Drosophila circadian clock by controlling TIMELESS oscillations.The ubiquitin ligases CUL-3 and SLMB collaborate to regulate the  Cul-3 RNAi or dominant negative forms of CUL-3 in the clock neurons alters locomotor behavior and dampens PER and TIM oscillations in light-dark cycles. In constant conditions, CUL-3 deregulation induces behavioral arrhythmicity and rapidly abolishes TIM cycling, with slower effects on PER. CUL-3 affects TIM accumulation more strongly in the absence of PER and forms protein complexes with hypo-phosphorylated TIM. In contrast, SLMB affects TIM more strongly in the presence of PER and preferentially associates with phosphorylated TIM. CUL-3 and SLMB show additive effects on TIM and PER, suggesting different roles for the two ubiquitination complexes on PER and TIM cycling. This work thus shows that CUL-3 is a new component of the Drosophila clock, which plays an important role in the control of TIM oscillations.Eukaryotic circadian clocks rely on transcriptional feedback loops. In Drosophila, the PERIOD (PER) and TIMELESS (TIM) proteins accumulate during the night, inhibit the activity of the CLOCK (CLK)/CYCLE (CYC) transcriptional complex, and are degraded in the early morning. The control of PER and TIM oscillations largely depends on post-translational mechanisms. They involve both light-dependent and light-independent pathways that rely on the phosphorylation, ubiquitination, and proteasomal degradation of the clock proteins. SLMB, which is part of a CULLIN-1-based E3 ubiquitin ligase complex, is required for the circadian degradation of phosphorylated PER. We show here that CULLIN-3 (CUL-3) is required for the circadian control of PER and TIM oscillations. Expression of either  Drosophila fruit fly, the PERIOD (PER) and TIMELESS (TIM) proteins coordinate the clock–they accumulate during the night, form a complex, and repress their own gene expression in the early morning. The temporal control of this oscillation involves the phosphorylation, ubiquitination, and proteasomal degradation of the PER and TIM proteins. The SUPERNUMERARY LIMBS (SLMB) ubiquitin ligase is known to play a key role in controlling the degradation of phosphorylated PER and TIM. In this study we investigated the role of another ubiquitin ligase, CULLIN-3 (CUL-3). We found that inhibition of CUL-3 activity results in the abolition of rest/activity rhythms in flies and flattens the PER and TIM oscillations. CUL-3 physically interacts and forms a complex with a lowphosphorylated version of TIM in the absence of PER, thereby allowing its accumulation during the night. In contrast, when PER is present SLMB preferentially interacts with phosphorylated TIM, favoring its degradation. The results suggest that CUL-3 and SLMB share the work to control the oscillations of the PER and TIM proteins during the day/night cycle.Circadian clocks adjust the physiology and behavior of organisms to the day/night cycle and rely on molecular feedback loops that generate daily oscillations of transcription. In the  The delayed accumulation, nuclear entry, and transcriptional activity of PER and TIM involve their phosphorylation by several kinases such as DOUBLE TIME (DBT) CK1ε Circadian clocks are present in most living organisms and control a variety of physiological and behavioral functions. Eukaryotic clocks stem from a transcriptional negative feedback loop where activators induce the expression of repressors, which then inhibit the activators S region The pace of the oscillation depends largely on the speed of PER/TIM accumulation during the early night and degradation in the late night. The stability of phosphorylated PER and TIM is controlled by the SUPERNUMERARY LIMBS (SLMB) E3 ubiquitin ligase In addition to SLMB that controls the circadian oscillations of TIM, the JETLAG (JET) E3 ligase targets TIM and CRYPTOCHROME (CRY) for the light-induced proteasome-dependent protein degradation that participates to the resetting of the clock Cul-3 mutants are homozygous lethal, we first used targeted expression of Cul-3 RNAi to test the possible role of CUL-3 in the control of behavioral rhythms. About 150 neurons express PER and TIM in the brain, and various studies have assigned specific behavioral contributions to defined neuronal subsets, depending on the environmental conditions Cul-3 RNAi in all clock cells under tim-gal4 control induced 100% lethality, but a limited number of adult flies could be obtained with the Clk-gal4 driver, whose expression is less broad than tim-gal4cry-gal4 and Pdf-gal4 drivers. In LD cycles, flies expressing Cul-3 RNAi under Pdf-gal4 or Clk-gal4 control displayed no or reduced morning anticipatory activity , supporting decreased CUL-3 activity in the RNAi flies. For RNAi genotypes, two copies of both the gal4 and UAS transgenes were required for generating arrhythmic behavior , suggesting that they were less efficient than RNAi for decreasing CUL-3 activity in the clock neurons. Driving their expression with tim-gal4 did not induce lethality, also supporting weaker effect compared to Cul-3 RNAi. We thus used gal1118 and tim-gal4 to test the effect of CUL3K717R on behavioral rhythms. LD morning anticipation was reduced and DD free running rhythms were lost or altered in flies expressing either one of two different CUL3K717R-encoding constructs . Altogether, the data indicated that the behavioral phenotype induced by CUL-3 downregulation was, at least for a large part, not a consequence of a developmental defect.Since proteins . Anti-PDw ; Pdf-gal4; UAS-Cul-3RNAi (hereafter Pdf>RNAi) flies showed dampened PER and TIM oscillations . Similarly dampened oscillations of the two proteins were also observed in the second day after transfer in DD  flies also showed altered PER and TIM oscillations, with a complete loss of TIM cycling on the third day of DD . In LD conditions, llations . The twoer in DD , providiay of DD . Althougive LNds .K717Rtim>Cul-3 flies. In LD conditions, mutant flies still showed robust PER (unpublished data) and TIM  might not produce sufficiently high levels of CUL-3K717R protein to affect LD oscillations, which are strongly driven by light. We then looked at protein oscillations in DD . Phosphatase treatment abolished the slow migrating TIM forms of K717Rtim>Cul-3 flies . At DD2, TIM cycling was strongly reduced, but the accumulation of phosphorylated TIM was less prominent than in flies expressing CUL-3K717R , with a stronger phenotype in the K717Rtim>Cul-3 flies  in the mutant, but peak levels were about 25% lower. At day 3 (right panels), per and tim mRNA oscillations were more severely dampened, with peak levels lowered by about 40%. Decreased mRNA thus likely contributed to the lower levels of unphosphorylated PER and TIM at the beginning of the night . However, mslmb mutants showed a slightly higher TIM molecular weight than K717Rtim>Cul-3 flies. In the double mutants, a progressive loss of unphosphorylated TIM was observed, leading to a large excess of phosphorylated protein at day 2, migrating similarly to the high molecular weight TIM of mslmb mutants. Moderately phosphorylated PER accumulated in the K717Rtim>Cul-3 flies, whereas hyper-phosphorylated protein was observed in both mslmb and double mutants, with even higher forms progressively accumulating in the double mutants. To confirm this interaction, PER was analyzed in LD , more subtle modifications would have to be involved or a yet uncharacterized TIM kinase would have to be destabilized by CUL-3. We thus believe that the simplest interpretation is a direct control of TIM by CUL-3. In this hypothesis, CUL-3 could either control the stability of phosphorylated TIM or control the stability of hypo-phosphorylated TIM, which would then be stabilized and subsequently phosphorylated more extensively. For example, accumulation of phosphorylated PER has been reported in long period dbt mutants Clk>Cul-3RNAi flies? Since they only show a 2-fold decrease of Cul-3 mRNA in head extracts, they are likely to display a weak phenotype compared to K717Rtim>Cul-3 flies. The absence of phosphorylated TIM in the RNAi flies would thus support CUL-3 acting on several forms of TIM, with phosphorylated TIM being more sensitive to degradation than hypo-phosphorylated TIM.Since phosphorylated TIM accumulates in +per flies indicate that CUL-3 preferentially binds to hypo-phosphorylated TIM , suggesting that TIM is targeted by another member of the large BTB-domain protein family. A recent study has revealed that the BTB domain protein encoded by the insomniac gene is involved in the control of sleep by specifically affecting sleep bout duration, but insomniac does not appear to have a circadian function Δ260–292 in both +Cul-3 and K717RCul-3 flies suggests that no phosphorylation can occur on this form of TIM or that phosphorylated TIMΔ260–292 cannot accumulate even with downregulated CUL-3. The absence of putative phosphorylation sites in TIMΔ260–292CUL-3 complexes recognize substrates through a BTB-domain Although both CUL-1 and CUL-3 complexes might control TIM indirectly, the possibility that they share the work to control TIM ubiquitination is interesting. For example, CUL-1/SLMB controls the proteolysis of highly phosphorylated CI, whereas CUL-3/RDX appears to control the degradation of a less phosphorylated form of the protein see . The preUAS-Cul-3RNAi line carries two independent insertions (CG11861-R1 and -R2) of the same RNAi construct, which are described at http://www.shigen.nig.ac.jp/fly/nigfly/index.jsp. The gft2Cul-3 loss-of-function allele has been described in ΔCUAS-Cul-3 in UAS-flag-Cul-3 and K717RUAS-flag-Cul-3 in K717RUAS-gfp-Cul-3 and UAS-gfp-Cul-3 have been described in UAS-gfp-Cul-3 expression shown to rescue Cul-3 mutant cellular clones Pdf-gal4gal1118cry-gal4-39Clk-gal4-6/1tim-gal4-62cry-gal80Δ260–292timm (slmb8 hs-slmb)slmb adults were produced by providing hs-slmb expression with daily heat-shocks during development, as described in tim-gal4 and K717Rtim-gal4 UAS-Cul-3 flies were in an s-tim background  and cloned into the pENTR/D-TOPO vector (Invitrogen). The ORF was recombined into pTFMW (https://dgrc.cgb.indiana.edu/vectors/store/vectors.html) containing a UASt promoter and N-terminal 3xFLAG and 6xMyc tags using LR clonase (Invitrogen). The construct was transformed into 1118w embryos using standard procedures and inserts on chromosomes 2 and 3 were selected. Line 23 was used for the anti-FLAG IP experiments.The ound see . For genDrosophila activity monitors (TriKinetics). Light was provided by standard, white, fluorescent, low-energy bulbs. Flies were entrained in 12 h∶12 h LD cycles for 4 d and then transferred to DD. Actograms are double-plotted graphs representing the absolute activity levels for each 0.5-h interval, averaged over the total number of flies of a given genotype. LD analysis was done on days 2–4 and DD analysis on days 6–12. Data analysis was done with FaasX software 1.16, which is derived from the Brandeis Rhythm Package . Index values were then averaged over 12–20 brain hemispheres for each time point.Experiments were done on whole-mounted brains as previously described PLUS SYBR green I mix with 500 nM of each primer, the reaction mix was loaded on the capillaries and submitted to 40 cycles of PCR , followed by a fusion cycle in order to analyze the melting curve of the PCR products. Negative control without the RTase was introduced to verify the absence of genomic DNA contaminants. Primers , measurement was made at least in duplicate .Total RNA were prepared from adult heads (about 35) or larvae (3 to 5) using the Promega SV total RNA isolation system. They were quantified using the Nanodrop ND-1000 spectrophotometer and the integrity of the RNA was verified using the Agilent 2100 bioanalyser with the eukaryote total RNA Nano assay. 1 µg of total RNA was reverse-transcribed in a 50 µl final reaction in presence of 0.4 µm oligodT(15), 8 mM dNTP, 40 units of RNasine, and 400 units of M-MLV RTase H-minus (Promega), during 3 h at 37°C. Quantitative PCR was performed with a Roche LightCycler using the SYBR green detection protocol of the manufacturer. 5 µl of a 25 times diluted cDNA were mixed with FastStart DNA master Primers  were deft values  were theProtein extracts were made from frozen heads homogenized in ice-cold HE extraction buffer , supplemented with 20 mM glycerophosphate, 0.1 mM DTT, phosphatase inhibitor cocktails 1 (0.5%) and 2 (1%) (Sigma), and protease inhibitor tablets (Roche) according to the manufacturer's instructions. Anti-FLAG immunoprecipitations were performed using EZview Red anti-FLAG affinity gel (Sigma) according to the manufacturer's instructions. For immunoprecipitations, 1 mg of total head proteins were extracted in supplemented HE buffer, and DTT was replaced by 0.1% NP40. For SDS-PAGE, 50 µg of extracts (or immunoprecipitated proteins from 1 mg of extracts) were separated on 3%–8% Tris-acetate (TIM) or 4% Tris-glycine . Top panels: averaged activity distribution of n flies in LD (see Locomotor activity of flies expressing n LD see . Dots inn LD see . Behavio(PDF)Click here for additional data file.Figure S2Cul-3 mRNA levels in flies expressing Cul-3RNAi. Quantitative RT-PCR was done as described in Cul-3 primers E7-E9. da-gal4 (daughterless) is a ubiquitously expressed gal4 driver, and da-gal4/+ ; UAS-Cul-3RNAi/+ flies were lethal after the larval stages. For each experiment, averaged normalized values of the mutant and control genotypes were expressed as a percentage of the maximum value set to 100. Means of three (top) or five (bottom) independent experiments are reported in the graphs. Error bars indicate s.e.m. Two other sets of Cul-3 primers were tested and gave similar results: Cul-3 mRNA levels were decreased to 46% (E7-E8 primers) or 45% (E3-E4 primers) of the control levels in Clk-gal4 ;; UAS-Cul-3-RNAi heads, and to 28% (E7-E8 primers) or 22% (E3-E4 primers) of the control levels in da-gal4/+ ; UAS-Cul-3-RNAi/+ larvae.(PDF)Click here for additional data file.Figure S3K717Rflag-Cul-3, ΔCCul-3, or gfp-Cul-3 transgenes. Flies were entrained for 4 d in LD 12∶12 and then transferred to DD. White and black/gray indicate lights-ON and lights-OFF, respectively. ZT is Zeitgeber Time (ZT0 corresponds to lights-ON). Top panels: averaged activity distribution of n flies in LD (see Locomotor activity of flies expressing n LD see . Dots inn LD see . Behavio(PDF)Click here for additional data file.Figure S4Cul-3 downregulated flies at different temperatures. Flies expressing Cul-3 RNAi and controls were grown at 25°C, and the adults were then either transferred at 20°C or kept at 25°C for 4 d in LD 12∶12 followed by DD. 25°C data are those already shown in Locomotor activity of (PDF)Click here for additional data file.Figure S5Cul-3 mutants and controls. Stacks of optical sections from adult brains immunolabeled with anti-PDF. Short arrow indicates slightly more defasciculated projections, and long arrow indicates reduced arborization in the medulla. Flies with a homozygous tim-gal4 insertion very often show some additional PDF-positive fibers that appear to derive from the Posterior Optic Tract (arrowheads). Scale bars, 50 µM.Morphology of PDF-positive s-LNvs in (PDF)Click here for additional data file.Figure S6K717Rtim>Cul-3 flies. Flies were entrained for 3 d and collected the fourth day of LD at ZT12, 16, or 20. Graphs represent quantifications of TIM immunolabeling in the four “morning” PDF-positive s-LNvs and the three “evening” CRY-positive LNds of K717Rtim>Cul-3 flies and controls. Error bars indicate s.e.m. Experiments were repeated twice with very similar results.TIM immunoreactivity in the LNs of (PDF)Click here for additional data file.Figure S7K717R and controls. Flies were entrained for 3 d in LD and collected every 3 h the fourth day of LD. White and black bars indicate day and night, respectively. ZT is Zeitgeber time. Phosphorylated (P-) and hypo-phosphorylated forms of TIM are indicated. Two independent Western blots were done for each genotype with very similar results.TIM Western blot of head extracts of flies expressing CUL-3(PDF)Click here for additional data file.Figure S8K717R. Phosphorylated (P-) and hypo-phosphorylated forms of PER and TIM were quantified with the Gel Analyzing Tool of Image J software (NIH), which compares the signal density and background of each track. Average values of at least three independent Western blots were used for each genotype/condition. The results are normalized to the maximum value of each blot, set to 100. Error bars indicate s.e.m. Gray and black bars indicate subjective day and subjective night, respectively. CT is circadian time.Quantification of PER and TIM in head extracts of flies expressing CUL-3(PDF)Click here for additional data file.Figure S9K717RCul-3 transgene and controls. Flies were entrained for 3 d in LD and transferred to DD for collection in the second day of DD. Gray and black bars indicate subjective day and subjective night, respectively. CT is circadian time. Phosphorylated (P-) and hypo-phosphorylated forms of TIM are indicated.TIM protein in head extracts of flies expressing another (PDF)Click here for additional data file.Figure S10K717Rtim>Cul-3 flies disappears after phosphatase treatment. Flies were entrained for 3 d in LD, transferred to DD, and collected the second day at CT39. Head extracts were treated (+) or not (−) with L-Phosphatase for 30 mn at 30°C and then subjected to electrophoresis and Western Blot analysis with anti-TIM antibodies. Untreated samples stored at 4°C were used to estimate endogenous phosphatase activities, which are not inhibited in the extraction buffer. Phosphatase treatment: protein extracts were made in Lysis Phosphatase buffer  and incubated with 1,000 units of L-phosphatase (New England Biolabs) for 30 mn at 30°C. The experiment was repeated twice with very similar results.Low mobility TIM observed in (PDF)Click here for additional data file.Figure S11K717R protein and controls. Flies were entrained for 3 d in LD and transferred to DD for collection in the second day of DD. Top: anti-TIM Western blot. Gray and black bars indicate subjective day and subjective night, respectively. CT is circadian time. Phosphorylated (P-) and hypo-phosphorylated forms of TIM are indicated. Two independent experiments were done with similar results for CUL-3 overexpression. A CUL-3K717R blot is shown here for comparison (see TIM protein in head extracts of flies overexpressing CUL-3 or CUL-3(PDF)Click here for additional data file.Table S1Anticipation phase score of fly group activity preceding Lights-ON/Lights-OFF transitions in flies with altered CUL-3 activity. Corresponding activity graphs with activity levels and number of flies are shown in (DOCX)Click here for additional data file.Table S2n+1 and n (1<E<2); R2, coefficient of determination of the calibration curve; Ct, Cycle threshold. The Ct values interval defines the linear range of the standard curve for each pair of primers.Primers specifications see . Efficie(DOCX)Click here for additional data file.


Proper hand hygiene is critical to prevent healthcare associated infections. Therefore, measurement of hand hygiene compliance is an important tool in infection prevention. After multi model hand hygiene campaign (HHC) compliance is known to remain low.Between October and December 2010, after an intensive HHC of ten years in our 1894-bedded tertiary care centre, hand hygiene opportunities (HHO) as defined according to the 5 categories from the WHO were measured by direct observation. Compliance rates (CR) were calculated as the number of hand hygiene activities (HHA) divided by the HHO.A total of 1007 HHO were documented: 134 in the protective isolation ward, 113 in the medical ward, 566 in the intensive care unit (ICU), and 161 in the surgical ward, resulting in CRs of 49% (66/134), 51% (58/113), 28% (159/566), and 51% (82/161), respectively. The CR in the ICU was statistically lower (p < 0.001) compared to the other wards and this was due to the absence of hand hygiene after glove use. In all wards except the protective isolation ward, CRs were statistically lower (p < 0.001) before patient contact (WHO indications 1 and 2) than after patient contact .Most increase in compliance can be achieved by focusing our new HHC on performing hand hygiene before aseptic tasks.None declared.


Traditional mattress sutures prove difficult to remove when the knot becomes buried in swollen tissue. We describe a suture with the benefits of a mattress suture that can be removed more easily:Starting on the ‘near-side’, cross the wound twice as per a regular horizontal mattress suture, leaving the loop proud.Pass one free end through the loop and pull to the near-side of the wound.Tie on the near-side, ensuring suture material of the loop bridges the outside of the wound.This will remain easily accessible for removal regardless of swelling .


Good morale among staff on inpatient psychiatric wards is an important requirement for the maintenance of strong therapeutic alliances and positive patient experiences, and for the successful implementation of initiatives to improve care. More understanding is needed of mechanisms underlying good and poor morale.We conducted individual and group interviews with staff of a full range of disciplines and levels of seniority on seven NHS in-patient wards of varying types in England.Inpatient staff feel sustained in their potentially stressful roles by mutual loyalty and trust within cohesive ward teams. Clear roles, supportive ward managers and well designed organisational procedures and structures maintain good morale. Perceived threats to good morale include staffing levels that are insufficient for staff to feel safe and able to spend time with patients, the high risk of violence, and lack of voice in the wider organisation.Increasing employee voice, designing jobs so as to maximise autonomy within clear and well-structured operational protocols, promoting greater staff-patient contact and improving responses to violence may contribute more to inpatient staff morale than formal support mechanisms. Psychiatric inpatient wards are potentially highly stressful places to work. In England, the shift towards community-based care in the post-deinstitutionalisation era has raised the threshold for admission, with more patients detained under section and shorter lengths of stay . Policy Staff morale in the NHS is important in several respects. Firstly, the NHS is one of the world's largest employers, and achieving the status of an exemplary employer has recently been defined as an important goal . SecondlUntil recently, there has been little comprehensive research on the morale of NHS inpatient mental health staff, with most studies employing small samples and confined to single sites or including only mental health nurses -12. The An understanding of the factors underlying good or poor morale on wards is likely to be required for effective strategies to improve morale to be designed, but empirical examinations of these are even rarer than studies of levels of morale . The quaQuantitative data of this type illuminate potential underlying mechanisms for good and poor morale to only a limited extent. Qualitative accounts have a major complementary role in allowing an understanding of how staff make sense of their experiences at work, their views about how to improve their experiences, and the mechanisms that might underlie their responses to particular sources of stress and satisfaction. A systematic review on staff morale in 2004 showed that 38 out of 39 qualitative studies included in the review were single site case studies . A qualiThe current study reports findings from a substantial multicentre qualitative investigation of inpatient staff views regarding the factors that influence their morale. Aims were to extend current understanding of the mechanisms underlying good and poor morale on inpatient wards, and to generate potential strategies for improving morale. This qualitative study was nested within a national multi-site quantitative study ,14: a seThis study reported here is the qualitative component of the multiple methods National Inpatient Staff Morale study, commissioned by the National Institute of Health Research Service Delivery and Organisation programme ,14. Mult1 [Seven wards in London and the Midlands were included in this qualitative investigation. They were a purposively selected sub-sample from the 100 wards participating in the quantitative questionnaire survey. The quantitative survey involved administering a questionnaire including measures of levels of morale to staff on 100 wards in 4 English regions, selected to represent all the main inpatient mental health sub-specialties and areas with a wide range of geographical and demographic characteristics. For practical reasons related to researcher location, the seven wards participating in the current qualitative study were selected from 6 of the 18 Trusts participating in the initial quantitative study. Purposive selection within these six Trusts was based on stratification of the sample by mean morale scores obtained in the initial quantitative surveys. For each ward participating in the quantitative study, we calculated a standardised mean morale score based on all the questionnaire measures of morale1 . We usedOn six of the seven wards, we conducted two focus groups with staff. One consisted of junior staff from a range of professional backgrounds, including ward nurses, junior doctors, nursing assistants and other staff without professional qualifications, and basic grade occupational therapists (OTs). The other consisted of senior staff who worked on the ward and also had some managerial responsibility for other staff on the ward, including the ward manager and deputy ward managers, consultant psychiatrists with responsibility for beds on the ward, and, where relevant, senior members of other professions such as consultant clinical psychologists and Head OTs. In one ward focus groups were not possible due to staffing constraints so extra individual interviews were carried out.On each ward, we also conducted individual interviews with members of staff of different seniority and professional backgrounds, and one interview with a more senior service manager not based on the ward, such as a lead nurse for a whole hospital or a service manager responsible for a group of wards. In the individual interviews, we sampled purposively to obtain the perspectives of staff from a full range of levels of seniority and professional backgrounds. The final data set consisted of 12 focus groups, 24 ward staff interviews , and 7 senior manager interviews.team, rather than individual, morale. The main questions for discussion among group participants were their views about which are the main positive and negative influences on team morale. All staff provided written informed consent prior to participating.Interviews followed a semi-structured format and were conducted by trained research workers supervised by GH, SJ and MP, using a topic guide that explored positive and negative aspects of work, perceptions of staff morale on the study ward, the factors affecting staff morale, and ideas for how morale might be maintained or enhanced. The main questions were very broad and open-ended, enquiring how staff felt about their jobs, what main factors they felt influenced their feelings about work, and how their working environment might be improved. A list of prompts was used to explore views about areas not spontaneously touched on - these were identified from two sources: (a) areas identified from the literature as potentially linked to morale; and (b) expert views from the large multidisciplinary steering group for the National Inpatient Staff Morale Study . They weInterviews were recorded and transcribed verbatim. Data were analysed using thematic analysis ,20 withiThe characteristics of the participants in individual interviews are shown in Table Identified themes relevant to staff morale and well-being were in four main categories: (a) the staff team; (b) the management and leadership context: (c) organisational structures and (d) being with patients. Below we describe themes within each of these categories. A fifth area, physical environment, will be briefly summarised here and more extensively described in a separate publication.Ward staff recurrently identified the composition of the front-line ward team and relationships within it as crucial for morale.Staffing levels were viewed as central to morale by staff on all wards, some describing them as intermittently and others as constantly very problematic:We need more staff desperately and yes, that's probably the one thing more than anything else really because that would free up everything. That would free up the off-duty and the annual leave, the morale, the pressure and people would enjoy their job more. Many front-line staff felt overworked, describing the physical and emotional toll of a busy shift. Staffing levels could make it difficult to find time for a break, and to organise supervision and training, particularly on acute wards, where the risk of incidents intensified the need for adequate staff presence:Just getting on with the day to day work means that some of the things that might actually be more supportive for people, like meeting together...get pushed to one side. A further concern on four wards was sickness absence and problems with recruitment leading to a perceived over-reliance on "bank" staff. Participants spoke of their uncertainty about the skills of bank staff, particularly regarding "control and restraint" procedures and adherence to ward routines and protocols. Staff also noticed that patients were generally reluctant to approach staff they did not know:I suppose the anxiety is if it kicks off, they're not going to know the best way to respond. Effective team working and good relationships with colleagues were the most highly valued positive influences on morale. Staff on two 'high morale' wards - the Rehabilitation ward and the Child & Adolescent Unit - were especially positive about a sense of shared responsibility and their reliance on peer support:It's probably one of the most important things that gets me out of bed in the mornings to come here, that, generally speaking, I have pretty good relationships with people here. Oh the team here are excellent. You couldn't wish for better people and everybody gets on well and there's a mixture of sort of staff and the ideas that everybody has, so we get on ever so well, yes definitely. A culture of openness and acceptance, where staff are encouraged to give their views regardless of seniority, was associated with good morale:Sometimes nursing assistants aren't seen as part of...domestics are not seen as part of the team. Consultants are put on a pedestal and that doesn't really happen here does it? Everyone seems to have an equal opinion and an equal say. But some tensions were also reported from such tight-knit ward communities, where very close relationships created the risk of fallouts and cliques.Themes emerged relating both to clinical leadership within the ward, generally perceived as originating from the ward manager and to some extent the lead psychiatrist or psychiatrists, and to the senior management team beyond the ward, such as those responsible for the hospital or mental health Trust as a whole.Senior staff stressed the importance of strong and effective leadership. Consistency in leadership, aided by effective communication within the managerial team, was thought to be reassuring for staff, whilst weak leadership was linked to ambiguity and uncertainty. On one ward, multiple references were made to the impact of a new consultant psychiatrist:This guy is very direct, very clear about what plans he wants in place, and he's very open and warm, and, you know, very good, so I think it's made a big difference. Several others on different wards made reference to the way inspiring individuals could boost morale, and the importance of a reliable leadership team:I think that the things that influence morale in a positive way are stability of the staff team, particularly in leadership functions... this kind of work brings its troubles but overall there's a leadership team which I think is very responsive, containing and supportive of the wider staff team and very good at its job... I think that's absolutely 90% of the whole thing. This was the most discussed issue among lead/managerial staff working on the ward. Managers and senior managers unanimously believed that formal support mechanisms and supervision are vital for a successful team. Formal supervision was said to help solidify roles and responsibilities and improve confidence. Four wards had staff support groups, on which views were mixed. Senior staff regarded them as a source of mutual emotional support but several front-line staff members said they found them uncomfortable.Front-line staff spoke more about the value of informal support from managers than about supervision. They appreciated the visible presence of leading staff on the "shop floor", their availability for guidance and reassurance, and their responsiveness to work-related problems. On all wards there was discussion of the importance of feeling valued, with frequent comments that praise and recognition could be more forthcoming.Support following violent incidents was seen as important by staff on every ward, not just for immediate reassurance but because it sent a message that staff were being looked after. One group talked about how they used to receive letters following an incident, which had since ceased. Although it was "the exact same letter" every time, said one person, "at least you felt they were thinking of you".They used to come down afterwards and check if everyone was alright and that's important, you know? The small things make a big difference. The availability of formal supervision and the extent to which staff felt supported in their roles varied between wards and between individuals on the same ward. Many staff reported good relationships with their immediate managers. The Rehabilitation ward emerged as maintaining a very supportive environment, with the staff support group also highly valued on this ward. On other wards comments were mixed, with some staff feeling under-valued:I think a major problem as well is that I think we're bending over backwards to look after the patients, but we're not being looked after. Breaks are really hard to take and it's just that more and more is being taken and it's a case of well, it's effective, and it's not really a case of 'you're doing a good job, so good on you'. A view that senior managers, who were rarely seen on the wards, had a poor understanding of front-line work emerged on all seven wards:I just think sometimes the managers are up there, they have their job we have our job, but I don't think they understand what we really do. They'd have to spend like two weeks solid working with us 12 hours a day to understand what's going on. However, on the Rehabilitation ward, relative independence from senior management was also seen as having some advantages, with staff valuing their insularity and internal community.Ward managers were also aware of the perceived remoteness of senior managers from front-line staff, feeling uncomfortably "sandwiched" between two tiers. They felt pressured from above by budgetary constraints and sometimes having to implement unpopular policies.Ward managers and other senior ward staff on every ward saw considerable benefits to morale of involving front-line staff in decision making, and described efforts to increase their currently limited 'voice' in the workplace:I think if the staff are not feeling contained and heard and as though they have a sense of agency, then it's almost as though they then can't give that to the patients that they're caring for and the whole thing falls apart - and I think, at times, it has felt very much like that. One nursing assistant was frustrated at being excluded from ward rounds despite spending a great deal of time with patients:...I feel like I'm just here to go through processes and the mechanics of the day... I don't feel that I have an opinion that's really valued, or taken into account. In general, for front-line staff, feeling unheard was more of an issue in relation to ward policies and organisation, especially workload, than clinical decisions.I would just like whatever issues I raise to be dealt with without me having to chase them up three or four times and it's really, like... You know, it kind of undermines... You feel, like, you know, no one cares. While negative comments predominated, some positive experiences were also reported. One ward has a system for lodging complaints or proposals to senior managers:We've got a good formal system management group with people high up in the Trust. So if you have a proposal it will be heard and taken seriously by management meetings. If they can't deliver at that meeting then it's certainly put on as an agenda item for another time. Experiences of feeling heard on other wards tended to be attributed to the approachability of particular senior ward staff.A further group of themes related to the definition of roles within ward teams and the protocols and guidelines in place for organising work on the ward.Role clarity was highly valued throughout the sample, though only a minority currently described a lack of this. Managers were especially concerned with coupling responsibility with role clarity, and described strategies such as delegation of clinical and domestic responsibilities and the use of visual aids such as notice boards:And, I think, that's the thing for me, as well: give people the responsibility. But, in order to do that you have to explain to them what the responsibility entails. Don't just expect them to do something because if they don't understand why they're doing it and what the benefit is, and all that, they'll never really put their heart into it. (Acute Care Service Manager)wanted greater responsibility. One nursing assistant described her contentment with her role facilitating cooking groups:As a caveat, staff did vary in the extent to which they I'm quite happy with my job, being a nursing assistant and I even got a chance to go and do my training, I said I didn't want to. I'm happy with this job. Consistent protocols and guidelines for organising work on the ward were found to help maintain clarity and confidence, whilst change was felt to create anxiety:If you can have cohesion in terms of a cohesive, communicating staff group and cohesion in the sense of structure, in terms of the way ward rounds (and) business meetings operate, that acts as a defence against the anxiety and chaos of psychosis. In my experience, that really assists the efficiency of the ward and that leads to pepping up and, sustaining morale. Formal frameworks were also seen as vital for the maintenance of regular supervision and team meetings, which otherwise tended to fall by the wayside. Flexibility within a well-organised system was also valued, particularly in relation to shift systems. Several staff complained about a lack of flexibility around shifts, whilst a more flexible system on the Rehabilitation ward was seen as contributing to high morale.On all seven wards there was discussion of recent structural or organisational changes and managers were aware that the frequent waves of change experienced by NHS staff, driven sometimes by central policy and sometimes by local reorganisation, have a considerable impact on staff.Opportunities for training were valued, and those in high-morale wards tended to be more positive about them. Ward managers also saw training positively as a way of improving standards, maintaining role clarity, imbuing confidence and maintaining morale. Good provision of mandatory courses was reported, but resource limitations restricted access to other courses, especially longer term ones, to which staff often had to dedicate their own time.A final group of themes related to staff experiences of direct contact with patients on the ward.The impact on staff of patients' severe disturbance was especially felt on acute and PICU wards:I think psychosis has a way of inducing chaos and fragmentation, and it's kind of like a manifestation of the condition but also, somehow that gets projected into structures and organisations and systems, in my experience, and there's plenty of room for chaos in a ward environment - especially within a busy ward environment. Staff who had worked in a variety of settings commented that this made acute work more stressful, but some also valued the intensity of the work and pace of change:I've always loved this ward and the challenge, the busy-ness of the ward, you know, the range of people here. there's no reparation really that can be made" . Staff described how one or two individuals could shift the whole atmosphere of the ward:The volatility of acute wards made violence frequent and risk highly salient to staff. A common sentiment running was that in cases of assault, "The worst time we had here was some time last year when we had a sort of gang mentality on the ward - like them and us, and that was pretty frightening sometimes really. Staff, especially on acute and PICU wards, appeared stoically to accept that some potential for violence was a given, but strategies for reducing risk were widely seen as inadequate. Higher staffing levels were seen as key, and some concerns were also raised about aspects of the physical environment, including locks and alarms:We haven't got enough staff, we haven't got enough time and we haven't got enough pagers and alarms to do it safely. That's the trouble. Knowing one's colleagues and feeling able to rely on them both for help in managing difficult situations and for emotional support was crucial. One nurse described how adverse incidents, when managed effectively, had the potential to enhance team morale:No incident is nice, but if we deal with it correctly and no one gets off really hurt or whatever and all the procedures are done, it's a good feeling. I think it's good because that shows we've got team work. Attitudes varied as to whether dealing with social problems was a legitimate role for ward staff. On the Rehabilitation ward, it was seen as rewarding:Here it's seeing people moving on and getting their own independence and living in their own flats and being a part of that really. On one acute ward however, suspicions were raised that some patients' problems were not really as they seemed: some service users were seen as 'using the system' to gain access to social resources, possibly preventing 'genuine patients' from accessing a bed.Spending time with patients was seen as a core source of satisfaction; on six wards, staff felt that inadequate staffing and excessive administrative duties were impediments:You can't spend enough time with them and you're stressed out, and then that makes it even more stressful, because they're telling you you're basically not doing your job properly, because you're not spending time with us as much as you should. On Rehabilitation and Child & Adolescent wards, staff valued having more time for this and for engaging in social and recreational activities with patients, building relationships with them. Staff who could find time for patients seemed to value their roles more and to see them as better defined.I was doing the cooking... and I remember one of the patients said, 'Today I felt like I'm a human being'. I said, 'Why are you saying that?' She said, 'You know the food that you gave me, it made me feel good, like I'm still alive.' Across all the wards, seeing patients get better was a positive influence on morale. Those working in Rehabilitation and Child & Adolescent units gained fulfillment from a long-term emotional investment in clients. For those in acute/intensive care, success was rated on a more short-term basis in terms of "stabilising" patients and discharging them home. Staff on these wards who maintained more consistent positive morale embraced the "challenge" of acute psychiatric care. Acute care staff were also more likely to see patients return to the ward. For a few, particularly on one acute ward, the "revolving door" phenomenon was a cause of frustration. Some felt disillusioned at the way factors beyond their control contributed to repeated readmission.Participants were also asked directly about the impact of the physical environment on their morale. Qualitative data pertaining to this topic will be reported elsewhere, so only a brief summary is given here.A comfortable and attractive environment was not surprisingly seen as conducive to a good atmosphere, especially where staff and patients had access to outdoor space. Problems identified varied from ward to ward and included insufficient staff areas, poor air quality and lighting and lack of designated spaces for group activities or one-to-one sessions with patients. Particularly demoralizing were enduring problems, which could lead staff to feel neglected, though several people also described the joining effect of having to make do in adverse circumstances. Improvements to the physical environment were viewed as highly morale enhancing, sending a message that staff were valued.The themes identified here both corroborate and extend previous findings on the factors affecting staff morale ,17,18, acan be experienced as highly supportive in the context of the right working relationship and organisational context, but that front-line staff experiences of formal support mechanisms vary widely between individuals and between wards.Participants emphasised the importance of role clarity and the structural and organisational factors needed to maintain it. Empowering staff and giving them greater autonomy was recognised as a way of enhancing morale, but only if responsibilities are clearly defined. These findings are congruent with and amplify those of our quantitative survey, where role clarity and autonomy, alongside strong team communication and support from colleagues, were closely associated with morale. Availability of supervision was not found to be associated with morale in our quantitative study. The qualitative findings suggest that supervision and training As in the quantitative study, our findings suggest that morale is influenced by a combination of individual job characteristics  and broader organisational and cultural factors. This is congruent with recent research showing that factors such as workplace norms, role clarity, staffing resources, team communication and training opportunities have independent predictive value beyond the job-related variables of the Karasek model -23.The emphasis placed on staffing in this qualitative study is at odds with the absence of any clear association between staffing and morale in our quantitative findings . ReasonsWhereas contacts with colleagues were for the most part described very positively, experiences with patients were more diverse. Staff on acute and PICU wards spoke about the challenges posed by working with severely unwell clients, including the risk of violence. A stoical acceptance seemed to characterise many staff's attitudes towards this risk, but accounts suggested considerable limitations both in precautions against violence and in response to it. Lack of staff-patient contact time was a common complaint. Across all wards staff spoke about the rewarding nature of client contact and being motivated by helping patients recover.The study's strengths are in the breadth of its sample, encompassing all levels of seniority and professions and several ward types. There is little previous relevant qualitative work and none of this scope. Constraints of space and the simple thematic nature of the analysis are limitations: we have had to focus only on the most prominent of the themes emerging from a large and rich data set. Selection of wards within the participating mental health Trusts was purposive, but considerations of convenience and feasibility also played a part in identifying the participating Trusts for this qualitative study.Ward team cohesion and mutual support and trust appear crucial to staff's ability to sustain and gain satisfaction from their roles in this potentially stressful environment. Much support seems to be informal, but trying to limit change in ward team composition or ensuring time and structures are in place for effective communication may reinforce it. Some hazards of such close-knit teams need also to be considered: high rates of bullying were reported in our quantitative study and staff who for any reason are perceived as outsiders by a very cohesive main group may be at risk from this.http://www.institute.nhs.uk/quality_and_value/productivity_series/productive_ward.html, aimed at redesigning processes across wards of a range of types, may support managers in this. The fit between a person's ideal conceptualisation of their role and the reality of that role in practice is important. Cahill et al [chosen to be where they were.As well as providing clear, sympathetic and flexible leadership, contributions that can be made by ward managers to staff well-being include implementing well organised ward routines and procedures, and attending to the clarity of staff roles and their ability to exercise some autonomy in carrying them out. Initiatives such as the recent Productive Ward initiative ll et al  identifiStaff's strong identification with the ward teams and their lack of a sense of 'voice' in the wider organisation may make new initiatives aimed at improving care difficult to implement, especially where they are seen as imposed by distant senior managers. Trade unions and professional bodies are a traditional conduit for worker voice, but no mentions were recorded of these in the qualitative study, suggesting they may not be prominent in the everyday life of the current NHS. Innovations for improving voice might include greater presence of senior managers on wards (the sense that they did not know what the life of the ward was like in practice was very strong among staff), opportunities for staff to be present at higher level Trust meetings, speak-up mechanisms allowing staff to get concerns and ideas for improvement heard by managers, more extensive consultations on important decisions and greater attention to the unions -26. The Staff were not as satisfied with their relationships with patients as with colleagues, feeling especially that they lacked time to spend with them. This suggests that initiatives such as the Productive Wards programme (see above) and the recently promoted "Protected Engagement Time" (PET) scheme, aimed at ring-fencing time for patient contact  may haveInpatient staff feel sustained in their potentially stressful roles above all by mutual loyalty and trust within cohesive ward teams. Clear roles, opportunities to work effectively with patients to achieve well-defined goals, supportive ward managers and well designed organisational procedures and structures also maintain good morale. Formal support mechanisms such as supervision and support groups are perceived as useful only to a limited extent by frontline staff. Perceived threats to good morale include staffing levels that are insufficient for staff to feel safe and able to spend time with patients, the high risk of violence, and lack of voice in the wider organisation.Potential strategies for improving morale include increasing employee voice, designing jobs so as to maximise autonomy within clear and well-structured operational protocols, promoting greater staff-patient contact and improving responses to violence. These may have more to contribute to staff morale than a focus on formal support mechanisms. Intervention studies exploring ways of improving morale and links between staff morale and patient experiences and outcomes would be valuable.The authors declare that they have no competing interests.JT contributed to data collection and then led on analysing the data and drafting the paper, GLH was qualitative methods lead for the study, commenting on draft instruments and methods at all stages, contributing to the analysis and commenting on drafts of this paper, EW contributed to development of methods and instruments for the paper, collected the majority of the data and commented on drafts of the paper. MP contributed to the development of the protocol and study instruments, supervised the researcher collecting data at three sites, contributed to the analysis and commented on drafts. SJ was Principal Investigator for the study and led on development of the original protocol, and she then contributed to methods and instruments, supervised EW and JT, contributed to data analysis and helped draft the paper. All authors have approved the final draft.1 Standardised mean morale scores were calculated by standardising each morale indicator so that scores were distributed on a 1 to 100 scale, reversing direction where appropriate so that a higher score indicated better morale, and then calculating for each ward the mean score for all the morale indicators. The morale indicators used in this calculation included measures of dimensions of burnout, intrinsic job satisfaction, work-related well-being, job involvement and general emotional well-being: details and references are given in the study final report [l report .The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/11/68/prepub


Although implementing brief intervention among risky clients by general practitioners (GPs) is part of public health efforts, a greater challenge is to evaluate the impact of such intervention. The authors re-examined the data elicited by a GP brief intervention project, which involved 308 risky drinkers among 2869 clients enrolled between 2005 and 2007 in two areas in and around Florence. Data presented in previous INEBRIA meetings showed that baseline risky drinkers had a 55.30 g/day average intake , which decreased to 37.61 g/day at the first follow-up . Regression to the mean methods (both uni- and multi-variate) were used to overcome possible statistical flaws. Applying the regression coefficient of the two series of measurements, the extent of regression to the mean was 6.77 g and 6.78 g for males and females. Therefore, the reduction of the consumption in grams between enrolment and the first follow-up has been transformed respectively into 11.67 and 8.80 g/day. This change does not appreciably alter the previous results. The GPs who identify between 8% and 20% of their patients as at-risk drinkers  had a statistically significantly higher number of patients showing up at follow-up (68.69%) compared to those who identify a lower (<8%) or higher rate of clients (> 20 %) (p <0.001). Women compared to men, and people aged 40-49 compared to the other age classes, appear to be most amenable to change. Identification of risky drinking by GPs is a good predictor of decreased alcohol consumption among clients. Being a woman and being middle-aged are good predictors of improved alcohol drinking patterns among clients with at-risk drinking.


WNT10A gene mutations are known to cause rare syndromes associated with tooth agenesis, including onycho-odontodermal dysplasia (OODD), Schöpf-Schulz-Passarge syndrome (SSPS), hypohidrotic ectodermal dysplasia (HED), and more than half of the cases of isolated oligodontia recently, the genotype-phenotype correlations and the mode of inheritance of WNT10A mutations remain unclear. The phenotypic expression with WNT10A mutations shows a high degree of variability, suggesting that other genes might function with WNT10A in regulating ectodermal organ development. Moreover, the involvement of mutations in other genes, such as EDA, which is also associated with HED and isolated tooth agenesis, is not clear. Therefore, we hypothesized that EDA mutations interact with WNT10A mutations to play a role in tooth agenesis. Additionally, EDA, EDAR, and EDARADD encode signaling molecules in the Eda/Edar/NF-κB signaling pathways, we also checked EDAR and EDARADD in this study.Dental agenesis is the most common, often heritable, developmental anomaly in humans. Although WNT10A, EDA, EDAR and EDARADD were sequenced in 88 patients with isolated oligodontia and 26 patients with syndromic tooth agenesis. The structure of two mutated WNT10A and two mutated EDA proteins was analyzed.WNT10A and EDA were identified in 2 of 88 (2.27%) isolated oligodontia cases and 4 of 26 (15.38%) syndromic tooth agenesis cases. No mutation in EDAR or EDARADD gene was found.Digenic mutations of both WNT10A and EDA digenic mutations could result in oligodontia and syndromic tooth agenesis in the Chinese population. Moreover, our results will greatly expand the genotypic spectrum of tooth agenesis. Permanent tooth agenesis is the most common developmental dental anomaly in humans, with an incidence rate of 2.2–10.1% in the general population  WNT10A gene are responsible for OODD, HED and SSPS Mutations of the WNT10A mutations were identified in more than half of isolated hypodontia cases WNT10A mutations shows a high degree of variability, ranging from only isolated hypodontia to various symptoms of ectodermal dysplasia. Moreover, the genotype-phenotype correlations and the mode of inheritance of WNT10A mutations remain unclear WNT10A mutations WNT10A, a 46.4-kDa protein with 10 putative α-helices and seven putative β-strands, belongs to Wnt proteins, a large family of secreted signaling proteins. Wnt10a is expressed in embryonic limb EDA is an important gene associated with tooth agenesis, it is located on chromosome Xq12–q13.1 and encodes for ectodysplasin-A (EDA) (MIM 300451), a member of the tumor necrosis factor (TNF) family EDAR and EDARADD encode the protein EDAR and EDARADD, belonging to the Eda/Edar/NF-κB signaling pathway. The binding of Eda-Edar (complex formed by EDA and its receptor) to the downstream adaptor Edaradd leads to activation of the transcription factor NF-κB and is essential for the development of hair follicles, teeth, exocrine glands and other ectodermal derivatives EDA gene mutations have been detected in 63–95% of X-Linked HED (XLHED) patients EDA mutations are also associated with cases of isolated tooth agenesis EDA mutations may interact with WNT10A mutations and play a role in the development of tooth agenesis.WNT10A, EDA, EDAR and EDARADD mutations in patients with isolated or syndromic tooth agenesis. Our results suggest that WNT10A and EDA digenic mutations could result in tooth agenesis. This is the first time such mutations have been reported in patients with tooth agenesis.In this study, we investigated the contribution of Written informed consent for DNA analysis and reproduction of the photographs was obtained from all the participants and the parents on the behalf of the minors or children participants. This study was conducted with the approval of the Ethics Committee of Peking University Health Science Center.The study participants were 88 non-consanguineous patients with isolated tooth agenesis, 26 non-consanguineous patients with syndromic tooth agenesis (24 with HED and 2 with OODD), and 451 non-consanguineous normal controls, who were referred to the Department of Prosthodontics, Peking University School and Hospital of Stomatology, or the Department of Prothodontics, Beijing Stomatological Hospital. Oral examinations for all participants were performed by a prosthodontist, who determined the status of the dentition. A panoramic radiograph was taken to confirm the diagnosis of tooth agenesis for these participants. The shape and size of the residual teeth were also observed.The patients with isolated dental agenesis reported normal sweating and lachrymal secretions. They had no complaints about dry mouth, intolerance to heat, or susceptibility to respiratory tract infections. The patients had hair on their body and scalp, and their facial features, skin, and nails were normal on observation.Patients with HED presented abnormalities of at least two of the three following ectodermal structures: teeth, hair and sweat glands. The OODD patients had abnormal teeth, nail dystrophy, palmoplantar keratoderma and other associated ectodermal abnormalities.Genomic DNA of the participants and their relatives was extracted from peripheral blood lymphocytes using a QIAamp DNA Blood Midi Kit (Qiagen). DNA samples of the normal participants recruited from the general population were extracted from buccal epithelial cells using the Chelex-100 (Sigma) method.WNT10A, EDA, EDAR, and EDARADD genes was performed by direct sequencing of five PCR fragments for WNT10A, eight PCR fragments for EDA, ten PCR fragments for EDAR, and eight PCR fragments for EDARADD, which cover the entire cDNA including exons and intron-exon junctions of more than 100 base pairs. We compared all primer sequences to the whole-genome assembly in the ENSEMBL database to verify their uniqueness against gene families. Primer sequences are available upon request.Screening of the WNT10A mutations (p.R171C and p.G213S) and two novel EDA mutations (p.G257R and p.I312M) that were identified in this study.We performed protein structure analysis on the two WNT10A, the conservation of residues in sequences was determined to predict the influence of the two mutations. The sequences of orthologs of human WNT10A protein were retrieved from the KEGG database For http://www.pymol.org) from PDB file 1RJ7.We used the crystal structure of EDA-A1 (PDB id 1RJ7 EDA and WNT10A were identified in 2 of 88 (2.27%) isolated oligodontia cases and 4 of 26 (15.38%) syndromic tooth agenesis cases. The patients in all four syndromic cases had HED  loci were detected  of the coding sequence in exon 3 of WNT10A, which results in the substitution of Arg at residue 171 to Cys. DNA sequencing of the parents' genome revealed that both mutant alleles were from their mother (EDA mutation (c.769G>C) and a heterozygous WNT10A c.511C>T mutation, and showed absence of only the left upper lateral incisor without other clinical abnormalities. No mutations in these genes were found in the father.“N1” in the non-syndromic group  represenr mother , who carEDA and heterozygous p.Arg171Cys (c.511C>T) mutation in WNT10A were detected. The coding sequence in exon 9 of EDA showed a C to G transition, which results in the substitution of Ile at residue 312 to Met; also, the coding sequence in exon 3 of WNT10A showed a C to T transition at nucleotide 511, which results in the substitution of Arg at residue 171 to Cys. Analyses of his parents' genome revealed that the mutant alleles were from his mother, who carried digenic heterozygous EDA and WNT10A mutations at the same locus as that of N2  of the coding sequence in exon 3 of WNT10A was detected, this leads to the substitution of Arg at residue 171 to Cys. Analyses of his parents' genome showed that the mutant EDA allele was from his mother  mutation was detected in exon 3 of WNT10A, this leads to the substitution of Gly at residue 213 to Ser. Sequence analyses revealed that both mutant alleles were from his mother  mutation was also detected in exon 3 of WNT10A, it results in the substitution of Gly at residue 213 to Ser. Sequence analyses of her parents' genome revealed that the mutant alleles were from her mother  mutation in exon 3 of WNT10A were detected. These mutations were not found in his father's genome, but because his mother's DNA sample was unavailable, the origin of the mutant alleles was not clear , patient S3 showed typical phenotypic expression of HED while her mother only had microdontia. Because the EDA mutation is the most frequent pathogenic cause of XLHED and the EDA gene is located on chromosome X, most female carriers of EDA mutations present with a normal or very mild phenotype Using phenotype-genotype analysis, we found that although patient S3 and her mother had the same mutation Click here for additional data file.


Gluteus Maximus (GMax) and Gluteus Medius (GMed) muscles are commonly weakened in hip osteoarthritis (OA), and functional weight-bearing (WB) exercises are frequently prescribed for gluteal strengthening. Muscle activity as an indicator of muscle recruitment can be measured using electromyography (EMG), with recruitment of >45% maximal voluntary isometric contraction (MVIC) required for strengthening. The aim of this study was to compare gluteal muscle activation during three WB exercises. As no participants with hip OA volunteered over the time-frame of this study, methods and results for the symptom-free group only are presented.+SD age, 48.68+6.66) were recruited. Surface EMG activity of GMax and GMed was measured bilaterally while participants performed 3 exercises: squat (SQ), right step up (RSU) and right step down (RSD). EMG data were normalized to each participant's MVIC. One way Analysis of variance was used to compare mean EMG activity across the three exercises for left and right GMax and GMed. Significance was set at p<0.05.Nineteen healthy, symptom-free participants  and R GMax (13.07+10.73) whilst RSD had higher recruitment for L GMed (16.66+6.74 %). Squat produced the lowest activation for both GMax and GMed. There was no significant difference between GMax activation across the three exercises. Significant differences occurred between step-up and squat for GMed. No significant gender difference existed in magnitude of muscle activation during the three exercises.All exercises produced mean EMG amplitudes less than 30% MVIC. RSU had the highest recruitment of R GMed (20.72Step-up was the most effective exercise for activating gluteal muscles. Our results suggest that these WB exercises may be beneficial for gluteal muscle endurance, rather than strength, training. These findings will be used to compare against activation levels for people with hip OA.


Olfactory circuits in both insects and rodents are wired according to a pattern of convergence in which axons from olfactory sensory neurons expressing a single receptor type (SRT) project into the same glomerulus. This permits the representation of individual olfactory receptors in a topographical map in the brain (, 2], , [


The relationship between histological chorioamnionitis and haematological and biochemical markers in mothers and infants at delivery, and in infants postnatally, is incompletely characterised. These markers are widely used in the diagnosis of maternal and neonatal infection. Our objective was to investigate the effects of histological chorioamnionitis (HCA) on haematological and biochemical inflammatory markers in mothers and infants at delivery, and in infants post-delivery.Two hundred and forty seven mothers, delivering 325 infants, were recruited at the only tertiary perinatal centre in Western Australia. Placentae were assessed for evidence of HCA using a semi-quantitative scoring system. Maternal high sensitivity C-reactive protein (hsCRP), procalcitonin, and umbilical cord hsCRP, procalcitonin, white cell count and absolute neutrophil count were measured at delivery. In infants where sepsis was clinically suspected, postnatal CRP, white cell count and absolute neutrophil count were measured up to 48 hours of age. The effect of HCA on maternal, cord and neonatal markers was evaluated by multivariable regression analysis.P<0.001). Histological chorioamnionitis was associated with higher umbilical cord hsCRP (75th percentile 2.91 mg/L (range 0–63.9) versus 75th percentile 0 mg/L (0–45.6); P<0.001) and procalcitonin (median 0.293 (range 0.05–27.37) versus median 0.064 (range 0.01–5.24) ug/L; P<0.001), with a sustained increase in neonatal absolute neutrophil count (median 4.5 (0.1–26.4)×109/L versus 3.0 (0.1–17.8)×109/L), and CRP up to 48 hours post-partum (median 10 versus 6.5 mg/L) (P<0.05 for each).The median gestational age was 34 weeks and HCA was present in 26 of 247 (10.5%) placentae. Mothers whose pregnancies were complicated by HCA had higher hsCRP (median 26 (range 2–107) versus 5.6 (0–108) mg/L; Histological chorioamnionitis is associated with modest systemic inflammation in maternal and cord blood. These systemic changes may increase postnatally, potentially undermining their utility in the diagnosis of early-onset neonatal infection. Intrauterine infection and inflammation (IUI) is an important determinant of spontaneous preterm birth Intrauterine infection and inflammation is most accurately diagnosed by histological examination of the placenta. Histological chorioamnionitis (HCA) is often asymptomatic and clinical signs, such as fever, uterine tenderness, maternal or fetal tachycardia and malodorous amniotic fluid, lack both sensitivity and specificity; over one third of women with these signs do not have histological evidence of placental inflammation C-reactive protein (CRP), procalcitonin (PCT) and absolute neutrophil count (ANC) are biomarkers that may be used as adjunctive tests in the diagnosis of inflammation, sepsis and infection. CRP is an acute phase reactant produced by the liver in response to pro-inflammatory cytokines, such as interleukin (IL)-6. Plasma levels increase 12–24 hours after the onset of inflammation, when diagnostic clinical signs may be non-specific, and remains elevated until after stimulus resolves Procalcitonin (PCT) may have advantages over CRP as a marker for inflammation or sepsis. PCT concentrations increase more rapidly than CRP (within 2–4 hours) in response to systemic inflammation or infection, remain elevated during the inflammatory process and rapidly return to baseline on resolution of the stimulus Most data on inflammatory biomarkers in HCA relate to C-reactive protein (CRP) and routine haematological parameters; there is less experience with newer markers such as PCT, especially in the maternal circulation The extent to which HCA may confound these diagnostic markers of neonatal sepsis is unknown, but HCA may affect inflammatory markers several days postpartum We hypothesised that HCA generates inflammatory changes that may be detected in the mother at the time of delivery and in the neonate up to 48 hours following delivery. Our aim was therefore to investigate the association of HCA and CRP, PCT and haematological parameters in the mother and baby at the time of delivery, and in the neonate up to 48 hours following delivery. We show that HCA has is associated with inflammatory responses in both the maternal circulation and cord blood and these may persist in the neonatal circulation postnatally. The presence of HCA should be considered when interpreting inflammatory indices in newborn preterm infants.This was a cross-sectional study of mother-infant pairs recruited at the only tertiary perinatal center  in Western Australia , between August 2003 and January 2006. We aimed to recruit women in preterm labour and those delivering at term in a ratio of approximately two to one. We enrolled women at deliveries attended by dedicated study personnel and consequently the study population was over-represented with multiple pregnancies and deliveries by caesarean section and under-represented with spontaneous vaginal deliveries of extreme preterm infants, whose deliveries frequently occur out-of-hours, when study personnel were unavailable. We excluded infants with major congenital abnormalities and mothers whose understanding of English did not allow fully informed consent. The study was approved by the King Edward Memorial Hospital for Women institutional ethics committee. All the subjects who were included in this study provided informed written consent.in utero ultrasound before 20 weeks, or in the absence of antenatal data, the Ballard score at delivery Gestational age was estimated from date of the last menstrual period, Demographic and clinical data were collected at delivery. Maternal data included age, ethnicity, reported smoking, parity, pregnancy complications, premature rupture of membranes, length of labour, intrapartum fever, antibiotic exposure, antenatal steroids, and mode of delivery. Clinical chorioamnionitis was defined by the presence of at least two of the following features: intrapartum fever, uterine tenderness, maternal or fetal tachycardia, or malodorous amniotic fluid. Neonatal data included gestational age, sex, birth weight, head circumference, length, 1, 5 and 10 minute Apgar scores, ventilatory support, neonatal sepsis  measurements. Placental specimens were examined by an experienced perinatal histopathologist, blinded to clinical and laboratory data. Tissue samples of HCA cases were cultured using standard media  for aerobic bacteria, selective agar for mycoplasma and ureaplasma, and selective agar and enrichment broth for listeria.Umbilical cord blood was collected in ethylene diamine tetra-acetic acid for full blood count and automated differential and lithium-heparin gel for plasma assays. Neonatal peripheral blood samples were collected when sepsis was clinically suspected by one or more suggestive clinical features: temperature instability, apnoea, bradycardia, or cardiovascular instability High sensitivity (hs)CRP assay was used to measure CRP in the maternal and cord blood samples as it was hypothesised that the effect of HCA on CRP would be modest and may not be detected using he conventional clinical assay, which has a lower limit of detection of 3–7 mg/L, depending on the assay used. High sensitivity CRP was measured by latex immunonephelometry . The lower limit of detection was 0.15 mg/L, with values <0.15 mg/mL set to zero for statistical analysis. Procalcitonin was measured by the BRAHMS PCT sensitive immunoluminometric assay on a Berthold Technologies Lumat LB9507 luminometer . The lower limit of detection was 0.01 ug/L and all samples had a value greater than this. The intra-assay co-efficient of variation was 4.8% and inter-assay co-efficient of variation was 5.6%. Cord and neonatal full blood counts were measured using a Beckman Coulter HmX analyzer .Postnatal CRP was measured only in neonates where clinically indicated up to the first 48 hours following delivery. For these samples, CRP was measured by enzymatic sandwich immunoassay using VITROS Chemistry Products CRP Slides . Until May 2005, the lower limit of detection for the assay was 7 mg/L. After this time, a more sensitive slide technique was used (lower limit of detection 3 mg/L). C-reactive protein values less than the lower limit of detection were analysed in two ways; with all values below the lower limit of detection set to 6.5 mg/L and 2.5 mg/L, respectively. The intra-assay co-efficient of variation was 3.1% and inter-assay co-efficient of variation was 2.5%. The maximum CRP value was the highest value for an infant within the first 48 h post-delivery, even if this was the only available CRP value.Continuous data were summarised using non-parametric statistics: medians, interquartile ranges (IQR) and ranges (R), and Mann-Whitney U tests were used to compare groups. Categorical data were summarised using frequency distributions. Pearson’s chi-square tests or Fisher’s exact test were used to compare frequency distributions between groups.2) was used to measure the proportion of variance explained by variables in the linear regression models. As there was no difference in parameters for multiple or singleton pregnancies, these were analysed together (data not shown). For all analyses, P<0.05 was considered statistically significant. Data was analysed using SPSS statistical software .Linear correlation and linear regression were used to explore the effect of HCA on maternal, cord and neonatal outcomes. Given the likely correlation between maternal and neonatal factors, analysis for maternal and neonatal outcomes used the number of infants delivered as the overall denominator. Regression models for maternal outcomes were adjusted for co-existing conditions associated with maternal inflammation; duration of PROM, intrapartum fever (>38°C), or duration of labour before delivery. Regression models for neonatal outcomes were adjusted for factors associated with elevated cytokine levels; gestational age, ‘small for gestational age’  A total of 343 mothers, delivering 421 infants, satisfied inclusion criteria, of whom 247 mothers and their 325 infants had placental samples; analysis was therefore based on these deliveries. Two hundred and twenty two (68%) neonates were delivered preterm, 90 (28%) at 28–32 weeks gestational age, and 19 (6%) <28 weeks gestational age. The median gestational age was 34 weeks (range 24–42 weeks). Approximately two thirds were delivered by elective caesarean section , and one third  were multiple pregnancies.Mycoplasma hominis (n = 5), Ureaplasma urealyticum (n = 2), and mixed bacterial species (n = 4)  had HCA with a maternal response graded more than 1, and/or a fetal response  (n = 4) .P = 0.004), and maternal self-reported smoking (10/26 (39%) versus 42/299 (14%); P = 0.001) (P<0.001). Fifteen of 26 (58%) HCA cases had spontaneous vaginal deliveries, compared to 72/299 (25%) of cases without HCA (P = 0.001). Antenatal steroids were more prevalent in mothers with HCA (15/26 (58%) versus 95/299 (32%); P = 0.009.Univariate comparison of mothers with (n = 26) and without HCA (n = 299) showed that HCA was associated with Aboriginal or Torres Strait Island descent (6/26 (23%) versus 20/299 (7%); = 0.001) . Seven (P = 0.028) and had a lower gestational age (median 30 (range 23–40) weeks versus 34 (26–42) weeks, respectively, P<0.001). More HCA cases were born at 28–32 weeks GA (7/26 (27%) versus 12/299 (4%); P<0.001), and <28 week GA (11/26 (42%) versus 79/299 (27%); P<0.001) (Newborns born after HCA-affected pregnancies were more likely to be preterm (23/26 (89%) versus 199/299 (68%); P<0.001) . The onlP<0.001), but there was no difference in median PCT . Multivariable linear regression, adjusting for duration of prolonged rupture of membranes, length of labour and intrapartum fever showed a significant association between HCA and maternal hsCRP (P<0.001), and maternal hsCRP was significantly higher in mothers with HCA compared to those without HCA . There was no statistically significant relationship between HCA and maternal PCT.Maternal hsCRP level was significantly higher in mothers with HCA  versus 5.6  mg/L; P<0.001), as was hsCRP  mg/L versus . For cord PCT, multivariable linear regression showed gestational age, being small for gestational age, and length of labour accounted for 30.7% of PCT variation, with HCA contributing a further 10.5% variance; HCA was a significant variable in the model (P = 0.001). For cord hsCRP, gestational age, small for gestational age and length of labour accounted for 3.7% of the increase in cord hsCRP and HCA contributed a further 9% variance; HCA was a significant variable in the model (P<0.001).The median cord PCT level was significantly higher in subjects with HCA  ug/L versus 0.064  ug/L; 9/L) than in those without HCA (2.0 (range 0.9–4.2)×109/L, P = NS). Gestational age, small for gestational age, length of labour, and antenatal steroid exposure affected white blood count and absolute neutrophil count, and the relationship between HCA and neonatal white blood count and absolute neutrophil count was further examined using multivariable linear regression. For neonatal white cell count, these factors accounted for 8.6% of variation, with HCA contributing a further 7.1%; HCA was significant in the regression model (P<0.001). For neonatal absolute neutrophil count, the same variables accounted for 23.8% of variation; HCA contributed a further 3.4% and remained significant (P = 0.002).No significant differences were observed between cord haematological parameters in subjects with and without HCA . NeonataC-reactive protein was measured in the peripheral blood of 217 neonates . Twenty-P<0.001). Analysis of the timing of maximal postnatal CRP in neonates exposed to HCA showed a significant relationship between HCA and maximal CRP at Day 1 (P<0.001) and Day 2 (P = 0.03), but no significant relationship with maximal CRP levels at birth.These findings highlight the possible relationship between HCA and inflammatory and haematological markers in the maternal, cord and neonatal circulation. In the majority of mothers, HCA was asymptomatic and associated with a modest increase in hsCRP, but not in PCT levels. In umbilical cord blood, HCA was associated with increased PCT and hsCRP, but did not affect cord white cell count or absolute neutrophil count. However, there was a significant increase in inflammatory and haematological markers in infants exposed to HCA without early onset sepsis, peaking at 24 h of age. Histological choriomanionitis may therefore result in sustained inflammatory changes in the neonatal circulation, in the absence of microbiologically diagnosed infection.The observation of higher CRP but no difference in PCT in the mothers with HCA is in keeping with many previous studies, although data are not uniformly consistent and most studies are on pregnancies complicated by prolonged rupture of membranes Perinatal inflammation is associated with increased umbilical cord inflammatory cytokines including interleukin (IL)-6 and acute phase reactants such as CRP Procalcitonin, like CRP, is regulated by cytokines that increase early in HCA Histological chorioamnionitis was associated with increased absolute neutrophil in the first 48 h of life, consistent with other data The strengths of our study include quantification of HCA by an experienced perinatal histopathologist, blinded to outcomes and analysis of parameters commonly used in clinical practice in maternal, fetal and post-natal samples. We acknowledge some limitations, most importantly the non-representative nature of our sample, which reflected the resources available to recruit mothers and infants and to process samples. We therefore enrolled a relative over-representation of moderately preterm infants and of those born by caesarean section. Therefore, and despite the overall sample size, there were fewer cases of HCA than we have previously observed in a more preterm cohort drawn from the same population Histological chorioamnionitis has differential effects on maternal and cord/neonatal inflammatory markers. In the newborn exposed to HCA, CRP, white cell count and absolute neutrophil count are increased when compared to newborns without HCA. This may confound the interpretation of these common diagnostic markers of early neonatal sepsis. Further investigations in larger unselected populations of preterm infants, with serial measurement for a longer period of hsCRP and PCT, irrespective of suspected sepsis, would be informative about the effects of HCA on postnatal inflammatory markers and would refine the use of plasma and haematological markers in the diagnosis of neonatal infection.Table S1Cord and neonatal haematological parameters in subjects with and without histological chorioamnionitis.(DOCX)Click here for additional data file.Table S2Availability of neonatal CRP measures within the first 48 hours of delivery.(DOCX)Click here for additional data file.


Hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels underlie these currents and are composed of homo- and hetero-tetramers of HCN channel subunits (HCN1–4), which confer distinct biophysical properties on the channel. Despite understanding the structure–function relationships of HCN channels with different subunit stoichiometry, our knowledge of their expression in defined neuronal populations remains limited. Recently, we have shown that HCN subunit expression is a feature of a specific population of dorsal horn interneurons that exhibit high-frequency AP discharge. Here we expand on this observation and use neuroanatomical markers to first identify well-characterised neuronal populations in the lumbar spinal cord and hippocampus and subsequently determine whether HCN4 expression correlates with high-frequency AP discharge in these populations. In the spinal cord, HCN4 is expressed in several putative inhibitory interneuron populations including parvalbumin (PV)-expressing islet cells , in addition to all putative Renshaw cells and Ia inhibitory interneurons. Similarly, virtually all PV-expressing cells in the hippocampal CA1 subfield  and the dentate gyrus  also express HCN4. This HCN4 expression profile in inhibitory interneurons mirrors both the prevalence of hI sub-threshold currents and high-frequency AP discharge. Our findings indicate that HCN4 subunits are expressed in several populations of spinal and hippocampal interneurons, which are known to express both hI sub-threshold currents and exhibit high-frequency AP discharge. As HCN channel function plays a critical role in pain perception, learning and memory, and sleep as well as the pathogenesis of several neurological diseases, these findings provide important insights into the identity and neurochemical status of cells that could underlie such conditions.Hyperpolarisation-activated ( For example, the discharge responses of rodent spinal dorsal horn neurons to depolarising current injection is highly variable and can be described as tonic firing, initial bursting, delayed firing or single spiking . Importacurrents . Hyperpote cells . Four gete cells . Importate cells . More rete cells .hI currents and pacemaker activity in neurons have focused principally on HCN1 and HCN2 subunits despite both HCN3 and HCN4 also being critical for establishing rhythmic firing in certain neuronal populations  Act 1986.A total of nine adult male Wistar rats  were deeply anaesthetised with pentabarbitone and perfused transcardially with 4% depolymerised formaldehyde. The brain and lumbar spinal cord were removed and post-fixed in the same solution for an additional 2 h. Spinal cord  and hippocampal sections  were cut on a Vibratome (60-μm thick) and subsequently incubated in 50% ethanol for 30 min to enhance antibody penetration.Free-floating spinal cord and hippocampal sections were incubated in either goat anti-parvalbumin (PV), rabbit anti-calbindin (CB) and mouse anti-HCN4 or goat anti-PV, rabbit anti-PKCγ and mouse anti-HCN4 for 72 h. All of these antibodies are available from commercial sources, are widely used and have been fully characterised  and goat anti-choline acetyltransferase (ChAT) before detecting immunolabelling for these markers using secondary antibodies conjugated to Alexa 488 and Cy5 respectively. All primary and secondary antibody cocktails were made up in 0.3 M phosphate-buffered saline with 0.3% Triton X-100. Sections were incubated in primary antibodies for 72 h and in secondary antibodies for 12–18 h.hI-type subthreshold currents and are therefore likely to express HCN channels . Image stacks from different regions of the spinal cord and hippocampus were collected using different powered objectives to (i) map the general distribution of HCN4-immunolabelling in spinal cord and hippocampal sections, or (ii) determine the expression of HCN4-immunolabeling in neurochemically defined populations. Montages of tiled images scanned with a ×20 lens were generated using Zen software  to illustrate patterns of HCN4-immunolabelling. HCN4 expression patterns in neurochemically defined populations was determined from image stacks taken with either ×40, ×60 or ×63 oil immersion lenses (0.3–0.5 μm z-separation) and analysed using Neurolucida for Confocal software  or Image J. Due to the punctuate nature of HCN4 labelling in some populations, the presence/absence of immunolabelling in individual cells was determined by analysing several optical sections from each cell.HCN4 immunolabelling in the spinal cord is found principally in the spinal grey matter, with some very weak and diffuse staining in the white matter a, b. In n = 43 and 63, respectively), none of the motor neurons analysed were immunolabelled for HCN4 (n = 102). These results are summarised in Most PV-IR cells were found in laminae IIi and III as reported previously . A totalstratum oriens, at the border of the stratum oriens and alveus, in the stratum pyramidale and in the cell body layer of the dentate gyrus  were immunopositive for HCN4. More specifically, in the stratum pyramidale 190 out of 204 PV-IR cells expressed HCN4 , compared to 175 out of 185  in the stratum oriens and six out of seven  in the stratum radiatum. Only one PV-IR cell was found in the stratum lacunosum-moleculare, however this cell also expressed HCN4. CB-expressing cells are found principally in the stratum pyramidale, with only occasional cells in other layers. A total of 643 CB-IR cells were analysed from CA1, of which only 15  were immunopositive for HCN4. More specifically, in the stratum pyramidale none of the 495 CB-IR cells expressed HCN4, compared to seven out of 80  in the stratum oriens and six out of 51  in the stratum radiatum. Of 17 CB-IR cells seen in the stratum lacunosum-moleculare, two expressed HCN4 . In the dentate gyrus, a total of 219 PV-IR cells were analysed from the granule cell and polymorphic layer, of which 199  were immunopositive for HCN4  populations of neurons also express HCNs in these regions. The selected populations have been implicated in specific functional roles within discrete neuronal circuits and, in light of recent reports implicating HCN channelopathies in neurological diseases such as epilepsy and pathological pain , warrantAlthough the terminology used to describe neuronal firing patterns in several regions of the CNS is often similar (e.g. fast spiking), the actual rates of AP discharge within physiologically defined groups from different anatomical areas vary considerably as illustrated in AI currents and have radial, vertical or central cell-like morphology  of PV-IR cells in this region are known to express both GABA and glycine (hI currents (hI currents (hI current in motor neurons. These differences in HCN4 expression are likely to reflect functional disparity in the various physiological properties of these channels between specific populations of cells, further highlighting the need to correlate channel composition and stoichiometry in individual cell types (In the spinal dorsal horn, combined electrophysiological and neuroanatomical studies have previously described relationships between morphology, neurotransmitter phenotype and firing patterns in dorsal horn neurons . Excitatrphology . In cont glycine , have is glycine  and are currents . We havecurrents . Similarcurrents . Interescurrents , suggestll types .stratum oriens and at the stratum oriens-alveus border and these also tend to have hI currents and fast-spiking discharge patterns (hI currents and fast-spiking discharge patterns in basket cells in these two regions (HCN4 expression also appeared to be a faithful marker of fast-spiking interneurons in both the CA1 and dentate gyrus of the hippocampus. Virtually all PV-expressing cells in the CA1 and dentate gyrus express GABA or its synthetic enzyme glutamic acid decarboxylase . Most ofpatterns . In the patterns , all of patterns . In contpatterns . Our fin regions .HCN4-expression in all immunolabelled cells is detectable in both the cell membrane and the cytoplasm. The cytoplasmic expression of HCN4-immunolabelling in hippocampal neurons is stronger than that observed in spinal interneurons, however the significance of this observation is unclear. HCN4 expression in the cell membrane also differs between cells and is likely to reflect subtle differences in the stoichiometry of channels in each subpopulation. For example, the intensity of HCN4-immunolabelling in the soma of PV-expressing cells in the spinal dorsal horn varies , whereashI currents in specific cell populations (hI currents. Although hI currents have been observed in both motor neurons and CA1 pyramidal cells (hI currents have been shown to influence both spike-firing probability and AP discharge (hI currents (hI in these regions (The four known HCN channel subunits display distinct biophysical properties, are activated at different membrane potentials and have different channel activation kinetics. These factors contribute to the variability of ulations . The expal cells , our finischarge . Furthercurrents , howevercurrents . Regardicurrents . In contcurrents . To furt regions .hI currents, exhibit fast-spiking AP discharge and express both HCN1 and HCN4 subunits (HCN channels have been implicated in both acute and chronic pain as well as a number of neurological diseases . We havesubunits . Althougsubunits . As chansubunits , it remasubunits , while Hsubunits . Given tsubunits .