The Notch-1 intracellular domain is found in sub-nuclear bodies in SH-SY5Y neuroblastomas and in primary cortical neurons Notch signalling affects most aspects of development , not least the determination of neural stem cell fate . Here , we describe the presence of the Notch-1 intracellular domain -LRB- N1ICD -RRB- in sub-nuclear bodies in SH-SY5Y neuroblastomas and in primary rat cortical neurons as well as several other mammalian cell lines . We also demonstrate that these N1ICD-positive sub-nuclear bodies are distinct from premyelocytic leukaemia -LRB- PML -RRB- and SC35 bodies . Furthermore , using Notch deletion constructs we determined that a region C-terminal of amino acid 2094 is involved in targeting the N1ICD into sub-nuclear bodies . These findings have ramifications for nuclear architecture and gene transcription . Notch signalling regulates many aspects of invertebrate and vertebrate development ranging from somite formation -LSB- 7 -RSB- to epithelial -- mesenchymal interactions -LSB- 20 -RSB- and the determination of neural stem cell fate -LSB- 23 -RSB- . Notch signalling is also active in adult tissues including the brain where it is involved in synaptic plasticity and memory -LSB- 3,19 -RSB- . There are four mammalian Notch receptors -LRB- Notch 1 -- 4 -RRB- , which undergo a series of sequential proteolytic cleavages . The first cleavage -LRB- S1 -RRB- occurs in the Golgi by a furin-like convertase generating two fragments that remain non-covalently attached to form the mature Notch receptor at the cell surface . Ligands of the Jagged and Delta families , expressed on neighbouring cells , bind to the Notch receptor leading to the second cleavage -LRB- S2 -RRB- by tumor necrosis factor alpha converting enzyme -LRB- TACE -RRB- , which results in the shedding of the extracellular domain . The second cleavage is followed by a constitutive cleavage -LRB- S3 -RRB- within the transmembrane domain of Notch by γ-secretase . This cleavage releases the Notch intracellular domain -LRB- NICD -RRB- , which migrates to the nucleus -LSB- 20,14,15 -RSB- where it interacts with the C-promoter binding factor-1 -LRB- CBF-1 -RRB- , the mammalian homologue of Suppressor of hairless -LRB- Drosophila -RRB- and Lag-1 -LRB- C. elegans -RRB- , also known as RBP-Jκ -LSB- 16 -RSB- . In the absence of NICD , CBF-1 forms a complex with repressor proteins , resulting in the suppression of gene transcription . Interaction with NICD displaces the repressor proteins and allows the entry of transcriptional activators such as p300/CBP and Mastermind , which convert the CBF-1 repressor complex into an activator of gene transcription -LSB- 8 -RSB- . Notch/CSL target genes include the hairy and enhancer of split -LRB- HES -RRB- family and the HES related proteins -LRB- HERP/Hey -RRB- that encode basic helix-loop-helix transcription factors , which suppress differentiation by antagonising the expression of down-stream lineage-specifying genes . In this report , we demonstrate the presence of the N1ICD in sub-nuclear bodies in SH-SY5Y neuroblastomas and in primary rat cortical neurons . To undertake this study the following cell lines , antibodies and plasmid constructs were used : SH-SY5Y neuroblastoma cells and chinese hamster ovary cells -LRB- CHO -RRB- were obtained from ECACC -LRB- UK -RRB- and human embryonic kidney 293a cells -LRB- HEK293a ; an adherent clone of HEK293 cells -RRB- were purchased from Quantum Biotechnologies -LRB- Canada -RRB- . Mouse monoclonal anti-splicing factor SC-35 antibody and rabbit polyclonal anti-FLAG tag antibody were from Sigma -LRB- UK -RRB- . Mouse monoclonal anti-myc antibody was from Cell Signalling Technology -LRB- UK -RRB- . Mouse monoclonal anti-promyelocytic leukaemia protein antibody , goat anti-Notch-1 , goat anti-mouse IgG and donkey anti-goat IgG were from Santa Cruz Biotechnology -LRB- USA -RRB- . Alexa Fluor 488 and 594 goat anti-mouse and donkey anti-goat IgG conjugates were from Molecular Probes -LRB- UK -RRB- . C-terminally tagged myc-His human N1ICD , ΔTAD-N1ICD -LRB- lacking the transactivation domain TAD ; the OPA domain and the proline -LRB- P -RRB- , glutamate -LRB- E -RRB- , serine -LRB- S -RRB- , threonine -LRB- T -RRB- rich sequence -- PEST -RRB- and ΔRA-N1ICD -LRB- lacking the RAM domain and the Ankyrin repeats -RRB- were gifts from Dr. Tom Kadesch -LRB- University of Pennsylvania , USA -RRB- -LSB- 13 -RSB- . Enhanced green fluorescent protein -LRB- EGFP -RRB- C-terminally tagged human N1ICD -LRB- N1ICD-EGFP -RRB- was from Dr. Allan Levey -LRB- Emory University School of Medicine , USA -RRB- -LSB- 11 -RSB- . The Notch-CBF-1 reporter , 4xwt-CBF-Luc , which contains four tandem repeats of the consensus CBF-1 DNA binding sequence , GTGGGAA and N-terminally tagged FLAG CBF-1 were generous gifts from Dr. Diane Hayward -LRB- Johns Hopkins University School of Medicine , USA -RRB- -LSB- 5 -RSB- . Cells were maintained in 5 % CO2 in air in a humidified incubator at 37 °C . All culture media and supplements were from Invitrogen , UK , unless otherwise stated . SH-SY5Y neuroblastomas were grown in a 50/50 mix of F12/Eagle 's minimal essential medium -LRB- EMEM -RRB- supplemented with 15 % -LRB- v/v -RRB- fetal bovine serum -LRB- FBS ; Autogen Bioclear , UK -RRB- , 2 mM l-glutamine , non-essential amino acids -LRB- Sigma -RRB- , 100 IU penicillin and 100 mg/ml streptomycin . HEK293a were cultured in low glucose Dulbecco 's modified Eagle 's medium -LRB- DMEM -RRB- containing 10 % -LRB- v/v -RRB- FBS , 2 mM l-glutamine , 100 IU penicillin and 100 mg/ml streptomycin . CHO cells were grown in F12 media supplemented as described above for HEK293a cells . Primary cortical neurons were prepared and cultured in Neurobasal media plus B27 supplement as previously described from E19 rat embryos -LSB- 17 -RSB- . Cells on coverslips were transfected with 500 ng of N1ICD-EGFP or 500 ng of myc-His N1ICD using FuGene 6 according to the manufacturer 's instructions . Cells were fixed in ice-cold methanol 24 h after transfection then stained according to standard protocols . In brief , cells were incubated with goat anti-Notch-1 antibody -LRB- 1:100 -RRB- , mouse monoclonal anti-promyelocytic leukaemia protein antibody -LRB- 1:100 -RRB- or mouse monoclonal anti-splicing factor SC-35 antibody -LRB- 1:250 -RRB- before being incubated with the appropriate fluorescent secondary antibody -LRB- 1:200 -RRB- . Nuclei were stained with Hoescht 33342 . Immunofluorescence was visualized and captured using a Zeiss LSM510 meta confocal microscope . Images were processed using LSM5 image examiner software -LRB- Zeiss -RRB- . All experiments were performed in triplicate . Figures shown are representative images from a single experiment . For luciferase reporter gene assays SH-SY5Y neuroblastomas were transfected with 400 ng of CBF-Luc -LRB- firefly luciferase based reporter DNA -RRB- and 500 ng of N1ICD-EGFP or 500 ng of myc-His N1ICD using FuGene 6 according to the manufacturer 's instructions -LRB- Roche , UK -RRB- . To control for transfection efficiency 50 ng of phTK-Renilla luciferase -LRB- Promega , UK -RRB- was also included in transfections . Empty vector DNA was included where necessary to maintain constant DNA concentrations . Twenty-four hours post-transfection the cells were lysed and firefly and Renilla luciferase activities were sequentially measured using Dual-Glo reagents -LRB- Promega -RRB- in a Wallac Trilux 1450 Luminometer -LRB- Perkin-Elmer , UK -RRB- . Values were normalized by dividing firefly values by the Renilla value from the same well . Data for each set of four replica transfections was averaged , the control in each set normalized to 1 and data presented as fold increases over control . All experiments were performed in triplicate . Endogenous S3 cleaved , nuclear , NICDs are expressed at very low levels and are difficult to detect in cell culture experiments -LSB- 9 -RSB- , therefore we expressed exogenous N1ICD-EGFP in SH-SY5Y neuroblastomas to model N1ICD in vitro . In these cells N1ICD-EGFP exhibited a diffuse nuclear staining pattern with intense sub-nuclear bodies -LRB- Fig. 1A and B -RRB- . In contrast , expression of exogenous p53 or β-catenin resulted in diffuse nuclear staining -LRB- data not shown -RRB- , providing evidence that the N1ICD-positive sub-nuclear bodies are specific . The sub-nuclear bodies were not attributable to the EGFP protein since SH-SY5Y cells transfected with the EGFP expression vector containing no insert did not display a speckled nuclear staining pattern -LRB- data not shown -RRB- . Furthermore , a myc-His tagged form of N1ICD was found in sub-nuclear bodies in SH-SY5Y neuroblastoma cells -LRB- Fig. 1C -RRB- as demonstrated using an anti-Notch-1 antibody , providing additional evidence that the observed speckled staining pattern is specific and not due to the presence of EGFP . Control cultures that were not exposed to primary antibody did not exhibit any positively stained cells -LRB- data not shown -RRB- . To ensure the presence of nuclear bodies was not an artefact of the fixation method used -LRB- ice-cold methanol -RRB- , SH-SY5Y cells were transfected with both EGFP and myc-His tagged Notch constructs and fixed with either 3.7 % formaldehyde or 4 % paraformaldehyde . With both methods very similar nuclear bodies were seen with both constructs -LRB- data not shown -RRB- . Of note , both N1ICD-EGFP and myc-His tagged N1ICD were functionally active as demonstrated using CBF-Luc -LRB- Fig. 1D -RRB- and we have previously shown that exogenous N1ICD expression promotes the inhibition of retinoic acid-induced differentiation in SH-SY5Y neuroblastomas -LSB- 4 -RSB- . N1ICD-positive sub-nuclear bodies were not cell-type specific . We observed identical sub-nuclear structures in primary rat cortical neurones -LRB- Fig. 2A -RRB- , HEK293a cells -LRB- Fig. 2B -RRB- and in CHO cells -LRB- Fig. 2C -RRB- with N1ICD-EGFP and myc-His tagged N1ICD -LRB- data not shown -RRB- . As the punctate structures were detected with both constructs in a variety of cell lines and in primary neurons they are unlikely to be an artefact of tagging or a result of cellular transformation . Furthermore , we found that exogenous CBF-1 exhibited a diffuse nuclear staining pattern in SH-SY5Y neuroblastomas -LRB- Fig. 2D -RRB- , but in the presence of N1ICD CBF-1 re-located into sub-nuclear bodies -LRB- Fig. 2E -RRB- , indicative of functional gene transcription within these nuclear domains . Several sub-nuclear structures , similar to those shown here , have been previously described including premyelocytic leukaemia -LRB- PML -RRB- bodies and SC35 bodies . These foci are believed to be areas of high transcriptional activity -LSB- 2 -RSB- and pre-mRNA splicing , respectively -LSB- 10 -RSB- . We therefore transfected SH-SY5Y cells with N1ICD-EGFP and used antibodies to detect endogenous PML and SC35 bodies . Both PML -LRB- Fig. 3A , mid left panel -RRB- and SC35 reactive bodies -LRB- Fig. 3B , mid left panel -RRB- were detected in SH-SY5Y cells . However , in both cases these formations were distinct from the N1ICD-EGFP positive foci -LRB- Fig. 3A and B , far right panels -RRB- . The PML and SC35 bodies were more consistent in size and typically smaller and more numerous than the formations in which N1ICD was detected . Next we examined which region of N1ICD was involved in targeting the protein to sub-nuclear bodies by using two myc-tagged N1ICD deletion constructs , ΔTAD-N1ICD and ΔRA-N1ICD . ΔTAD-N1ICD runs from a.a. 1760 to 2093 and lacks the transactivation domain , the OPA domain and the proline -LRB- P -RRB- , glutamate -LRB- E -RRB- , serine -LRB- S -RRB- , threonine -LRB- T -RRB- rich sequence -LRB- PEST -RRB- . ΔRA-N1ICD extends from a.a. 2094 to 2556 and lacks the RAM domain and the Ankyrin repeats -LRB- Fig. 4A -RRB- . ΔTAD-N1ICD was detected diffusely throughout the nucleus in SH-SY5Y neuroblastomas -LRB- Fig. 4B , left panel -RRB- , while ΔRA-N1ICD was located in sub-nuclear bodies very similar in appearance to those seen with N1ICD -LRB- Fig. 4B , right panel -RRB- . This indicates that a region C-terminal of a.a. 2094 , lying within the TAD , the OPA domain or the PEST domain or an unidentified motif within the intervening regions , is involved in targeting N1ICD into sub-nuclear bodies . In summary , we demonstrate that exogenous N1ICD is expressed in sub-nuclear bodies in SH-SY5Y neuroblastomas , primary rat cortical neurons as well as other mammalian cell lines and these sub-nuclear structures were distinct from SC35 or PML bodies . N1ICD foci were detectable as early as 5 h after transfection -LRB- cells were routinely fixed 24 h post-transfection in all other experiments described in this report -RRB- , which suggests that the nuclear inclusions are not merely a consequence of protein aggregation with time -LRB- data not shown -RRB- . The presence of the N1ICD in sub-nuclear bodies has previously been demonstrated when associated with the Epstein-Barr virus derived protein , RPMS -LSB- 24 -RSB- and the Mastermind family of Notch/CBF -1 co-activators -LSB- 18,21,22 -RSB- . Here , we report that the N1ICD appeared in foci independently of other exogenously expressed proteins . A number of endogenous Notch associated proteins , including CBF-1 , have recently been observed in similar nuclear foci in primary hippocampal neurons -LSB- 12 -RSB- , although the presence of NICDs was not examined . Interestingly , we found that exogenously expressed CBF-1 exhibited a diffuse nuclear staining pattern , however in the presence of exogenous N1ICD CBF-1 was found in sub-nuclear bodies . A number of previous reports -LSB- 1,6 -RSB- concerning Notch cytochemistry contain figures in which N1ICD appears to be in punctate formations , which with greater resolution could have been interpreted as distinct sub-nuclear bodies . It is feasible then that the N1ICD-positive bodies form part of a physiologically relevant , multimeric , protein complex involved in gene transcription . Moreover , the presence of such sub-nuclear bodies provides evidence that the nucleus contains defined domains and is not merely a homogenous pool of nucleic acids and proteins .

O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


How does the self-reported clinical management of patients with low back pain relate to the attitudes and beliefs of health care practitioners ? A survey of UK general practitioners and physiotherapists Guidelines for the management of low back pain -LRB- LBP -RRB- have existed for many years , but adherence to these by health care practitioners -LRB- HCPs -RRB- remains suboptimal . The aim of this study was to measure the attitudes , beliefs and reported clinical behaviour of UK physiotherapists -LRB- PTs -RRB- and general practitioners -LRB- GPs -RRB- about LBP and to explore the associations between these . A cross-sectional postal survey of GPs -LRB- n = 2000 -RRB- and PTs -LRB- n = 2000 -RRB- was conducted that included the Pain Attitudes and Beliefs Scale -LRB- PABT.PT -RRB- , and a vignette of a patient with non-specific LBP -LRB- NSLBP -RRB- with questions asking about recommendations for work , activity and bedrest . Data from 1022 respondents -LRB- 442 GPs and 580 PTs -RRB- who had recently treated patients with LBP were analysed . Although the majority of HCPs reported providing advice for the vignette patient that was broadly in line with guideline recommendations , 28 % reported they would advise this patient to remain off work . Work advice was significantly related to the PABS.PT scores with higher biomedical -LRB- F1 ,986 = 77.5 , p < 0.0001 -RRB- and lower behavioural -LRB- F1 ,981 = 31.9 , p < 0.001 -RRB- scores associated with advice to remain off work . We have demonstrated that the attitudes and reported practice behaviour of UK GPs and PTs for patients with NSLBP are diverse . Many HCPs held the belief that LBP necessitates some avoidance of activities and work . The attitudes and beliefs of these HCPs were associated with their self-reported clinical behaviour regarding advice about work . Future studies need to investigate whether approaches aimed at modifying these HCP factors can lead to improved patient outcomes . 1 Introduction Low back pain -LRB- LBP -RRB- is common , affecting 38 % of adults in any one year , of whom 1 in 4 experience significant disability -LSB- 37 -RSB- . Only 25 % of patients consulting in primary care will be symptom free 12 months later -LSB- 18 -RSB- . The last two decades have also seen dramatic rises in work loss and sickness benefit payments , attributed to recurrent and persistent LBP -LSB- 16,36 -RSB- . Guidelines for the clinical management of patients with LBP encourage health care practitioners -LRB- HCPs -RRB- to advise patients to stay active , avoid bed rest , stay at or return to work , and stress simple messages about self-management -LSB- 3,31,45,47,49,50 -RSB- . Previous studies have identified that HCPs do not always follow guideline recommendations for LBP -LSB- 10,20,24,26 -RSB- and so despite the abundance of guidelines for practice , the management of LBP poses considerable challenges and frustrations for both patients and practitioners -LSB- 14 -RSB- and it is increasingly clear that it is insufficient to study patient factors alone -LSB- 25,48 -RSB- . A potentially important but relatively unexplored influence on patients ' pain experiences is the attitudes and beliefs of the HCPs with whom they come into contact . HCPs are frequently asked to provide advice and recommendations about physical activities , work , and rest and HCPs ' attitudes and beliefs may be an integral part of the health care process , influencing the success or failure of treatment . HCPs hold a range of attitudes and beliefs about back pain -LSB- 17,19,28,32,39,42 -- 44 -RSB- , and these attitudes appear to be associated with the work and activity recommendations that HCPs give to patients -LSB- 17,28,42,44 -RSB- . In the UK , approximately 98 % of the population is registered with a National Health Service general practitioner -LRB- GP -RRB- -LSB- 13 -RSB- . GPs serve as gatekeepers to secondary care , selecting and referring patients for specialist investigations and treatment services . Physiotherapy is one of the most common services to which patients are referred , or which patients seek out privately -LSB- 36 -RSB- , and LBP accounts for more than half of physiotherapists ' workload in the UK -LSB- 24 -RSB- . Few studies have explored HCP factors in the UK , but it has been shown that many physiotherapists -LRB- PTs -RRB- continue to advise limitations of work and activity levels , despite identifying when patients with LBP are at risk of chronicity -LSB- 11 -RSB- and an important proportion of therapists continue treating patients with LBP even when they fail to improve -LSB- 41 -RSB- . The aim of this study was to measure , in national random samples , the attitudes , beliefs and reported clinical behaviour of GPs and PTs about LBP , explore their associations and evaluate the implications for both clinical practice and future research . 2 Methods 2.1 Design and setting We conducted a cross-sectional , nationwide postal survey of UK GPs and PTs , involved in the management of patients with LBP , between April and November 2005 . Ethical approval for the study was obtained from the West Midlands Multi-centre Research Ethics Committee -LRB- MREC -RRB- . Written consent was not sought from each participant for use of survey data , but consent of respondents was assumed if they completed and returned the questionnaire . 2.2 Questionnaire sample and mailing process We used simple random sampling to obtain details of GPs -LRB- n = 2000 -RRB- and PTs -LRB- n = 2000 -RRB- from national databases -LRB- Binleys database for GPs , n = 46,000 GPs on the list ; Chartered Society of Physiotherapy membership database , n = 32,000 PTs on the list -RRB- . In the UK , all GPs working in the National Health Service are included on the Binleys database -LSB- 7 -RSB- , which is produced in conjunction with the Royal College of General Practitioners . The Chartered Society of Physiotherapy -LRB- CSP -RRB- is the professional , educational and trade union body representing the UK 's chartered physiotherapists and 98 % of all PTs are members of the CSP . A sample size calculation indicated that a sample of 900 responders -LRB- 450 GPs and 450 PTs -RRB- was required to allow us to find a minimum difference of 10 % in the proportion of respondents with ` helpful ' to ` unhelpful ' beliefs by important practitioner characteristics at a significance level of 0.05 and a power of 90 % -LSB- 2 -RSB- . A questionnaire package containing the questionnaire , a cover letter , an information sheet and a pre-paid envelope was mailed to each HCP . A single reminder was sent to all non-responders four weeks after the first mailing . In order to allow assessment of non-response bias within the survey estimates , a brief questionnaire was mailed to a random sample of non-responders . No incentives for completing the questionnaire were offered . 2.3 Questionnaire A filter question was used to identify those HCPs who had treated at least one patient with non-specific LBP -LRB- NSLBP -RRB- in the previous six months , so that only respondents with recent experience of managing patients with LBP were included in the analysis . 2.3.1 Demographics and practice information A number of demographic and practice questions , relevant to each profession , were included . Some items were pertinent to both professions : gender ; years since qualification ; postgraduate training in LBP ; clinical interests/speciality and personal experience of back pain . Data gathered exclusively from GPs included whether they worked only in general practice and whether the practice was a single-handed or a group practice . Data gathered exclusively from PTs included how much of their clinical practice was based in the NHS , what proportion of their caseload was primary care patients , whether they worked alone or in a team , and grade of current job . 2.3.2 Attitudes and beliefs measure The Pain Attitudes and Beliefs Scale -LRB- PABS.PT -LSB- 28,39 -RSB- -RRB- was included as a measure of HCPs ' attitudes about LBP . This was selected following a systematic review of available tools for assessing the attitudes and beliefs of HCPs about LBP -LSB- 12 -RSB- , in which the PABS.PT fared well on pre-defined quality criteria -LSB- 34 -RSB- . This tool was originally developed for use in physiotherapists , but more recently has been applied to a cohort of Dutch general practitioners -LSB- 30 -RSB- . In addition , the members of a multi-disciplinary clinical advisory group confirmed face and content validity of the PABS.PT for both GPs and PTs after recommending that the term ` therapy ' was changed to ` treatment ' in two of the items of the PABS.PT . The resulting minimally amended PABS.PT was used for both GPs and PTs . The PABS.PT assesses the strength of treatment orientation on two subscales , ` biomedical ' and ` behavioural ' . The biomedical orientation is described as one in which the HCP believes in a biomechanical model of disease , where disability and pain are a consequence of a specific pathology within the spinal tissues and treatment is aimed at treating the pathology and alleviating the pain . The behavioural orientation is where the HCP believes in a biopsychosocial model of disease in which pain does not have to be a consequence of tissue damage , and can be influenced by social and psychological factors . We used the amended PABS.PT -LSB- 28 -RSB- , which consists of 19 items , each rated on a six point Likert scale -LRB- ` Totally disagree ' = 1 to ` Totally agree ' = 6 -RRB- , with ten items on the biomedical subscale -LRB- score range : 10 -- 60 -RRB- and nine on the behavioural subscale -LRB- score range : 9 -- 54 -RRB- . Higher scores on each subscale indicate a stronger biomedical or behavioural treatment orientation , respectively . 2.3.3 Clinical behaviour measures Clinical behaviour was elicited by asking the HCPs about diagnostic investigations and for their recommendations about work , activity levels , and bedrest , for a patient with NSLBP described in a vignette . The vignette described a patient with uncomplicated NSLBP who was not at work as a result of their symptoms -LRB- Appendix A -RRB- . Vignettes have been shown to be a useful measure of clinicians ' practice behaviour and a more accurate assessment of clinical behaviour than data extracted from case notes when measured against the gold standard of standardised patients -LSB- 40 -RSB- . The clinical behaviour question regarding work was as follows : `` The patient described in the vignette asks what your advice would be about her work . I would recommend this patient to : -LRB- Please tick the one response that best describes what you would recommend this patient to do -RRB- a.Be off work until pain has completely disappearedb.Return to part time or light dutiesc.Be off work for a further ... weeks -LRB- please state number of weeks -RRB- d.Return to normal worke.Be off work until pain has improved '' Responses for each of the work , activity and bedrest questions were subsequently classified by the authors as being ` strictly in line with guideline recommendations ' , ` broadly in line with guideline recommendations ' and ` not in line with guidelines ' . For the work question given above , we considered option ` d' to be strictly in line with guideline recommendations , option ` b ' to be broadly in line with guideline recommendations and options ` a ' , ` c ' and ` e ' to be not in line with guideline recommendations . This classification was based on a previously published expert consensus carried out on similar practice recommendations in a postal survey of physiotherapists , osteopaths and chiropractors in the UK -LSB- 22 -RSB- . 2.4 Brief questionnaire The brief questionnaire sent to a sample of non-responders contained the filter question to ensure that respondents recently involved in the management of patients with LBP could be identified . Alongside key demographic questions , we included four items from the PABS.PT -LRB- two from each subscale chosen on the basis of factor loadings described by the tool 's developers and data from a pilot study -RRB- , the vignette patient and the clinical behaviour questions related to work , activity and bedrest . 2.5 Statistical analysis Scores for the PABS.PT were calculated according to methods specified by the questionnaire developers , i.e. a simple summation of the items in each subscale -LSB- 39 -RSB- . No method for dealing with missing data on this measure has been published so a pragmatic decision was made that if one value was missing from a subscale , a mean score based on the remaining values was substituted . If more than one value was missing the score for the whole subscale was classed as missing . A Pearson 's correlation coefficient was calculated between the scores on the two subscales of the PABS.PT as previous work has shown that they are not totally independent -LSB- 28,39 -RSB- . We used descriptive statistics to summarise , by professional group , demographic , and practice data for both subscales of the PABS.PT . In addition , in response to the reviewer 's suggestions , we conducted a subgroup analysis of work , activity and bedrest recommendations for those respondents who had high biomedical scores and low behavioural scores and vice versa . Unless differences occurred by profession , analyses were performed on the combined GP and PT dataset . The relationship between attitudes and beliefs and clinical behaviour was examined using ANOVA to test for an overall relationship with clinical behaviour and , when appropriate , for a linear trend across clinical behaviour groups -LRB- strictly in line , broadly in line and not in line with guidelines -RRB- . The effect of non-response was examined by comparing responses from all responders to the full questionnaire to those completing the brief questionnaire . All analyses were carried out using the Statistical Package for Social Scientists for Windows -LRB- SPSS Inc. , Chicago , IL , version 13 -RRB- . 3 Results The overall response rate was 38 % -LRB- n = 1534 -RRB- , 22 % -LRB- n = 443 -RRB- for GPs and 55 % -LRB- n = 1091 -RRB- for PTs . Of the respondents , 580 PTs and 442 GPs reported treating at least one patient with LBP in the previous six months and were included in the analysis . 3.1 Characteristics of respondents The demographic and professional characteristics of the respondents are summarised in Table 1 . The majority of GPs worked exclusively in general practice , within group practices and had at least one specialist clinical interest . The majority of PTs worked within the NHS , with other HCPs , were of senior clinical grade or above , and had a patient caseload of more than 50 % primary care patients . The PTs were qualified for a shorter length of time than GPs , were more likely to be female and to have postgraduate training in LBP . 3.2 Attitudes and beliefs Scores for both of the PABS.PT subscales could be calculated for the majority of the 1022 responders -LRB- biomedical n = 1010 , behavioural n = 1004 -RRB- . Mean -LRB- standard deviation , range -RRB- score for the biomedical subscale was 31.0 -LRB- 6.4 , 12 -- 50 -RRB- overall : GPs 30.9 -LRB- 5.3 -RRB- ; PTs 31.1 -LRB- 7.2 -RRB- , and for the behavioural subscale was 33.0 -LRB- 4.6 , 15 -- 48 -RRB- overall : GPs 33.7 -LRB- 4.2 -RRB- ; PTs 32.5 -LRB- 4.8 -RRB- . For both subscales and both professional groups , the mean observed scores were in the middle of the possible ranges . The Pearson 's correlation coefficient -LRB- r = − 0.38 ; p < 0.0001 -RRB- showed a statistically significant level of dependence between the two subscales , suggesting that respondents who score higher on one subscale tend to score lower on the other subscale . 3.3 Diagnostic investigations In response to the vignette patient , most HCPs reported that they would not want the patient described to have any diagnostic investigations . Of the GPs 33 % -LRB- n = 142 -RRB- reported that they would request at least one investigation , compared with 24 % -LRB- n = 134 -RRB- of PTs -LRB- Table 2 -RRB- . GPs were more likely to want laboratory tests and PTs were more likely to want an X-ray or special imaging procedure such as an MRI . 3.4 Clinical behaviour The responses to the clinical behaviour questions were classified according to whether these were ` strictly in line ' , ` broadly in line ' or ` not in line ' with guideline recommendations and the responses and the classifications are summarised in Table 3 . The majority of respondents reported advice that was either ` strictly in line ' or ` broadly in line ' with guideline recommendations ' . Very small proportions of respondents reported they would provide advice that was ` not in line ' with guideline recommendations for activity and bedrest , however , this figure was considerably higher for recommendations regarding work , with 28 % of respondents reporting that they would recommend the patient in the vignette to remain off work . The summary of responses of the two subgroups of high biomedical and low behavioural scores -LRB- n = 187 -RRB- and low biomedical and high behavioural scores -LRB- n = 137 -RRB- , compared to the total sample , is also presented in Table 3 . The proportion of practitioners recommending that the patient in the vignette remain off work , i.e. not in line with guideline recommendations , was substantially higher in those with high biomedical and low behavioural scores -LRB- 44.9 % -RRB- than those with high behavioural and low biomedical scores -LRB- 11.9 % -RRB- . Similar differences were also seen for recommendations regarding activity and bedrest . 3.5 Relationship between attitudes and beliefs and clinical behaviour Given the very small proportion of respondents whose advice was ` not in line with guidelines ' for both activity and bedrest , associations with the PABS.PT scores were not examined . Fig. 1 shows the distributions of the PABS.PT biomedical and behavioural subscale scores for each of the reported work recommendation groups . With increasing disparity with guidelines , biomedical scores increased -LRB- mean scores : 28.3 , 30.6 , 33.5 -RRB- and behavioural scores decreased -LRB- mean scores : 34.1 , 33.3 , 31.8 -RRB- . These associations were shown to have a significant linear trend for both the biomedical -LRB- F1 ,986 = 77.5 , p < 0.001 -RRB- and behavioural -LRB- F1 ,981 = 31.9 , p < 0.001 -RRB- subscale scores . 3.6 Effect of non-response In order to assess the impact of non-response bias within the survey estimates , a brief questionnaire was mailed to a random sample of non-responders -LRB- GPs n = 414 , PTs n = 243 -RRB- , and responses were received from 14 % of GPs -LRB- n = 59 -RRB- and 17 % PTs -LRB- n = 40 -RRB- . For the GPs , gender mix and years in practice were similar for those completing the full and brief questionnaire . For the PTs , those completing the brief questionnaire were slightly less experienced -LRB- mean of 12 years experience versus 15 years -RRB- and more likely to be male compared to the full questionnaire responders -LRB- 25 % vs. 19 % male -RRB- . Responses to both behavioural subscale PABS.PT items and one of the two items from the biomedical subscale were similar to those for the full questionnaire . Responders to the brief questionnaire , from both professions , were more likely to agree with the statement that ` patients with back pain should preferably practice only pain free movements ' , indicating a more biomedical orientation . The responses to the items regarding work and activity advice were similar for responders to the full and brief questionnaires . GPs responding to the brief questionnaire reported bedrest advice that was less in line with guideline recommendations than the responders to the full questionnaire -LRB- 19.3 % strictly in line with guidelines compared to 38.4 % , respectively -RRB- , whereas the PTs completing the brief questionnaire reported bedrest advice that was more in line with guideline recommendations than the initial responders -LRB- 35.0 % strictly in line with guidelines compared to 21.8 % , respectively -RRB- . 4 Discussion 4.1 Main findings This is the first national UK survey of LBP related attitudes , beliefs and reported clinical behaviour of GPs and PTs and results show that responses are diverse . The majority of respondents reported advice that was strictly or broadly in line with guideline recommendations about activity and bedrest , however , over a quarter of HCPs recommended that the vignette patient with NSLBP should remain off work . Reasons why adherence to guideline recommendations for work is lower than for activity and bedrest are unclear , but may be due to the complex nature of the clinical consultation , and previous studies have shown that GPs see sickness certification as a potential threat to the doctor -- patient relationship -LSB- 15,29 -RSB- . The attitudes and beliefs of HCPs were significantly associated with reported work advice for the patient described , i.e. HCPs with stronger biomedical and weaker behavioural treatment orientations were more likely to report advice , regarding work , which was ` not in line with clinical guidelines ' . The subgroup analysis supports this , although only a third of respondents could be categorized into these subgroups i.e. high biomedical and low behavioural scores on the PABS.PT or vice versa . The differences in the PABS.PT scores were small , and although statistically significant , no guidance is currently available to suggest whether these represent a clinically relevant difference . A considerable proportion of HCPs in the UK continue to provide advice to patients about work that is not in line with guideline recommendations . The associations between attitudes , beliefs and reported clinical behaviour suggest that some HCPs continue to practice predominantly within a biomedical model , placing most importance on the severity of tissue damage when determining a patient 's level of pain and functional disability . Others have adopted a more behavioural approach to management , embracing the notion that the level of pain and functional loss may be influenced by psychological and social factors in addition to biomechanical factors . 4.2 Comparison to other studies HCPs in this study had similar attitudes and beliefs to therapists in the Netherlands -LSB- 28 -RSB- , with Dutch therapists having similar mean biomedical scores -LRB- 29.5 vs. 31.0 -RRB- , but slightly higher behavioural scores -LRB- 35.6 vs. 33.0 -RRB- on the PABS.PT . Direct comparison of subscale scores with studies using the original PABS.PT is not possible due to a different number of items -LSB- 30,39 -RSB- . The attitudes and beliefs of HCPs were significantly associated with reported work advice for the vignette patient . Respondents reporting advice ` strictly in line with guidelines ' demonstrated stronger behavioural and weaker biomedical orientations than those reporting advice ` not in line with guideline recommendations ' . Using a variety of measures , previous studies have demonstrated that advice to restrict work or activities is also associated with a biomedical treatment orientation -LSB- 28 -RSB- , patho-anatomical focus of training courses -LSB- 39 -RSB- , higher fear avoidance beliefs of HCPs -LSB- 17,32,42 -RSB- and a strong belief that pain and impairment are invariably linked -LSB- 28,44 -RSB- . Our study adds to this body of literature by showing a significant association between attitudes and beliefs and reported work advice in HCPs in the UK . 4.3 Implications for clinical practice and future research The results suggest that the attitudes and beliefs of HCPs are linked to clinical practice and the recommendations provided to patients . These practitioner factors are thus part of the dynamic interaction within LBP care episodes , along with the LBP problem itself and the patient 's own perceptions about their problem . This may help explain patient outcomes , although the mechanisms behind this are likely to be complex . It is probable that HCPs ' attitudes and beliefs are expressed to patients in a variety of ways , with a range of possible consequences . By restricting activities and work , HCPs may reinforce patient 's unhelpful illness perceptions and increase spinal vigilance . Alternatively , they may over-direct the patient by providing strict advice to perform only specific activities and exclude others , encouraging an over-reliance on the HCP -LSB- 35 -RSB- , which may make it difficult to foster the patients ' self-management skills , something recommended as part of best practice for patients with LBP . The reported clinical behaviour of HCPs illustrates that the majority would provide advice that is strictly or broadly in line with guideline recommendations , however , nearly 30 % reported they would advise the described patient to remain off work . Staying at work or an early return to work with NSLBP is recommended -LSB- 50 -RSB- , as the longer someone is off work the likelihood of them returning steadily diminishes , with a 20 % risk of long term disability for those off work for four to six weeks -LSB- 51 -RSB- . Although the management of LBP , in terms of advice about activity and bedrest , seems to be broadly in line with guideline recommendations , our results show that adherence about advising early return to work is suboptimal . Attitudes and beliefs held by HCPs may help explain why implementation of current LBP guidelines has been slow and difficult -LSB- 6,8,20,23,33 -RSB- . Changing clinical behaviour is recognised to be a challenge -LSB- 27 -RSB- . Evidence from recent clinical trials suggests that although modest intervention strategies can result in moderate changes in reported adherence to guideline recommendations -LSB- 8 -RSB- , this does not lead to a corresponding improvement in patient outcomes -LSB- 9,21,30 -RSB- . A better understanding of the attitudes and beliefs of HCPs , what influences these and how these relate to outcomes of patients with LBP is needed to inform development of future implementation strategies . Future work should further test the psychometric properties of the PABS.PT to assess responsiveness and determine appropriate cut offs for ` high ' and ` low ' scores on the subscales and what constitutes a clinically relevant change . Methods to assess HCP attitudes , beliefs and behaviours warrant further study . For example , the validity of using methods to measure implicit attitudes about LBP , such as those employing automatic responses , could be explored in an attempt to overcome potential social desirability bias in survey responses as HCPs become more aware of clinical guidelines . 4.4 Strengths and limitations The strengths of this study include the large sample sizes , simple random sampling of UK GPs and PTs , use of a validated beliefs measure , and investigation of potential non-response bias . The response rate of GPs was low , but comparable to other postal surveys of GPs in the UK -LSB- 4,5,38 -RSB- . The sample size calculation took this into account and yielded the required sample size for the planned analyses . The response rate of PTs was in keeping with other studies -LSB- 11,39,41 -RSB- . Responses to the brief questionnaire were broadly similar to those completing the full questionnaire in terms of attitudes , and recommendations for work and activity . However , some differences in the advice for bedrest suggest that we can not rule out non-response bias in our survey . Responses to one PABS.PT item showed a stronger biomedical treatment orientation for responders to the brief questionnaire . Also , GPs responding to the brief questionnaire reported advice for bedrest that was less in line with guideline recommendations than responders to the full questionnaire , so for GPs , where the potential for non-response bias is greatest , our survey may underestimate the strength of a biomedical treatment orientation and the numbers providing advice not in line with guideline recommendations . This study captured self-reported behaviour rather than real clinical practice , which is very difficult to measure . To provide a context for the clinical behaviour questions we used a vignette of a patient with NSLBP , an approach shown previously to have acceptable validity -LSB- 40,46 -RSB- . Although we used established tools to assess attitudes , beliefs and clinical behaviours , there may be some overlap in the constructs they measure . We attempted to address this by the wording of instructions and the order of the tools within the questionnaire . The PABS.PT attitudes measure came first with instructions to respond to the general attitudinal type statements . The vignette and the behaviour questions came later with the instruction to consider the specific management of the patient described . 5 Conclusion This study shows the diversity of the attitudes and self-reported practice behaviour of UK GPs and PTs for patients with NSLBP . Many HCPs believed LBP necessitates some avoidance of activities and the need to be off work . For a patient with a history of being off work since onset of LBP four weeks previously , over a quarter of HCPs recommended further time off work . The attitudes and beliefs of HCPs were associated with their advice about return to work . Future studies need to investigate the associations between HCP factors and patient outcomes , and test if approaches aimed at modifying attitudes , beliefs and clinical behaviours of HCPs can be successful .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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