In silico identification and in vivo validation of miR-495 as a novel regulator of motivation for cocaine that targets multiple addiction-related networks in the nucleus accumbens.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression and are implicated in the etiology of several neuropsychiatric disorders, including substance use disorders (SUDs). Using in silico genome-wide sequence analyses, we identified miR-495 as a miRNA whose predicted targets are significantly enriched in the Knowledgebase for Addiction Related Genes (ARG) database (KARG; http://karg.cbi.pku.edu.cn). This small non-coding RNA is also highly expressed within the nucleus accumbens (NAc), a pivotal brain region underlying reward and motivation. Using luciferase reporter assays, we found that miR-495 directly targeted the 3'UTRs of Bdnf, Camk2a and Arc. Furthermore, we measured miR-495 expression in response to acute cocaine in mice and found that it is downregulated rapidly and selectively in the NAc, along with concomitant increases in ARG expression. Lentiviral-mediated miR-495 overexpression in the NAc shell (NAcsh) not only reversed these cocaine-induced effects but also downregulated multiple ARG mRNAs in specific SUD-related biological pathways, including those that regulate synaptic plasticity. miR-495 expression was also downregulated in the NAcsh of rats following cocaine self-administration. Most importantly, we found that NAcsh miR-495 overexpression suppressed the motivation to self-administer and seek cocaine across progressive ratio, extinction and reinstatement testing, but had no effect on food reinforcement, suggesting that miR-495 selectively affects addiction-related behaviors. Overall, our in silico search for post-transcriptional regulators identified miR-495 as a novel regulator of multiple ARGs that have a role in modulating motivation for cocaine.

Adaptation of Rhodopseudomonas acidophila strain 7050 to growth at different light intensities: what are the benefits to changing the type of LH2?

Typical purple bacterial photosynthetic units consist of light harvesting one/reaction centre 'core' complexes surrounded by light harvesting two complexes. Factors such as the number and size of photosynthetic units per cell, as well as the type of light harvesting two complex that is produced, are controlled by environmental factors. In this paper, the change in the type of LH2 present in the Rhodopsuedomonas acidophila strain 7050 is described when cells are grown at a range of different light intensities. This species contains multiple pucBA genes that encode the apoproteins that form light-harvesting complex two, and a more complex mixture of spectroscopic forms of this complex has been found than was previously thought to be the case. Femto-second time resolved absorption has been used to investigate how the energy transfer properties in the membranes of high-light and low-light adapted cells change as the composition of the LH2 complexes varies.

Biochemical and genetic predictors and correlates of response to lamotrigine and folic acid in bipolar depression: Analysis of the CEQUEL clinical trial.

OBJECTIVES: CEQUEL (Comparative Evaluation of QUEtiapine plus Lamotrigine combination versus quetiapine monotherapy [and folic acid versus placebo] in bipolar depression) was a double-blind, randomized, placebo-controlled, parallel group, 2×2 factorial trial that examined the effect of adding lamotrigine and/or folic acid (FA) to quetiapine in bipolar depression. Lamotrigine improved depression, but its effectiveness was reduced by FA. We investigated the baseline predictors and correlates of clinical response, and the possible basis of the interaction. METHODS: The main outcome was change in depressive symptoms at 12 weeks, measured using the Quick Inventory for Depressive Symptoms-self report version 16 (QIDS-SR16). We examined the relationship between symptoms and lamotrigine levels, and biochemical measures of one-carbon metabolism and functional polymorphisms in catechol-O-methyltransferase (COMT), methylene tetrahydrofolate reductase (MTHFR) and folate hydrolase 1 (FOLH1). RESULTS: Lamotrigine levels were unaffected by FA and did not differ between those participants who achieved remission and those with persisting symptoms. When participants with subtherapeutic serum levels were excluded, there was a main effect of lamotrigine on the main outcome, although this remained limited to those randomized to FA placebo. None of the biochemical measures correlated with clinical outcome. The negative impact of FA on lamotrigine response was limited to COMT Met carriers. FOLH1 and MTHFR had no effect. CONCLUSIONS: Our results clarify that FA's inhibition of lamotrigine's efficacy is not a pharmacokinetic effect, and that low serum lamotrigine levels contributed to lamotrigine's lack of a main effect at 12 weeks. We were unable to explain the lamotrigine-FA interaction, but our finding that it is modulated by the COMT genotype provides a starting point for follow-on neurobiological investigations. More broadly, our results highlight the value of including biochemical and genetic indices in randomized clinical trials.

Towards quantification of vibronic coupling in photosynthetic antenna complexes.

Photosynthetic antenna complexes harvest sunlight and efficiently transport energy to the reaction center where charge separation powers biochemical energy storage. The discovery of existence of long lived quantum coherence during energy transfer has sparked the discussion on the role of quantum coherence on the energy transfer efficiency. Early works assigned observed coherences to electronic states, and theoretical studies showed that electronic coherences could affect energy transfer efficiency--by either enhancing or suppressing transfer. However, the nature of coherences has been fiercely debated as coherences only report the energy gap between the states that generate coherence signals. Recent works have suggested that either the coherences observed in photosynthetic antenna complexes arise from vibrational wave packets on the ground state or, alternatively, coherences arise from mixed electronic and vibrational states. Understanding origin of coherences is important for designing molecules for efficient light harvesting. Here, we give a direct experimental observation from a mutant of LH2, which does not have B800 chromophores, to distinguish between electronic, vibrational, and vibronic coherence. We also present a minimal theoretical model to characterize the coherences both in the two limiting cases of purely vibrational and purely electronic coherence as well as in the intermediate, vibronic regime.

The evolution of novelty in conserved genes; evidence of positive selection in the Drosophila fruitless gene is localised to alternatively spliced exons.

There has been much debate concerning whether cis-regulatory or coding changes are more likely to produce evolutionary innovation or adaptation in gene function, but an additional complication is that some genes can dramatically diverge through alternative splicing, increasing the diversity of gene function within a locus. The fruitless gene is a major transcription factor with a wide range of pleiotropic functions, including a fundamental conserved role in sexual differentiation, species-specific morphology and an important influence on male sexual behaviour. Here, we examine the structure of fruitless in multiple species of Drosophila, and determine the patterns of selective constraint acting across the coding region. We found that the pattern of selection, estimated from the ratio of non-synonymous to synonymous substitutions, varied considerably across the gene, with most regions of the gene evolutionarily conserved but with several regions showing evidence of divergence as a result of positive selection. The regions that showed evidence of positive selection were found to be localised to relatively consistent regions across multiple speciation events, and are associated with alternative splicing. Alternative splicing may thus provide a route to gene diversification in key regulatory loci.

Evaluation of the Edmonton Functional Assessment Tool (EFAT2) within palliative care: a pilot study.

BACKGROUND: There is increasing need for rehabilitation in both cancer and palliative care. However, there are few validated outcome measures that are suitable for measuring functional performance in this population. The present study evaluated the validity, sensitivity and reliability of the Edmonton Functional Assessment Tool (EFAT2) within a UK palliative care setting. METHODS: Eleven participants aged 65 years and over (mean age 76.5 ± 6.7 years) receiving rehabilitation in a palliative care inpatient setting were studied. Concurrent validity was assessed by comparing EFAT2 with the Barthel Index. Inter-rater reliability of EFAT2 was examined using a sample of four participants recruited from a cancer care ward. RESULTS: A significant negative correlation was observed between the Barthel Index and EFAT2 (r = -0.765, p = 0.01) and both measures were found to be sensitive as determined by Cohen's effect size (EFAT2 = 0.60, Barthel Index = 0.72). High inter-rater reliability was noted for EFAT2 (ICC3, 1 = 0.85) and the agreement between scores was confirmed by Bland-Altman analysis. CONCLUSIONS: EFAT2 showed concurrent validity with the Barthel Index when used to assess the effects of rehabilitation on participants with advanced cancer. The tool was sensitive to change and was found to be reliable when used by different raters. The findings indicate that EFAT2 might be an appropriate outcome measure to use within the palliative care setting. However, the feasibility of using EFAT2 needs to be explored and larger studies are required to confirm its reliability.

Randomized controlled trial of patient-controlled sedation for colonoscopy: Entonox vs modified patient-maintained target-controlled propofol.

AIM: Propofol sedation is often associated with deep sedation and decreased manoeuvrability. Patient-maintained sedation has been used in such patients with minimal side-effects. We aimed to compare novel modified patient-maintained target-controlled infusion (TCI) of propofol with patient-controlled Entonox inhalation for colonoscopy in terms of analgesic efficacy (primary outcome), depth of sedation, manoeuvrability and patient and endoscopist satisfaction (secondary outcomes). METHOD: One hundred patients undergoing elective colonoscopy were randomized to receive either TCI propofol or Entonox. Patients in the propofol group were administered propofol initially to achieve a target concentration of 1.2 µg/ml and then allowed to self-administer a bolus of propofol (200 µg/kg/ml) using a patient-controlled analgesia pump with a handset. Entonox group patients inhaled the gas through a mouthpiece until caecum was reached and then as required. Sedation was initially given by an anaesthetist to achieve a score of 4 (Modified Observer's Assessment of Alertness and Sedation Scale), and colonoscopy was then started. Patients completed an anxiety score (Hospital Anxiety and Depression questionnaire), a baseline letter cancellation test and a pain score on a 100-mm visual analogue scale before and after the procedure. All patients completed a satisfaction survey at discharge and 24 h postprocedure. RESULTS: The median dose of propofol was 174 mg, and the median number of propofol boluses was four. There was no difference between the two groups in terms of pain recorded (95% confidence interval of the difference -0.809, 5.02) and patient/endoscopist satisfaction. There was no difference between the two groups in either depth of sedation or manoeuvrability. CONCLUSION: Both Entonox and the modified TCI propofol provide equally effective sedation and pain relief, simultaneously allowing patients to be easily manoeuvred during the procedures.

Artificial neural networks to predict presence of significant pathology in patients presenting to routine colorectal clinics.

AIM: Artificial neural networks (ANNs) are computer programs used to identify complex relations within data. Routine predictions of presence of colorectal pathology based on population statistics have little meaning for individual patient. This results in large number of unnecessary lower gastrointestinal endoscopies (LGEs - colonoscopies and flexible sigmoidoscopies). We aimed to develop a neural network algorithm that can accurately predict presence of significant pathology in patients attending routine outpatient clinics for gastrointestinal symptoms. METHOD: Ethics approval was obtained and the study was monitored according to International Committee on Harmonisation - Good Clinical Practice (ICH-GCP) standards. Three-hundred patients undergoing LGE prospectively completed a specifically developed questionnaire, which included 40 variables based on clinical symptoms, signs, past- and family history. Complete data sets of 100 patients were used to train the ANN; the remaining data was used for internal validation. The primary output used was positive finding on LGE, including polyps, cancer, diverticular disease or colitis. For external validation, the ANN was applied to data from 50 patients in primary care and also compared with the predictions of four clinicians. RESULTS: Clear correlation between actual data value and ANN predictions were found (r = 0.931; P = 0.0001). The predictive accuracy of ANN was 95% in training group and 90% (95% CI 84-96) in the internal validation set and this was significantly higher than the clinical accuracy (75%). ANN also showed high accuracy in the external validation group (89%). CONCLUSION: Artificial neural networks offer the possibility of personal prediction of outcome for individual patients presenting in clinics with colorectal symptoms, making it possible to make more appropriate requests for lower gastrointestinal endoscopy.

Patient satisfaction with lower gastrointestinal endoscopy: doctors, nurse and nonmedical endoscopists.

AIM: Assessment of patient satisfaction with lower gastrointestinal endoscopy (LGE) comprising colonoscopy and flexible sigmoidoscopy is gaining increasing importance. We have now trained non healthcare professionals such as nonmedical endoscopists (NMEs) to perform LGE to overcome shortage of trained endoscopists. The aim of this study was to prospectively determine patient satisfaction, factors affecting satisfaction with LGE and to compare with nurses, NME and medical endoscopists, in terms of patient satisfaction. METHOD: Consecutive patients undergoing LGE answered specially developed patient satisfaction questionnaire at discharge and 24 h thereafter. This questionnaire was a modification of m-Group Health Association of America questionnaire. Construct and face validity of questionnaire were tested by an expert group. Demographic and clinical data was prospectively collected. Multivariate regression analysis was performed to determine factors influencing patient satisfaction. RESULTS: Some 503 patients were surveyed after LGE. Examinations were performed by nurse (n = 105), doctor (n = 191), or NMEs (n = 155). There were no differences between three groups in terms of completion rates/complications. No differences were detected between endoscopists in patient rating for overall satisfaction (P = 0.6), technical skills (P = 0.58), communication skills (P = 0.61) or interpersonal skills (0.59). Multivariate regression analysis showed that higher preprocedure anxiety, history of pelvic operations/hysterectomy and higher pain scores were associated with adverse patient satisfaction and preprocedure anxiety, history of hysterectomy and female gender were associated with higher pain scores. CONCLUSION: This study has shown that there are no differences in patient satisfaction with LGE performed by nurse, doctor or NME. The most important factor affecting patient satisfaction is degree of discomfort/pain experienced by patient.

Randomized clinical trial of Entonox versus midazolam-fentanyl sedation for colonoscopy.

BACKGROUND: Intravenous sedation for colonoscopy is associated with cardiorespiratory complications and delayed recovery. The aim of this randomized clinical trial was to compare the efficacy of Entonox (50 per cent nitrous oxide and 50 per cent oxygen) and intravenous sedation using midazolam-fentanyl for colonoscopy. METHODS: Some 131 patients undergoing elective colonoscopy were included. Patients completed a Hospital Anxiety and Depression questionnaire, letter cancellation tests and pain scores on a 100-mm visual analogue scale before, immediately after the procedure and at discharge. They also completed a satisfaction survey at discharge and 24 h after the procedure. RESULTS: Sixty-five patients were randomized to receive Entonox and 66 to midazolam-fentanyl. Completion rates were similar (94 versus 92 per cent respectively; P = 0.513). Patients receiving Entonox had a shorter time to discharge. They reported significantly less pain (mean score 16.7 versus 40.1; P < 0.001), and showed better recovery of psychomotor function immediately after the procedure and at discharge. Patient satisfaction was higher among patients who received Entonox (median score 96 versus 89; P = 0.001). CONCLUSION: Entonox provides better pain relief and faster recovery than midazolam-fentanyl and so is more effective for colonoscopy.

Can the use of a rapid polymerase chain screening method decrease the incidence of nosocomial meticillin-resistant Staphylococcus aureus?

Rapid detection of MRSA may be important for the control of MRSA spread in hospitals. The aim of this investigation was to compare the use of a rapid polymerase chain reaction (PCR) screening method with standard culture for the detection of meticillin-resistant Staphylococcus aureus (MRSA) colonisation and to determine its impact on the incidence of MRSA in two hospital wards. During the first phase of the investigation (four months), patients in a surgical ward were screened using the rapid PCR technique and patients in a medical/cardiology ward were screened with standard culture methods. During the second phase of the investigation (four months), MRSA screening methods were switched between the two wards. An audit of infection control practices on each ward was made at the end of each phase in order to check whether any changes had occurred that might influence the risks of MRSA transmission. Use of the rapid PCR method significantly reduced the median time between swabs being taken, to the results being telephoned to the wards (excluding weekends), from 47 to 21 h (P<0.001). However, comparison of MRSA incidence during use of PCR (20/1000 bed-days) and culture methods (22.1/1000 bed-days) revealed no significant difference in incidence on the surgical ward (P=0.69). Regarding the medical/cardiology ward, analysis of data was complicated by an increase in the detection of MRSA during the PCR phase (P<0.05). The study demonstrated that rapid PCR can significantly reduce the turnaround times but reducing the time between swabs being taken to results being telephoned to the ward is still not sufficient to limit the transmission of MRSA.

Results of the United Kingdoms first pilot study for nonmedical endoscopy practitioners.

OBJECTIVE: Spencer and Ready published the first description of nonmedically qualified endoscopists in 1977 [Dis Colon Rectum 1977; 20: 94]. Since then there has been an explosion of interest in this concept. Duthie et al. [Gut 1998; 43: 711] first described Nurse Endoscopy (NE) in the UK in 1998. In recent years, endoscopy services in England have been subject to review as a consequence of perceived inadequacies in service provision and delivery. METHOD: Increasing pressure on endoscopic services led the Modernisation Agency to commission a pilot study evaluating nontraditional individuals to practice flexible sigmoidoscopy (FS). A science graduate, basic grade nurse and nonclinical member of staff were successfully trained. As a result, a second pilot scheme was introduced, whereby nine nonmedically qualified individuals received education and training in FS. A curriculum was designed, developed and implemented to create an endoscopic practitioner (EP) who was both safe and competent to undertake FS. The student backgrounds included administration, health-care assistants, phlebotomist, junior doctor assistant and clinical physiologist. RESULTS: In the context of academic achievement, prescribed learning outcomes were achieved at a rate of 93% (101 from 108) of success in first attempt at assessment. In terms of practical achievement, each student demonstrated competency in FS. CONCLUSION: The curriculum designed provides a suitable framework to develop individuals with the necessary knowledge, skills and attitudes required for safe, competent practice within FS. This indicates that the curriculum provides a broad range of learning, teaching and assessment strategies befitting to a range of individuals.

A comprehensive genetic and histopathologic analysis identifies two subgroups of B-cell malignancies carrying a t(14;19)(q32;q13) or variant BCL3-translocation.

The biologic and pathologic features of B-cell malignancies bearing a translocation t(14;19)(q32;q13) leading to a fusion of IGH and BCL3 are still poorly described. Herein we report the results of a comprehensive cytogenetic, fluorescence in situ hybridization (FISH), molecular and histopathological survey of a large series of B-cell malignancies with t(14;19) or variant translocations. A total of 56 B-cell malignancies with a FISH-proven BCL3 involvement were identified with the translocation partners being IGH (n=51), IGL (n=2), IGK (n=2) and a non-IG locus (n=1). Hierarchical clustering of chromosomal changes associated with the t(14;19) indicated the presence of two different groups of IG/BCL3-positive lymphatic neoplasias. The first group included 26 B-cell malignancies of various histologic subtypes containing a relatively high number of chromosomal changes and mostly mutated IgVH genes. This cluster displayed three cytogenetic branches, one with rearrangements in 7q, another with deletions in 17p and a third one with rearrangements in 1q and deletions in 6q and 13q. The second group included 19 cases, mostly diagnosed as B-cell chronic lymphocytic leukemia (B-CLL), and characterized by few additional chromosomal changes (e.g. trisomy 12) and unmutated IgVH genes. In conclusion, our study indicates that BCL3 translocations are not restricted to B-CLL but present in a heterogeneous group of B-cell malignancies.

Cerberus regulates left-right asymmetry of the embryonic head and heart.

BACKGROUND: Most of the molecules known to regulate left-right asymmetry in vertebrate embryos are expressed on the left side of the future trunk region of the embryo. Members of the protein family comprising Cerberus and the putative tumour suppressor Dan have not before been implicated in left-right asymmetry. In Xenopus, these proteins have been shown to antagonise members of the transforming growth factor beta (TGF-beta) and Wnt families of signalling proteins. RESULTS: Chick Cerberus (cCer) was found to be expressed in the left head mesenchyme and in the left flank of the embryo. Expression on the left side of the head was controlled by Sonic hedgehog (Shh) acting through the TGF-beta family member Nodal; in the flank, cCer was also regulated by Shh, but independently of Nodal. Surprisingly, although no known targets of Cerberus are expressed asymmetrically on the right side of the embryo at these stages, misexpression of cCer on this side of the embryo led to upregulation of the transcription factor Pitx2 and reversal of the direction of heart and head turning, apparently as independent events. Consistent with the possibility that cCer may be acting on bilaterally expressed TGF-beta family members such as the bone morphogenetic proteins (BMPs), this result was mimicked by right-sided misexpression of the BMP antagonist, Noggin. CONCLUSIONS: Our findings suggest that cCer maintains a delicate balance of different TGF-beta family members involved in laterality decisions, and reveal the existence of partially overlapping molecular pathways regulating left-right asymmetry in the head and trunk of the embryo.

Investigation and treatment of faecal incontinence.

Faecal incontinence is a debilitating condition affecting people of all ages, and significantly impairs quality of life. Proper clinical assessment followed by conservative medical therapy leads to improvement in more than 50% of cases, including patients with severe symptoms. Patients with advanced incontinence or those resistant to initial treatment should be evaluated by anorectal physiology testing to establish the severity and type of incontinence. Several treatment options with promising results exist. Patients with gross sphincter defects should undergo surgical repair. Those who fail to respond to sphincteroplasty and those with no anatomical defects have the option of either sacral nerve stimulation or other advanced procedures. Stoma formation should be reserved for patients who do not respond to any of the above procedures.

Longitudinal copy number, whole exome and targeted deep sequencing of 'good risk' IGHV-mutated CLL patients with progressive disease.

The biological features of IGHV-M chronic lymphocytic leukemia responsible for disease progression are still poorly understood. We undertook a longitudinal study close to diagnosis, pre-treatment and post relapse in 13 patients presenting with cMBL or Stage A disease and good-risk biomarkers (IGHV-M genes, no del(17p) or del(11q) and low CD38 expression) who nevertheless developed progressive disease, of whom 10 have required therapy. Using cytogenetics, fluorescence in situ hybridisation, genome-wide DNA methylation and copy number analysis together with whole exome, targeted deep- and Sanger sequencing at diagnosis, we identified mutations in established chronic lymphocytic leukemia driver genes in nine patients (69%), non-coding mutations (PAX5 enhancer region) in three patients and genomic complexity in two patients. Branching evolutionary trajectories predominated (n=9/13), revealing intra-tumoural epi- and genetic heterogeneity and sub-clonal competition before therapy. Of the patients subsequently requiring treatment, two had sub-clonal TP53 mutations that would not be detected by standard methodologies, three qualified for the very-low-risk category defined by integrated mutational and cytogenetic analysis and yet had established or putative driver mutations and one patient developed progressive, therapy-refractory disease associated with the emergence of an IGHV-U clone. These data suggest that extended genomic and immunogenetic screening may have clinical utility in patients with apparent good-risk disease.

Frequent homozygous deletions of the D13S25 locus in chromosome region 13q14 defines the location of a gene critical in leukaemogenesis in chronic B-cell lymphocytic leukaemia.

Cytogenetic studies of B-cell chronic lymphocytic leukaemia show structural abnormalities involving the 13q14 chromosome region as the only karyotypic change in a significant proportion of tumours. This observation suggests the location of a gene important in leukaemogenesis. A series of 68 BCLL tumours have been analysed for allele loss using a series of probes from 13q14. Using intragenic polymorphic markers from the retinoblastoma predisposition gene LOH was observed in 25% of tumours including 3/6 showing cytogenetically obvious deletions of the 13q14 region and 3/6 showing translocations involving 13q14. However, three deletions with proximal breakpoints in 13q14 did not show allele loss, demonstrating that the breakpoint lay distal to RB1. Using the D13S25 locus, which lies 1.6 cM distal to RB1, allele loss was seen in 90% of tumours with structural rearrangements of 13q14 and 75% of tumours with an apparently normal karyotype. 50% of these tumours showed homozygous loss of D13S25, suggesting that a 'tumour suppressor gene' lies in this region. The more distal D13S31 locus, 1 cM distal to D13S25, was infrequently involved in allele loss demonstrating that the minimum region of overlap for homozygous deletions is approximately 1 Mbp around the D13S25 locus.

Cloning of two candidate tumor suppressor genes within a 10 kb region on chromosome 13q14, frequently deleted in chronic lymphocytic leukemia.

Previous studies have indicated the presence of a putative tumor suppressor gene on chromosome 13q14, commonly deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). We have previously defined a minimally deleted region of 130 kb centromeric to the marker D13S272, and constructed a PAC and cosmid contig encompassing this area. In the present study we have made a detailed restriction and transcriptional map of the region of interest. Using these tools we have screened a panel of 206 primary CLL clones and three cell lines. In five CLL cases we found limited deletions defining the region of interest to an area of no more than 10 kb. Two adjacent genes, termed Leu1 and Leu2 (leukemia-associated gene 1 and 2), were mapped to the minimally deleted region, with several patients showing deletion borders within these genes. The Leu1 and Leu2 genes show little homology to previously published genes at the nucleotide and expected translated amino acid sequence level. Mutational analysis of the Leu1 and 2 genes in 170 CLL samples revealed no small intragenic mutations or point mutations. However, in all cases of 13q14 loss examined, the first exon of both genes, which are only 300 bp apart, were deleted. We conclude that the Leu1 and Leu2 genes are strong candidates as tumor suppressor gene(s) involved in B-CLL leukemogenesis.