and 5 years after randomization. Automated, objective measures of size and severity of perfusion abnormalities on rest-dipyridamole PET images and of stenosis severity on arteriograms were made by computer algorithms. RESULTS: Size and severity of perfusion abnormalities on dipyridamole PET images decreased (improved) after risk factor modification in the experimental group compared with an increase (worsening) of size and severity in controls. The percentage of left ventricle perfusion abnormalities outside 2.5 SDs of those of normal persons (based on 20 disease-free individuals) on the dipyridamole PET image of normalized counts worsened in controls (mean +/- SE, + 10.3% +/- 5.6%) and improved in the experimental group (mean +/- SE, -5.1% +/- 4.8%) (P = .02); the percentage of left ventricle with activity less than 60% of the maximum activity on the dipyridamole PET image of normalized counts worsened in controls (+13.5% +/- 3.8%) and improved in the experimental group (-4.2% +/- 3.8%) (P = .002); and the myocardial quadrant on the PET image with the lowest average activity expressed as a percentage of maximum activity worsened in controls (-8.8% +/- 2.3%) and improved in the experimental group (+4.9% +/- 3.3%) (P = .001). The size and severity of perfusion abnormalities on resting PET images were also significantly improved in the experimental group as compared with controls. The relative magnitude of changes in size and severity of PET perfusion abnormalities was comparable to or greater than the magnitude of changes in percent diameter stenosis, absolute stenosis lumen area, or stenosis flow reserve documented by quantitative coronary arteriography. CONCLUSIONS: Modest regression of coronary artery stenoses after risk factor modification is associated with decreased size and severity of perfusion abnormalities on rest-dipyridamole PET images. Progression or regression of coronary artery disease can be followed noninvasively by dipyridamole PET reflecting the integrated flow capacity of the entire coronary arterial circulation.

Studies with [11C]alprazolam: an agonist for the benzodiazepine receptor.

We have built a system for the synthesis of high specific activity carbon-11 alprazolam (Xanax), a high affinity agonist for the benzodiazepine receptor. The system produces 30-40 mCi of the compound with a specific activity of > 12,000 Ci per millimole. Using this compound we have performed PET studies on 6 normal subjects and studied the cerebral influx and efflux of the compound. The uptake in the brain was low, approx. 1% of the administered dose. However, the levels of the compound in the circulation at early time points are heavily affected by the specific activity of the tracer, i.e. when pharmacologically active doses are used as blocking doses the concentration of radioactive material is higher in the circulation and more material enters the brain. We attribute this to a depot effect where the compound is trapped in saturatable sites in an organ, probably the lungs, and is slowly released over time. In the presence of blocking doses of agonist, the compound washes out of the brain more quickly suggesting that some blockade of the receptors is occurring. However, the pharmacological activity of the compound does not permit the administration of enough material to ensure complete receptor blockade. The compound shows definite signs of acting as a receptor binding ligand but the unusual pharmacokinetics complicate the interpretation of the data.