Even though nanometer size resolution information was not required, the large signal enhancement of TERS was employed here to detect a monolayer coverage of weakly scattering analytes that were not detectable with normal Raman spectroscopy, emphasizing the usefulness of TERS.Full spectroscopic imaging by means of tip-enhanced Raman spectroscopy (TERS) was used to measure the distribution of two isomeric thiols (2-mercaptopyridine (2-PySH) and 4-mercaptopyridine (4-PySH)) in a self-assembled monolayer (SAM) on a gold surface. From a patterned sample created by microcontact printing, an image with full spectral information in every pixel was acquired. The spectroscopic data is in good agreement with the expected molecular distribution on the sample surface due to the microcontact printing process. Using specific marker bands at 1000 cm The chemical characterization of surface adsorbates is of great interest in several areas of research. The composition of biological membranes or of artificially structured surfaces, used in molecular electronics, determines their properties as well as their function. However, characterization is difficult due to the small size and the low number of the molecules that comprise these structures. Most techniques such as nuclear magnetic resonance (NMR), infrared (IR) spectroscopy and Raman spectroscopy (RS) lack the necessary spatial resolution, while others such as scanning tunneling microscopy (STM) or scanning electron microscopy (SEM) do not provide enough chemical information. Furthermore, the limited quantity of analyte results in weak signals rendering characterization even more difficult. Ideally, information should be gathered with minimal disturbance of the molecules, which rules out any kind of labeling, and also emphasizes the need for an ambient pressure technique.Tip-enhanced Raman spectroscopy (TERS) has been developed to obtain chemical information with very high spatial resolution –4, or chThiols are used for several purposes. They can form a very thin protective layer on metal surfaces  or can bTo produce patterned SAM structures on a gold surface, microcontact printing is the tool of choice. The technique originated in the lab of Whitesides in 1993  and provFor our experiments we chose 2- and 4-mercaptopyridine (2-PySH and 4-PySH), which have been used to modify electrode surfaces in protein electrochemistry . In a no2 [Previous studies on self-assembled thiol films were conducted using AFM , STM 1515,24–25,2 . As showIn this article, we show that TERS can be used to image chemically heterogeneous surfaces without the need for labeling, even when the different adsorbates have very similar properties, i.e., a monolayer consisting of both 2-PySH and 4-PySH. With TERS, both isomers could be localized on the surface by their spectroscopic signature and approximate information on the surface coverage could be obtained from the intensity of characteristic marker bands within the spectra. Due to the strong enhancement, this could even be done with a (sub-)monolayer of non-resonant molecules. Similarly to AFM and STM, TERS images could be acquired with different step sizes to gather information about objects or structures on the nanometer or micrometer scale.Flat template-stripped gold thin films with a thickness of 60–200 nm on glass were used as substrates for the experiments and functionalized with thiols using micro contact printing . In a first step, the efficiency and coherence of the transferred 2-PySH thiol monolayer was investigated using AFM. Attempts to spectroscopically visualize the thiol layer with confocal Raman spectroscopy failed due to the intrinsically weak cross section of the Raman process and the small number of molecules forming the investigated monolayer. No typical Raman bands were seen during excitation for 6 × 10 s at 3 mW by a 632.8 nm laser in confocal measurements. The absence of signals from decomposition products  leads to the conclusion that the SAM was not destroyed by the high laser power, but that the intensity of Raman signals from the intact monolayer was too weak to be detected.In experiments on Ag SERS substrates , the printed patterns could not be detected and localized by confocal RS. Either no Raman signals at all or homogeneous signals from all over the substrate were detected (data not shown). Due to the strong but inhomogeneous enhancement by single sites on a typical SERS substrate, it is most likely that a very small fraction of molecules diffusing on the Ag surface during production of the samples dominated the spectra, preventing a localization of the molecules. Another possible explanation is that the roughness of the SERS substrate interfered with the patterning process used compare .2, with 156 nm/pixel, and full spectral information at every pixel, was acquired with a 632.8 nm laser at a power of 300 μW and an acquisition time of 2 s per spectrum. −1 indicating the presence of 2-PySH on the surface. The circular structures from the microcontact printing can be seen clearly and distinguished from the pure metal background. The experiment did not destroy the thiol on the surface due to the low laser power and the non-contact nature of the STM. The AFM phase image in Results of a tip-enhanced Raman experiment are presented in A microcontact printed 2-PySH surface was then incubated in a 10 mM ethanolic solution of 4-PySH for 10 s, in order to fill the bare gold surfaces between the covered areas with 4-PySH. The result was a mixed monolayer of two thiols, with very similar properties, distributed on the surface in a well-defined pattern. The AFM (a) phase and (b) topography images in −1 and 1100 cm−1, respectively. To exclude the possibility that changes in enhancement during the experiment are responsible for the contrast , the ratio between the two marker bands is also shown in −1 from the strongest band of 4-PySH. Considering that during preparation the entire sample is immersed in 4-PySH to fill the gaps from the microcontact printing, a certain amount of 4-PySH is expected to be embedded in the area printed with 2-PySH. Additionally, the 60 s confocal background spectrum (navy blue) from the 2-PySH thiol monolayer shows that the Raman signal of the monolayer is too weak to be picked up by a confocal measurement.One possible but very tedious way to distinguish the two thiols would have been to use high resolution STM  to search for typical molecular patterns in the SAM structure. However, both thiols can assemble in several different structures ,24 and a5–106.Full spectral imaging using TERS can be used to visualize the distribution of two very similar non-resonant thiols within a single monolayer on a gold film. Two isomeric thiol species were differentiated and localized on the sample surface using their spectroscopic signatures. The investigation of monolayers could be useful in the analysis of catalytic processes in heterogeneous catalysis , where tAll spectra were acquired by a combined AFM/STM connected to a quadruple grating Raman spectrometer  coupled to an EMCCD . This top-illumination TERS setup has been described in detail in .−5 mbar, with gold  evaporated by resistive heating at a rate ≤ 0.1 nm/s. The deposited gold film was bonded to clean microscope slide fragments using NOA61 . The gold films were mechanically stripped from the Si wafers and used immediately.Template-stripped gold films were created using a similar method to that described in , by coat2 STM scan in STM was used to characterize the properties of the surface before sample preparation. The 10 × 10 μmMicrocontact printing can be used to transfer a monolayer of thiols onto a noble metal substrate as described in –37. A siThe master for microcontact printing stamps was fabricated by standard photolithography ,38–39. BFor microcontact printing, a droplet of a 10 mM ethanolic solution of 2-PySH was placed on the stamp for 1 min, and washed with copious amounts of ethanol. The pattern was then printed onto the gold film by placing the stamp with the pressure of its own weight onto the gold film for 10 s followed by careful lift-off.The filling of the bare gold areas by 4-PySH was achieved by covering the entire printed gold film with a 10 mM ethanolic solution for 10 s followed by thorough cleaning with ethanol.TERS tips were fabricated by electrochemical etching of silver tips  similarly to ,40 in a For the TERS experiments, tips were carefully approached to the sample and checked for Raman activity using a 632.8 nm Helium–Neon laser. Selected tips were then aligned using the laser scanning mirrors  and subs


The dropout rate was high with 19 participants completing the treatments and 12 remaining at followup. One participant from the SSEAP group had confirmed cessation at week 8 and end of followup (5%), but there was no difference between groups for confirmed cessation or NWS. Adverse events were few and minor.This study investigated the efficacy and safety of ear acupressure (EAP) as a stand-alone intervention for smoking cessation and the feasibility of this study design. Adult smokers were randomised to receive EAP specific for smoking cessation (SSEAP) or a nonspecific EAP (NSEAP) intervention which is not typically used for smoking cessation. Participants received 8 weekly treatments and were requested to press the five pellets taped to one ear at least three times daily. Participants were followed up for three months. Primary outcome measures were a 7-day point-prevalence cessation rate confirmed by exhaled carbon monoxide and relief of nicotine withdrawal symptoms (NWS). Intention-to-treat analysis was applied. Forty-three adult smokers were randomly assigned to SSEAP ( Smoking is the largest single preventable cause of death and disease worldwide . It is eVarious forms of NRT are often combined with behavioural support to assist smoking cessation . Other pn = 141) compared a combination of true acupuncture and education versus sham acupuncture plus education and reported a higher cessation rate for true acupuncture which was still evident at a 3-month followup [n = 38) with sham ear plus body acupressure (n = 32) in active smokers for 3 weeks with a followup at 3 months. Both groups reduced smoking but there were no significant differences between groups [n = 59) showed a significant decrease in NWS [Positive results have been reported for the use of ear acupuncture/acupressure for smoking cessation. One study  was done on 5 points as recommended in the literature for smoking cessation and used in previous trials (see Protocol ). The coACTRN12611000761965), and a protocol has been accepted for publication [The study was conducted at RMIT University, Australia, approved by the RMIT Human Research Ethics Committee, and registered with the Australian New Zealand Clinical Trials Registry  group or the nonspecific ear acupressure (NSEAP) group. Participants selected a quit date at the first treatment which had to be before the third treatment.During the 8-week treatment, participants received either SSEAP or NSEAP once per week. All participants received stainless-steel press-pellet tapes  on five EAP points. Each pellet was 1.2 mm in diameter and attached to a round adhesive tape (7 mm in diameter) in a tan colour. The skin was cleaned and sterilised with a 70% alcoholic swab. Then, pellets were taped onto one of the participants' ears with no skin penetration. Then, the acupuncturist pressed each pellet for 10 seconds or until the ear became reddish and/or slightly sore. Participants were instructed on how to press the pellets and were requested to press all pellets three times daily throughout the week and whenever they felt a craving for cigarettes. At the subsequent visit the remaining pellets were removed by the acupuncturist and new pellets were placed on the opposite ear. Consultations lasted approximately for 10 minutes.In the SSEAP group the following EAP points were used: Shenmen (TF4), Fei “lung” (CO14), Kou “mouth” (CO1), Hunger (extra), and Gan “liver” (CO12) , 24. TheThe Fagerstrom Test for Nicotine Dependence was administered at baseline to assess the equivalence of groups . To assePrimary outcome 1 was smoking cessation. Successful cessation was defined as a 7-day point prevalence (no smoking over 7 consecutive days)  measuredPrimary outcome 2 was NWS assessed using the Mood and Physical Symptoms Scale (MPSS) in which participants rate the severity of depression, irritability, restlessness, hunger, poor concentration, anxiety, insomnia, frequency and levels of smoking urges, and physical symptoms such as mouth sores, constipation, and cough or sore throat . All queSecondary outcome measures included (1) NRT usage throughout the trial; (2) daily consumption of cigarettes; (3) body weight (measured each time the participant was on site); and (4) quality of life using the Short-Form 36v2 Health Survey  at initial assessment, end of treatment, and end of followup periods.All participants were asked to record in their CRFs any unpleasant experience throughout the trial and to assess whether the AE was related to the EAP. Participants answered a question on the CRF at the end of the first and the last treatment weeks regarding which group they perceived that they had been allocated into.Statistical Analysis. Statistical Package for the Social Sciences software version 18.0  was used for analyses. Quality Metric Health Outcomes Scoring Software 4.0 was used for SF36 scores . Intention-to-treat analysis was applied with missing data being managed using expectation maximisation. Baseline characteristics of the groups were assessed using unpaired t-tests and χ2 tests for equivalence. For primary and secondary outcome measures, unpaired t-tests or general linear model (GLM) was used as appropriate to compare group differences. The randomisation code was broken after the analysis of outcome measures was completed.Ninety-five (95) smokers were screened for eligibility for this study, eighteen did not meet inclusion criteria, 10 declined to participate, 12 were unable to participate due to various reasons , and 12 participants were not contactable after the screening process .n = 20) or the NSEAP group (n = 23). At the end of treatment week 8, 19 participants remained in the study and 12 remained till the end of followup. Thirteen dropouts gave no reason and were lost to contact. For those who gave a reason (n = 18), the most common was “too much going on” (n = 8) followed by 5 who dropped out because of travel. Four people in the NSEAP group reported that the treatment was ineffective, and one person in this group gave discomfort with ear pellets as the reason for dropping out . By the end of treatment, the two confirmed quitters in the NSEAP group had dropped out and were lost to followup. At three-month followup, the one participant from the SSEAP group had continued to abstain from smoking (CO confirmed), so the cessation rates were 5% for SSEAP and 0% for NSEAP at end of treatment and at end of followup. A post hoc analysis using a CO threshold of 7 ppm, which was at the lower end of the manufacturer's recommended range for the Smokerlyser, excluded one quitter from the NSEAP group but did not change the overall result.During the treatment period, the CO-confirmed (10 ppm) 7-day-point prevalence cessation was three participants, one from the SSEAP group and two from the NSEAP group with no significant difference between groups  and Question  9 “magnitude of urges to smoke” . These two scores were used as covariates in a GLM analysis for these two questions to adjust to this difference. At the end of treatment and at the end of followup, there were no significant differences between the two groups for any of the NWS measured by MPSS.All MPSS scores were low at baseline. At baseline, there were significant differences between groups on the mean MPSS scores for Question  8 “frequency of urges to smoke” . At the end of followup, consumption had increased compared with the end of treatment (SSEAP 14.5 ± 4.57 versus NSEAP 17.76 ± 5.03), but both groups remained lower than their respective baselines. A small but significant difference was found between groups  at the end of followup.Based on participants' self-report, the mean daily cigarette consumption decreased from baseline to the end of treatment in both the SSEAP  and NSEAP  groups. The decreases were 30.0% and 35.5%, respectively. At the end of treatment, there was no significant difference between groups in cigarette consumption  or at the end of followup .The mean individual change in body weight compared to baseline was not significantly different between groups at the end of treatment  or mental component scores  of SF36v2 at end of treatment. Also, at the end of followup, no group differences were found on the physical  or mental component scores .There were no significant differences between the SSEAP and NSEAP groups on the physical  or at the end of treatment week 8 . Also, participant's belief regarding their group allocation did not significantly affect whether they dropped out or not.There was no significant difference between the two groups in participants' perception of group allocation at the end of the first treatment week (All AEs (1 event from one SSEAP participant and 5 from four NSEAP participants) were mild or moderate, but one participant dropped out for discomfort with the ear pellets. The most frequent AEs were mild to moderate discomfort on the ear (1 in SSEAP and 4 in NSEAP). Participants reported that these AEs were “probably due to the EAP.” One participant in the NSEAP group reported slight headache and dizziness (1 event). All AEs were resolved without any medical intervention.This study compared the effects of an SSEAP intervention to an NSEAP intervention in adult smokers who wished to stop smoking. We employed the 7-day point-prevalence method to calculate cessation rate. One participant from the SSEAP group (5%) and 2 participants from the NSEAP group had quit smoking (8.7%) by the end of treatment. However, the only confirmed quitter at the end of treatment and at 3 months of followup was the SSEAP participant, since the two NSEAP participants did not continue in the study and all dropouts were considered as smokers in the ITT analysis.Randomised trials of over-the-counter NRT patch versus placebo patch have reported successful quit rates of 5.6% to 15% in the treatment groups , 29–31. High dropout rates in smoking cessation studies are not unusual . Bier etn = 3) showed short-term increases in their scores for questions which measured cigarette cravings, whereas the known nonquitters showed little change. Therefore, change in the MPSS indicated the presence of individual cessation attempts at particular time points, so MPSS was found to be sensitive to changes in cigarette cravings. However, these changes were not detectable in the consolidated data because they lasted for short periods and occurred at different time points. In future trials a different approach to assessing NWS between groups should be considered.No differences in NWS were found between groups. The mean NWS scores were very low at baseline and throughout the trial. This could mean that the participants were not diligent in recording their NWS in the CRFs or that many participants did not make attempts to quit on their designated quit day. Post hoc investigation of the individual data found that the known quitters (n = 11), one participant reduced his/her daily cigarette consumption by 75% compared to baseline, two participants reduced it by 47%, and one participant reduced it by 42%. In the NSEAP group (n = 8), one participant reduced his/her daily cigarette consumption by 32%, while another participant achieved a reduction rate of 15%. It is interesting to note that although most participants did not quit smoking, those receiving the SSEAP treatment showed a greater reduction in the number of cigarettes they smoked during the treatment period and a less pronounced rebound during followup. Cutting down is a step towards complete cessation [There was an overall reduction in cigarette consumption. For those participants who completed the treatment period, in the SSEAP group  could have been perceived as onerous, and participants may not have carefully considered their responses to questions. Therefore, reduction in the number of secondary outcome measures may assist in compliance and may also provide better accuracy in reporting. No support or counselling was provided during the trial, although this was available to participants via helplines or their GPs. This aimed to isolate the effect of the EAP and not to introduce an additional source of variation, but future studies should use a more pragmatic intervention that includes a form of support provided by an independent person blind to treatment allocation.The ear points were selected based on existing literature and use in other clinical trials . In geneThis study followed a rigorous methodological protocol, and blinding of participants was successful. Consistent with previous acupressure studies, only minor adverse events were reported. Dropout rates were high in both groups. ITT analysis found that after 8 weeks of treatment, the 7-day point prevalence, CO-confirmed quit rate in the SSEAP group was 5%, and this continued at three months after treatment, but there was no significant differences between groups. Cigarette consumption declined in both groups over the treatment period. The decline appeared more pronounced in the SSEAP group and there was a small statistical difference at followup. NRT usage was low throughout. There was no apparent effect on NWS in either group, but the method used appears unsuitable for detecting between group changes. Future studies should address the design issues found in this trial and investigate continuous abstinence and long-term cessation rate at 6 months.


Screening and treating newly arriving immigrants for latent tuberculosis infection (LTBI) in low-incidence countries could be promising to reduce the tuberculosis incidence among this population. The effectiveness of screening with the tuberculin skin test (TST) is unknown.To estimate the risk of progression to tuberculosis within two years after entry, stratified by TST result at entry.In a case-base design, we determined the prevalence of TST positives (10 mm and 15 mm) among a representative cohort of immunocompetent immigrants (n = 643) aged ≥18 years who arrived between April 2009 and March 2011 in the Netherlands (base cohort). Immigrants who progressed to tuberculosis within two years after arrival in 2005, 2006 or 2007 were extracted from the Netherlands Tuberculosis Register (case source cohort). The prevalence of TST positives from the base cohort was projected on the case source cohort to estimate the risk of progression to active tuberculosis by using Bayesian analyses to adjust for the sensitivity of the TST and Poisson regression analyses to take into account the random error of the number of extracted cases.The prevalence of TST positives was 42% and 23% for a cut-off value of 10 mm and 15 mm, respectively. The overall risk of progression to tuberculosis if TST positive was 238 per 100,000 population (95% CI 151–343) and 295 per 100,000 population (95% CI 161–473) for a cut-off value of ≥10 mm and ≥15 mm, respectively. The corresponding risk for TST negatives was 19 (95% CI 0–59) and 58 (95% CI 25–103).The TST has the discriminatory ability to differentiate between individuals at low and high risk of disease. Mycobacterium TB (MTB), and the ones who belonged to a cluster  were often not epidemiologically linked In countries with a low-incidence of tuberculosis (TB), TB is primarily prevalent among first generation immigrants. In the Netherlands, 73% of the new patients with TB in 2010 were first generation immigrants, corresponding to an incidence of 45.6 per 100,000 persons, whereas this incidence was 1.6/100,000 for the native population All immigrants aged >12 years from high–incidence countries intending to stay more than three months in the Netherlands, currently only undergo a mandatory screening for TB by chest x-ray (CXR) Interferon gamma release assays (IGRAs) were developed to improve the diagnosis of LTBI. These are based on identifying cellular production of interferon gamma (IFN-γ) in response to MTB specific antigens and do therefore not react on BCG and most environmental mycobactaria. One of those IGRAs is the QuantiFERON®-TB Gold In-Tube (QFT-GIT). In a recent study we have shown that immigrants with a positive QFT-GIT at entry had a considerable higher risk of progression to active TB within two years compared to immigrants with a negative QFT-GIT at entry The effectiveness of using the TST in screening immigrants for LTBI is still unknown since it has never been used in the Dutch setting. Several studies found that, among immigrant contacts of TB patients, the TST and QFT-GIT were both indicative in predicting the risk of developing TB Ethical approval was obtained from the Netherlands Central Committee on Research Involving Subjects.This study is a post-hoc analysis of data derived from the study assessing the relationship between QFT-GIT result and risk of active TB in immigrants within two years of time of entry screening All immigrants aged ≥18 years who visited the PHS for their entry screening, reported to be immunocompetent and provided written informed consent, were eligible for enrolment in the base cohort. We excluded immigrants who were diagnosed with active TB within six months after entry, because in the NTR these are reported as detected at entry screening and not during follow-up. Regarding the sample size, in our previous study 1570 immigrants consented. Out of these, 643 received a TST. The sample of 643 gave us enough power to find a prevalence of TST positives of 30% with a precision of 7%, and to do stratified analyses . The TST was performed according to the Mantoux method with 0.1 ml purified protein derivative RT23 . Experienced medical staff read the induration in millimetres after 48–120 hours .To calculate the risk of progression to active TB per 100,000 population we used a Bayesian approach with non-informative priors. The numerator (number of cases identified from the case source cohort) was modelled using the Poisson distribution to allow for uncertainties in the number of identified patients. The denominator (number of immigrants with positive/negative TST) took into account differences in sensitivity of the TST reported in the published data The risk estimates of progression to active TB enabled us to calculate the numbers needed to treat (NNT), and the numbers needed to screen (NNS) to prevent one TB patient within two years, for TST ≥10 mm and TST ≥15 mm. We assumed a 60% efficacy rate of the preventive treatment 2. Baseline characteristics were sex, age , region of origin , incidence of TB in country of origin (<100 (low), 100–199 (intermediate) and ≥200 (high) per 100,000 population), BCG vaccination (yes if BCG-scar was present and/or participant indicated being vaccinated), previous diagnosis of TB (yes/no), smoking (yes if ever smoked/no) and time in the Netherlands before screening (yes if ≤3 months in the Netherlands before screening/no). Associations were considered statistically significant when p-values were ≤0.05. Statistical analyses were conducted using SPSS 17.0  and WinBUGS 1.4.3 .Eligible patients could have 1 of 3 reasons for not being included in the present study. First, they could not have consented to participate in the original study; second, they did participate but did not want to have a TST administered, or third, they had a TST administered but there was no available result (test failure or no return for test reading). Given the post-hoc analysis, there is a need to carefully check for selection bias at each of these instances. We therefore compared the baseline characteristics of the base cohort with the case source cohort, the non-consenters and the participants without a TST result by Pearson χDuring the study period, 2,569 immigrants were approached for study enrolment at the seven PHSs. Out of 1,570 consenters, 1,559 were eligible for TST testing since 11 were excluded because they were HIV-positive (n = 4), used immunosuppressives (n = 5), or were diagnosed with active TB within 6 months (n = 2) . In 753 Overall, 273 out of 643 (42%) were TST positive when the cut-off value was set at 10 mm and 145 (23%) when the cut-off value was set at 15 mm .In the case source cohort we identified 30 immigrants who developed active TB within two years. Ten cases were clustered, but none were epidemiologically linked to a TB patient in the Netherlands according to the registries. When the TST cut-off value was set at 10 mm, the expected number of TST positives in the case source cohort based on the prevalence found among the base cohort, was 11,173 (42%) . The medFor the cut-off value of 15 mm we estimated a sensitivity of 59% (95% CI 37%–82%). The overall risk of progression to TB for TST positives was 295 per 100.000 population (95% CI 161–473)  and 58 pFor both cut-off values of the TST, no marked differences in risk of progression to TB were estimated between the males and females., while minor differences were seen with respect to strata of age (15 mm) or TB-incidence in country of origin (10 mm and 15 mm)s , 4.The NNT and NNS to prevent one TB patient within two years after entry for a TST cut-off value of 10 mm were 700 and 1649, respectively. The corresponding NNT and NNS for a TST cut-off value of 15 mm were 565 and 2505, respectively.This study shows that the TST had the discriminatory ability to identify high and low risk groups for progression to active TB among newly arriving immigrants. This ability was irrespective of the cut-off value of 10 mm or 15 mm, but did not apply for the highest age group  and highest incident category with respect to country of origin . The estimated risks for TST positives to progress to TB within two years were considerably higher than the incidence of 50/100,000 which is used in the Netherlands as a cut-off value to define a risk group. Appropriate preventive interventions to lower the incidence of TB among newly arriving immigrants should therefore be undertaken.In a previous study Screening immigrants at entry will only be effective if positive test results are followed by adequate interventions like prescribing preventive treatment. The Dutch preventive treatment regimen is three months daily isoniazid plus rifampicin, or six months daily isoniazid. The duration and the risk of severe side effects, such as hepatotoxicity, influence adherence and could harm the efficacy of the treatment We did not observe marked differences in the risk of progression to disease for both TST-positives and TST-negatives between the strata of sex, age and incidence in country of origin. The overlapping credibility intervals are a result of the small number of immigrants who progressed to disease.There were several limitations of this study. Firstly, more than half of the immigrants eligible for TST testing refused. However, there were no marked differences in baseline characteristics between the participants and the ones who refused. The main reason for refusing was a lack of time for a second visit and the fact that it was not mandatory. This highlights that the willingness of immigrants to become screened by TST is limited. There is some controversy whether physician’s and patient’s adherence to guidelines is determined by the diagnostics used. Grinsdale et al. showed that contacts of culture-confirmed TB patients were more likely to complete evaluation and to complete isoniazid preventive therapy when tested with IGRA compared to the TST In conclusion, in this study we found that newly arriving immigrants with a positive TST result at entry were at considerable risk of progression to active TB, whereas for TST negatives this risk was limited. Nevertheless, the discriminatory ability of the TST was somewhat lower than that of the QFT-GIT. This suggests that the QFT-GIT, either used alone or in combination with the TST, is the favorable diagnostic tool for screening newly arriving immigrants coming to low-incidence countries. Further study is needed with respect to the cost-effectiveness for the TST and the QFT-GIT in the immigrant screening program to gain further evidence for considering screening immigrants for LTBI.Text S1Calculation of the risk of progression to tuberculosis.(DOC)Click here for additional data file.


The purpose of this study is to evaluate the functional outcome, union and complication rates after surgical treatment of unstable or displaced proximal humeral fractures using the Polarus intramedullary nail, by reviewing our institutional experience and the relevant current literature.Twenty-seven patients were treated operatively for proximal humeral fracture using the Polarus nail. Fractures were classified according to Neer’s classification. A number of parameters including patient demographics, mechanism of injury, operative time, time to union and complications were recorded. Functional outcome was evaluated using the Constant Shoulder Score. A comparison among functional outcomes in patients >60 years in relation to the younger ones was performed. Moreover, a review of the literature was carried out to evaluate the overall union and complication rates.p<0.05). From the literature review and from a total of 215 patients treated with a Polarus nail, the mean union rate was 95.8%, the overall reported complication rate, including both minor and major complications, ranged widely from 9.3% up to 70%.Two patients lost to follow-up were excluded from the analysis. For the twenty-five patients (mean age: 61 years), the mean follow-up was 36 months. There were 7 complications (28%), including one fixation failure, four protruded screws, one superficial infection and one case of impingement. The union rate was 96% (mean time to union: 4.2 months). The mean Constant score was 74.5 (range: 48–89). Patients under the age of 60 had a better functional outcome compared to patients >60 years of age (The Polarus nail was found to be an effective implant for stabilisation of proximal humeral fractures. Functional outcome is for the vast majority of the cases excellent or good, but in elderly patients a lower Constant score can be expected. Proximal humeral fractures account for 5% of all fractures and constitute the third most frequent fracture in elderly patients, with substantial economic effect on health care systems -4. The rNon-operative treatment of unstable or displaced proximal humeral fractures may result in malunion and stiffness of the shoulder ,5. DiffeThe Polarus intramedullary nail  is one of the available locked antegrade intramedullary (IM) devices for the surgical treatment of proximal humeral fractures and it allows screw stabilisation of the humeral head and tuberosities. There are scarce reports in the literature reporting on functional and radiological outcome and complication rate after its clinical use ,14-20.In the present study, our aim is to report on our clinical experience from the surgical treatment of unstable or displaced proximal humeral fractures using this intramedullary device. Moreover, a review of the current literature was carried out to evaluate the overall union rates and incidence of reported complications.From January 2007 to December 2009, all consecutive patients that were treated in our institution with the Polarus nail for stabilisation of fractures of the proximal humerus were included in this retrospective study. Institutional board approval was obtained. Using true anteroposterior (AP) and y-views radiographs of the shoulder and, when necessary, CT scan to accurately assess the fracture pattern (in cases of comminuted fracture patterns), fractures were classified according to Neer’s classification . InclusiIn all cases, a short Polarus nail was used. This device is a cannulated locked antegrade intramedullary humeral rod with a tapered profile to reduce distal stress concentration, and it has a spiral array of four 5-mm proximal screw-holes and two 3.5-mm distal interlocking holes ,14,16,17Passive physiotherapy was initiated from the 1st postoperative day, including pendulum motion and passive elevation and rotation. At the same time, all patients were encouraged to actively exercise the wrist and elbow joints. Subsequently, active assisted exercises after the 2nd week and active motion of the shoulder joint at 4 weeks was encouraged. Patients were trained in self-assisted shoulder abduction and elevation using the uninjured extremity as an assistant. Outpatient physiotherapy was initiated 2 weeks postoperatively. Exercises under resistance were restrained until the 6th postoperatively week or until fracture union was radiologically confirmed.Following discharge from the hospital, patients were followed up in the outpatient clinic at 2 and 6 weeks, and at 3, 6, 12 months and yearly thereafter (maximum 3 years) as indicated. Anteroposterior and axillary radiographs were taken at each follow-up in order to evaluate the progress in fracture union and the presence of implant related complications. The time to union as well as all minor or major complications and their management were documented. Numerous other parameters were recorded and analysed including patient demographics, mechanism of injury, associated injuries, days to surgery and operative time. The mean follow-up time was 36 months (range of 27–43).U-test. Statistical analysis was performed using SPSS version 13.00  and a p-value of 0.05 or less was considered as statistically significant.The functional outcome was assessed using the Constant shoulder scoring system  at the fFinally, the current literature was reviewed and all relevant studies reporting on the clinical use of this fixation method were collected. Data regarding total number and mean age of patients, fracture patterns, union, functional outcome and scoring system used as well as complications rate were documented and analysed.Institutional board approval was obtained from the local Trauma & Orthopaedic Directorate and the research carried out was in compliance with the Helsinki Declaration. Every patient consented to participate in this study for publication of this report and any accompanying images.Out of 27 patients that met the inclusion criteria, 2 patients were lost to follow up and therefore were excluded from the final analysis. From the remaining twenty-five patients, there were 18 women and 7 men, with a mean age of 50 years (range: 16–81) and 66 years (range: 33–92), respectively. The overall patients’ mean age was 61 years (range: 18–92). Mechanisms of injury included 18 simple falls, 5 pedestrian versus automobile impacts, and other causes in 2 cases. Three patients presented with other associated injuries as shown in Table The fractures were stabilised either acutely or after failure of conservative management in three cases Figures , 2 and 3The overall mean Constant score was 74.5 (range: 46–99). Fifteen patients (65%) had excellent results, eight patients 30%) had satisfactory results and two patients (5%) had poor results Table . Patient0% had saIn terms of complications Table , no majoFinally, the literature review revealed eight studies with a total of 215 patients that had been treated with the Polarus nail for proximal humeral fractures ,14-20. TProximal humeral fractures occur frequently and when displaced they may require operative treatment. Operative treatment options include open reduction and internal fixation with plates and screws, staples and wires, percutaneous pinning, intramedullary nails, and shoulder hemiarthroplasty ,6-9. In We retrospectively evaluated the indications, functional outcome and union and complication rates in patients with displaced and unstable proximal humeral fractures who underwent operative treatment with the Polarus intramedullary nail in our institution within a 2-year period. We acknowledge limitations within our study. Only 2-part, 3-part or 4-part (suitable to nailing) proximal humeral fractures were included; whereas specific fracture patterns, such as head split fractures or fractures extending in the humeral shaft, were not included. Furthermore, the number of cases was relatively small. Nevertheless, our results represent the experience of one single unit. Other strengths of the study include a long follow up and the evaluation of the functional assessment of the patients.The first published study used the Polarus nail for 2-part and 3-part displaced proximal humeral fractures , but itsFrom the complications that we have encountered, there were six minor ones , and only one major (4%) including failure of the fixation requiring revision surgery and conversion to shoulder hemiarthroplasty. However, none of the patients developed avascular necrosis of the humeral head, which is one of the major complications after displaced fractures of the proximal humerus ,24. The Proximal humeral fractures constitute a significant cause of morbidity in the elderly and the good functional scores achieved with the Polarus nail are promising together with the excellent results in younger patients. Additionally, it can be used in more complex fracture patterns like 4-part with very good results. Other advantages include the short intra-operative time, the limited exposure and soft tissue damage and the preservation of periosteal blood supply. Moreover, it facilitates rehabilitation and it requires short post-operative hospital stay. Union is achieved in 96% of proximal humeral fractures; but a recent study has reported a 100% union rate even when the Polarus nail was used for proximal humerus established non-unions in combination with autologous bone grafting .The overall functional outcome from our case series and the analysed studies is for the vast majority of the cases graded as excellent or good. In elderly patients however, a lower Constant score can be expected. Further studies are desirable to evaluate in more detail its role in 3- and 4-part fracture patterns.All authors declare that they have no competing interests.FNX, RD and NM contributed to the retrospective collection of the data and the documentation, evaluation and analysis of the clinical, radiological and functional outcome of the patients. They also performed the literature review and writing of specific sections of the manuscript. RH performed the operations and evaluated the clinical and functional outcome of the patients. He was also involved in the interpretation of the data and in drafting the manuscript. PVG has made substantial contributions to the conception of the study and he critically revised the manuscript for its intellectual content. All authors read and have given final approval of the final manuscript.


Only few longitudinal studies on the course of asthma among adults have been carried out.The aim of the present prospective study, carried out between 2000 and 2009 in Italy, is to assess asthma remission and control in adults with asthma, as well as their determinants.All the subjects with current asthma (21–47 years) identified in 2000 in the Italian Study on Asthma in Young Adults in 6 Italian centres were followed up. Asthma remission was assessed at follow-up in 2008–2009 (n = 214), asthma control at baseline and follow-up. Asthma remission and control were related to potential determinants by a binomial logistic and a multinomial logistic model. Separate models for remission were used for men and women.The estimate of the proportion of subjects who were in remission was 29.7% . Men who were not under control at baseline had a very low probability of being in remission at follow-up  when compared to women . The estimates of the proportion of subjects who were under control, partial control or who were not under control in our sample were 26.3% , 51.6%  and 22.1% , respectively. Female gender, increasing age, the presence of chronic cough and phlegm and partial or absent asthma control at baseline increased the risk of uncontrolled asthma at follow-up.Asthma remission was achieved in nearly 1/3 of the subjects with active asthma in the Italian adult population, whereas the proportion of the subjects with controlled asthma among the remaining subjects was still low. Asthma is a major chronic respiratory disease Progress in asthma treatment has reduced the symptoms of asthma and the frequency of severe life–threatening exacerbations of the disease. Asthma is now a treatable disease, but it is not curable. At present, most discussions on asthma treatment goals revolve around disease control Detecting the factors related to asthma remission or control could have clinical and public health implications. Few longitudinal studies have focused on these implications and many of them have been limited to short follow-up studies The aim of the present prospective study, carried out between 2000 and 2009 in Italy, was to assess asthma remission and control in adult subjects with asthma and their determinants.A screening questionnaire investigating the presence of respiratory symptoms and asthma management was sent to random samples of people aged 20–44 years at the time of recruitment, in the frame of the Italian Study on Asthma in Young Adults (ISAYA) carried out between 1998 and 2000 All the subjects in the 6 Italian centres , who reported current asthma at the screening questionnaire (self-reported physician's diagnosis of asthma and at least one asthma attack in the last 12 months and/or current use of anti-asthma drugs), between April and June 2000, were invited to participate in a phone interview aimed at collecting information on symptoms and treatment Local ethics committees at each center approved the local study protocols and the follow-up study was approved by the ethic committee of the University of Pavia.Remission was defined as no current use of asthma medication, no asthma-like symptoms  and no asthma attacks in the last 12 months, as reported by the subjects at follow-up.2-agonists twice or less per week in the last 3 months and no use of oral steroids in the last 12 months. Asthma was considered to be partly controlled if 1 or 2 of the above features were absent. Asthma was considered to be uncontrolled if more than 2 features were absent or if asthma, shortness of breath, or wheezing had caused hospital/emergency department admissions in the last 12 months; oral steroids were used in short courses or continuously in the last 3 months; or the subject had more than 12 asthma attacks in the last 3 months.Asthma control was defined by using a customized version of the asthma control definition provided in the Global Initiative for Asthma (GINA) guidelines Information on lung function was not available.Treatment was classified in one of five steps according to the average daily dose of inhaled corticosteroids (ICS) based on the last 3 months STEP 1: As needed short-acting β2-agonist;STEP 2: Sustained release theophylline OR Leukotriene modifier OR budesonide <400 μg (low-dose) OR an equipotent dose of other ICS;STEP 3: Budesonide < = 400 μg or an equipotent dose of other ICS plus long-acting β2-agonist ORBudesonide < = 400 μg or an equipotent dose of other ICS plus leukotriene modifier ORBudesonide < = 400 μg or an equipotent dose of other ICS plus sustained release theophylline ORBudesonide >400 μg (medium or high dose) or an equipotent dose of other ICS;STEP 4: Budesonide >400 μg or an equipotent dose of other ICS plus long-acting β2-agonist ORBudesonide >400 μg or an equipotent dose of other ICS plus leukotriene modifier ORBudesonide >400 μg or an equipotent dose of other ICS plus sustained release theophylline;STEP 5: Oral corticosteroids or Anti-IgE treatmentThe equipotent doses of other ICS – beclomethasone dipropionate, flunisolide, fluticasone, mometasone, ciclesonide and triamcinolone – were the ones reported in the GINA guidelines 2 test for categorical variables and the t-test or the Wilcoxon test for continuous variables. The conventional 5% level of statistical significance was used.Data were summarized as means with standard deviations (s.d.), or as medians with interquartile ranges (IQRs), when appropriate, for continuous variables and as percentages for categorical variables. Comparison of variables across strata was performed by using the χWe addressed the problem of unit non-response by the assignment of statistical weights to the participants To identify the factors associated with asthma remission, a logistic regression model was fitted to the data, using a dummy indicator as the dependent variable (presence/absence of remission at follow-up). The logistic regression model was weighted by using the mentioned non-response adjustment. We assessed the association between several determinants  measured at baseline and asthma remission at follow-up, adjusting for the effect of the type of survey (telephone/face to face). Moreover, we assessed the interactions between gender and the other covariates, and the presence of interactions was assessed using the Wald test. The interactions between gender and smoking habits (p for the interaction = 0.023), gender and control at baseline (p = 0.003) and gender and age at asthma onset (p = 0.023) were statistically significant. The complete model included the significant interaction terms, and 2 separate models were used for men and women. Multivariate associations of potential determinants with asthma remission were expressed by odds ratios (OR) and their 95% Confidence Intervals (95%CIs).To identify the factors associated with asthma control, a multinomial regression model was fitted to the data, using a 3-level dependent variable . The multinomial regression model was weighted by using the inverse of the predicted response propensity. Multivariate associations of potential determinants with asthma control were expressed by relative risk ratios  and their 95%CIs.The multivariate models identified centers as clustering factors, and the Huber/White sandwich estimate of variance was used to adjust for within-center correlation The statistical analyses were performed using STATA software, release 12.0 .Gender, age and smoking habits were not significantly different between subjects who did participate in the follow-up and subjects who did not . ParticiAsthma remission could not be defined for 4 subjects at follow-up due to missing information on current asthma drug use, asthma symptoms or attacks in the last 12 months. After adjusting for non-response and for the effect of covariates, the estimate of the proportion of subjects who were in remission was 29.7% . A regression model for remission at follow-up was estimated separately for men and women  and 3. TSmoking was associated with asthma remission in women  of them had controlled, 60 (40.8%) partially controlled and 46 (31.3%) uncontrolled asthma.Asthma control was defined for 138 subjects with active asthma at follow-up: 38 (27.5%) of them had controlled, 69 (50.0%) partially controlled and 31 (22.5%) uncontrolled asthma.The distribution of asthma control at follow-up was significantly associated with asthma control at baseline (p = 0.016) : the perAfter adjusting for non-response and for the effect of covariates, the estimates of the proportion of subjects who were under control, partial control or who were not under control were 26.3% , 51.6%  and 22.1% , respectively.At follow-up, women were more likely to have partially controlled  or uncontrolled asthma  than controlled asthma with respect to men. Increasing age, the presence of chronic cough and phlegm and partial or absent asthma control at baseline increased the risk of uncontrolled asthma at follow-up .2-agonists when needed, was 68%, 58% and 42% in subjects with controlled, partially controlled and uncontrolled asthma, respectively  was not following a therapy aimed at keeping asthma under control: such large proportions of subjects with partially controlled or uncontrolled asthma who were not using anti-inflammatory drugs raise some concern. This evidence is supported from the results of an Italian study, where the authors found that Italian general practitioners did not use ICS to treat a large number of patients with persistent asthma Some caveats should be considered in the interpretation of our results because of the impossibility to assess the level of the control of asthma through objective measures, as the baseline and follow-up surveys were based on telephone interviews To our knowledge, this is one of the few cohort studies evaluating asthma control according to the GINA classification over a nine-year period in a relatively large, random sample of subjects with current asthma in Italy. Furthermore, we had the opportunity to simultaneously evaluate changes in asthma control and asthma remission. Unlike the studies carried out on hospital patients or outpatients, this study was based on subjects with asthma sampled from the general population. Therefore, even subjects with mild forms of the disease could have been included in the survey.In conclusion, in the present study we found that nearly 1/3 (29.7%) of the subjects with current asthma sampled from the Italian adult population aged 20–44 years had recovered from their asthma after about 10 years, whereas only 26.3% of the subjects with active asthma showed controlled asthma. Asthma control at baseline seems to be an important predictor of asthma remission and asthma control at follow up.Clinical asthma control is highly relevant at individual and societal levels, and measures of control (which to some extent reflect severity) are related to remission probability.


This segregated data structure likely limits our ability to investigate and understand complex biological systems. To facilitate research, therefore, we have built HIVToolbox, which integrates much of the knowledge about HIV proteins and allows virologists and structural biologists to access sequence, structure, and functional relationships in an intuitive web application. HIV-1 integrase protein was used as a case study to show the utility of this application. We show how data integration facilitates identification of new questions and hypotheses much more rapid and convenient than current approaches using isolated repositories. Several new hypotheses for integrase were created as an example, and we experimentally confirmed a predicted CK2 phosphorylation site. Weblink: [ Human immunodeficiency virus type 1 (HIV-1) is a recently emerged human virus that over the past thirty years has ignited the worldwide AIDS pandemic To understand the complex processes involved during viral infection, we assert that it is beneficial to consider all available knowledge to effectively select targets for therapeutic intervention. These include molecular information about protein sequence and structure, protein-protein interactions, protein modifications, protein localization, protein domains and phylogenetic information. Several databases have emerged which focus on subsets of the aforementioned areas and are routinely used by scientists to study HIV These and other bioinformatic databases and applications generally focus on a specific area of knowledge, and are federated with some information from other databases. These segregated data sources likely limit the ability to investigate and understand complex biological systems. Here, we compile existing informatics relating to HIV-1 infection into an intuitively accessed database. We propose that integrated data management has distinct advantages over existing data repositories in hypothesis-generated science and experimental interpretation. We have built HIVToolbox, a database/web application that integrates information about HIV protein sequence, structure, and function. This tool facilitates hypothesis generation, experimental design, and interpretation as demonstrated by example analyses of HIV integrase.–, To demonstrate different types of analysis supported by HIVToolbox, integrase (IN) was analyzed as a case study. IN is a well-studied multidomain and oligomeric viral protein that is essential for integrating viral DNA into the host genome, for viral infectivity, and for which potent inhibitors of its strand transfer function are chemotherapeutically available. Examples of how HIVToolbox can assist with hypothesis generation, experimental design, interpretation of results, and evaluation of structures and structural models are in Examination of the residues making contacts in different dimer structures of HIV-1 IN reveals that the relevant residues are largely clustered on one face of IN and are >98% conserved in 3787 IN sequences from the Los Alamos HIV Sequence Database Fig. 1A A number of different studies have identified different sets of IN residues that bind DNA . Karyopherin α5 binds three regions on the surface of IN dimers , Analysis of IN with HIVToolbox also reveals that there is a striking overlap of clusters of DNA binding residues with several nuclear import motifs , 2C.The spatial arrangement of the nuclear import motifs on the surface of the IN dimer is striking. The five known nuclear import motifs are spatially contiguous like a ‘zipper; along the surface of the dimer, with two Karyopherin α5 sites located on one subunit, in trans with one Karyopherin α5 and two Importin 7 motifs on the other subunit Fig. 2C.While Karyopherin α5 and Importin 7 both serve roles in nuclear import, they likely would compete with binding of IN to the HIV-1 LTR and to LEDGF. Presumably, these karyopherins would block these functional sites in the cytosol, but become activated after import of IN into the nucleus. It is not surprising given so many IN nuclear import motifs, which are likely redundant, that a recent re-evaluation found none to be required for nuclear import Summarizing these analyses with HIVToolbox suggests that it is important to determine 1) if LEDGF competes with Karyopherin α5 for binding IN, 2) if Importin 7 and/or Karyopherin α5 compete with binding of viral LTRs and 3) the stoichiometry of binding of IN dimers and tetramers with LEDGF and importins and how this is affected by DNA binding.in vivo by the host DNA repair machinery, restoring the integrity of cellular genome. Our IN tetramer model contains an asymmetric dimer of dimers, a symmetry evident from the 1K6Y structure , yellow line) are aligned nearly perpendicular to proposed DNA binding channel. When this molecule is flipped  IN model with viral DNA is based on the structure of a 3 domain IN PFV monomer dimerized with an IN catalytic domain and bound to viral DNA We generated a tetramer model of HIV-1 IN with two viral DNA fragments, by superposition of the HIV-1 and PFV domains and superposition of the structure of the LEDGF:IN complex; interdomain linkers were ignored in this model see Fig. 4. Based on these observations we must consider the possibility that while the integration reaction is conserved, the oligomeric structures of the catalysts may differ between HIV-1 and HPV. The TN5 transposase has a different tetramer structure and the PFV IN and may also have a structure of its active tetramer that is distinct from HIV-1 IN Alternatively, differences in the HIV-1 and PFV structures may represent differences between those states required for completion of the IN reactional set. Our new model is based on the apo form lacking DNA, while the HFV model in complex with viral DNA. There are also several possible intermediates in the strand transfer reaction. None of the existing models, including ours, is consistent with the recently determined low-resolution electron microscopy (EM) tomography image of the HIV-1 IN tetramer: DNA complex in vitro, but is not known to phosphorylate IN 32P-γ-ATP. Serial dilution of CK2 showed phosphorylation of recombinant HIV-1 IN substrate with as little as 0.02 U/mL of kinase  gave the strongest signal for phosphorylation when compared to N-terminal domain  and the catalytic core domain , both of which were weakly phosphorylated Fig. 5B.in vitro CK2 phosphorylation site in IN. Despite efficient phosphorylation of S283 in vitro, mutation of this phosphorylation site (S283A) in a recombinant virus showed no impairment of IN nuclear import, syncytia formation, or detectable accumulation of p24 when assayed for growth through immortalized cell lines (data not shown), nor when the C-terminal amino acids containing the CK2 consensus minimotif (275–288) were deleted To further investigate the sites of IN CK2-mediated phosphorylation, we generated all possible permutations of Ser/Thr to Ala point mutations in the 4 putative CK2 phosphorylation sites . His-tagged IN and these mutants were purified, normalized for their protein content, and tested in kinase reactions using purified CK2. The S283A substitution severely compromised CK2 phosphorylation, reducing it by 90% when compared to wild type IN, whereas other mutants were without effect Fig. 5B.Since one purpose of HIVToolbox is to assist with interpretation, the CK2 sites within IN are discussed within this context. The CK2 site at 283–286 was phosphorylated by CK2. These residues were poorly conserved in the IN sequences of 3787 isolates, as determined with a position-specific scoring matrix (PSSM) in HIVToolbox. However, when we examined conservation in different strains, IN 283–288 was highly conserved in >1500 viruses from Group M, clades B and D Table 1.trans in other subunits. One possibility is that this unstructured region is not disordered in the tetramer and blocks the DNA binding channel before IN is transported into the nuclear compartment. This may help to repress access to other molecules, but would likely be dispensable in assays that assess replication, as was previously observed Examination of the spatial relationship of this CK2 phosphorylation site to other functional IN regions shows this site is in an intrinsically unstructured segment on the C-terminus extending 18 residues from D270, the last structured residue in 1EX4. In the structure of the IN monomer the phosphorylation site would be expected to lie away from the active site and likely only have access to some lysine acetylation sites and some of the DNA interacting residues in vitro. Examination of IN structures showed that these sites are on the surface of the monomeric unit . HIVToolbox was implemented as a website in order to minimize usage barriers; a standalone application requiring downloading and installation would discourage many prospective users from trying HIVToolbox. In designing the application, we considered common analysis workflows for investigating proteins. For example, new functional regions can be identified by plotting sequence conservation onto protein structure surfaces using tools such as VENN, ConSurf, and Evolutionary Trace HIVToolbox processes and presents this data in an easy-to-use open-access web application ; model structures are indicated by dotted lines. Directly under the protein sequence, horizontal thin black lines mark known host protein interaction sites. Clicking a line causes the interaction site to be colored in the appropriate structure window. Under the protein interaction site lines are a series of figures representing putative minimotifs predicted from consensus sequences or instances in the Minimotif Miner database


Analyses aimed at identifying genes that have been targeted by past selection provide a powerful means for investigating the molecular basis of adaptive differentiation. In the case of crop plants, such studies have the potential to not only shed light on important evolutionary processes, but also to identify genes of agronomic interest. In this study, we test for evidence of positive selection at the DNA sequence level in a set of candidate genes previously identified in a genome-wide scan for genotypic evidence of selection during the evolution of cultivated sunflower. In the majority of cases, we were able to confirm the effects of selection in shaping diversity at these loci. Notably, the genes that were found to be under selection via our sequence-based analyses were devoid of variation in the cultivated sunflower gene pool. This result confirms a possible strategy for streamlining the search for adaptively-important loci process by pre-screening the derived population to identify the strongest candidates before sequencing them in the ancestral population. Identifying the molecular basis of phenotypic differentiation and understanding the role of selection in producing such differences is a major goal of evolutionary genetics A complementary approach to the above map-based methods is to use patterns of population genetic variation to identify putative targets of selection in the genome. Strong selection is known to influence patterns of diversity and, in the case of crop domestication, the molecular targets of selection are expected to exhibit reduced polymorphism in the crop gene pool (as compared to levels in the wild or landrace gene pools) and skewed allele frequencies relative to non-selected loci Genotypic scans for selection have been performed in a variety of crop species In sunflower, which is a globally-important oilseed crop and also an important source of edible seeds, Chapman et al. H. petiolaris). This was the same panel of individuals that was used to investigate patterns of DNA sequence variation in the original six genes, as well as in an analysis of selection on genes in the fatty acid biosynthetic pathway (see http://compgenomics.ucdavis.edu/), comparing them against genomic sequences from Arabidopsis, rice, grape, and poplar to infer the likely intron positions, and then using primer3 This study focuses on 36 candidates for genes targeted by selection during sunflower domestication/improvement that were identified by Chapman et al. hway see . BrieflyLoci were amplified via PCR with each reaction containing 10 ng of template DNA, 30 mM Tricine pH 8.4-KOH, 50 mM KCl, 2 mM MgCl2, 100 µM each deoxynucleotide triphosphate, 0.1 µM each primer, and one unit of Taq DNA polymerase. PCR conditions used a touchdown protocol to minimise spurious amplification as follows: initial denaturation at 95°C for 3min; 10 cycles of 30 s at 94°C, 30 s at 65°C , and 45 s at 72°C; followed by 30 cycles of 30 s at 94°C, 30 s at 55°C, and 45–90 s at 72°C; and a final extension time of 20 min at 72°C. Amplification was confirmed using agarose gel electrophoresis. Primer sequences are listed in PCR products were treated with 4 units Exonuclease I and 0.8 units Shrimp Alkaline Phosphatase  at 37°C for 45 min followed by enzyme denaturation at 80°C for 15 min to prepare for sequencing. BigDye v3.1 (Applied Biosystems) was used for the DNA sequencing reaction following the manufacturer's protocol, except that a reduced volume of BigDye was used in each reaction. Unincorporated dyes were removed from the sequencing reactions via Sephadex clean-up (Amersham), and the sequences were resolved on an ABI 3730xl (Applied Biosystems).Escherichia coli, and PCR-screened for the presence of an insert. Four or five positive colonies were then sequenced as above except that vector primers (T7 and SP6) were used.Where individuals were heterozygous for an insertion/deletion (indel), the PCR product was cloned into pGEM-T vector (Promega), transformed into competent S), nucleotide diversity (p), number of haplotypes, and Watterson's θ). In order to distinguish the loss of genetic diversity that is due to the domestication bottleneck from true events of positive selection, sequence diversity at each of the 27 genes was compared to that of the seven putatively neutral genes within the ML-HKA framework. Before doing this, however, we first tested each of the putatively neutral loci against the other six loci, as follows. First, a strictly neutral model was run, followed by a model in which each gene was compared to the other six genes. These tests were carried out separately for the wild, landrace, and improved datasets. Two times the difference in log-likelihoods of the models was then used in a Chi-square (χ2) test with two degrees of freedom to test for statistical significance. Importantly, none of the neutral loci showed evidence of selection, establishing their validity as control loci for the investigation of selection on the candidate genes. Each of the 27 genes was then tested against the neutral loci using the approach outline above. By carrying out the tests for wild, landrace, and improved gene pools separately, we were also able to investigate the timing of selection  in cases where selection was detected. The parameters employed in the ML-HKA analyses are listed in Tests for evidence of positive selection were performed using the maximum-likelihood (ML) version of the Hudson-Kreitman-Aguade HKA;  test ML as previθ) in wild sunflower and the lowest levels in the improved cultivars  in at least one of the comparisons  and 10 of the 14 (71%) candidate improvement genes. Applying an FDR correction http://genomics.princeton.edu/storeylab/qvalue/) in the R statistics package (http://www.r-project.org/) reduced this to ten loci at FDR <0.05, including four domestication-related and six improvement-related genes, with a three additional loci exhibiting marginal significance for selection during sunflower improvement after FDR correction (0.05< P<0.10). In all cases, genetic diversity was severely reduced as compared to the neutral control genes in the selected population(s) – i.e., landrace and improved for the domestication-related genes or improved only for the improvement-related genes . For c1533, the outgroup allele only exhibited one SNP relative to the most common allele in cultivated sunflower, potentially impacting our ability to detect selection. Similarly, in the study of sorghum domestication referenced above, low divergence of the out group from sorghum was one of the reasons given for the small number of loci that showed departure from neutrality Interestingly, regardless of our initial classification of these genes, there was a tendency to detect selection more frequently during improvement vs. domestication. Thus, while our initial SSR screen suggested a roughly 50∶50 split between domestication and improvement genes, the sequence-based analyses described herein suggest a bias toward selection during improvement . This diWhile our analyses provide clear statistical evidence of the role of selection in shaping sequence diversity in a number of genes, it must be kept in mind that the effects of selective sweeps can extend into linked, neighbouring regions. It thus remains possible that the genes showing evidence of selection are linked to the actual targets of selection as opposed to having been targeted by selection themselves. In this light, it is worth noting that the initial studies of linkage disequilibrium (LD) in sunflower found evidence for relatively rapid decay For the loci with significant evidence of selection after applying the FDR correction, we identified SNPs that differentiated the alleles in different gene pools, specifically looking for what appeared to be novel variants or fixed, non-synonymous differences. Two loci  exhibited at least one fixed non-synonymous mutation in the improved gene pool that was found to be at low frequency (<20%) in the wild. Two additional loci (c1649 and c2963) had at least one non-synonymous polymorphism  that showed fixed differences between the wild and improved gene pools. Finally, for one locus (c5898), a single cultivated line (RHA801) contained an amino acid insertion that was not present in the sampled landrace lines, but was present at low frequency in the wild, possibly suggesting introgression from the wild into this line. While it is possible that some of these non-synonymous differences could be adaptive, it must be kept in mind that these findings are based on relatively limited sampling and that we also lack data from the full lengths of these genes. As such, care should be taken to avoid reading too much into these results.As for why a subset of the loci identified as being under selection in the original SSR screen did not show evidence of selection at the sequence level, it should be kept in mind that the tests employed in that study were not, for the most part, formal molecular evolutionary analyses. Rather, they were largely based on the identification of extreme outliers, an approach that may have been more prone to false positives. Also, as noted above, the sequence-based tests for selection employed smaller sample sizes. As such, one or two highly divergent alleles could produce a non-significant ML-HKA test result, whereas this effect could have been diluted in the larger screen of SSR polymorphism.In addition to confirming the effects of selection on population genetic diversity at the majority of loci that we had previously identified as bearing the signature of selection in sunflower, our results also provide methodological insights. Our results highlight a potential means for increasing the efficiency of sequence-based screens for selection in a pool of candidate genes. Because all 10 genes that showed sequence-based evidence of positive selection were devoid of sequence variation in the selected population(s), it should be possible to enrich for selectively important loci by performing a pre-screen of the derived population to identify loci with exceptionally low levels of diversity. This subset of loci can then be assayed in the ancestral population to produce the data necessary for formal tests of selection. In fact, this general approach has been successfully applied in two studies of maize Table S1Accessions from which the individuals employed in the DNA sequence analyses were sampled.(DOCX)Click here for additional data file.Table S2Polymerase chain reaction (PCR) primer sequences.(DOCX)Click here for additional data file.Table S3Extension of  to contain the parameter estimates used in the ML-HKA test.(XLSX)Click here for additional data file.


Thymic epithelial tumours (thymoma and carcinoma) are exceptionally rare in children. We describe a national multicentre series with a view to illustrating their clinical behaviour and the results of treatment.rare paediatric tumours" were centrally registered by the Italian centres participating in the TREP project (Tumori Rari in Età Pediatrica [Rare Tumours in Paediatric Age]). The clinical data of children with a thymic epithelial tumour registered as at December 2009 were analyzed for the purposes of the present study.From January 2000 all patients under 18 years of age diagnosed with "Our series comprised 4 patients with thymoma and 5 with carcinoma . The tumour masses were mainly large, exceeding 5 cm in largest diameter. Based on the Masaoka staging system, 3 patients were stage I, 1 was stage III, 1 was stage IVa and 4 were stage IVb.All 3 patients with stage I thymoma underwent complete tumour resection at diagnosis and were alive 22, 35 and 93 months after surgery. One patient with a thymoma metastasizing to the kidneys died rapidly due to respiratory failure.Thymic carcinomas were much more aggressive, infiltrating nearby organs (in 4 cases) and regional nodes (in 5), and spreading to the bone (in 3) and liver (in 1). All patients received multidrug chemotherapy (platinum derivatives + etoposide or other drugs) with evidence of tumour reduction in 3 cases. Two patients underwent partial tumour resection (after chemo-radiotherapy in one case) and 4 patients were given radiotherapy (45-54 Gy). All patients died of their disease.Children with thymomas completely resected at diagnosis have an excellent prognosis while thymic carcinomas behave aggressively and carry a poor prognosis despite multimodal treatment. Primary thymic epithelial neoplasms, thymomas and thymic carcinomas, are uncommon tumours with an annual incidence of approximately 1-5 per million population .Thymoma is classified in two main types, depending on whether the neoplastic epithelial cells and their nuclei are uniformly bland (type A thymoma) or have a predominantly round or polygonal appearance (type B) . Type B Thymic carcinomas are termed according to their differentiation  .In adults, the main histological subtypes in most published series are type B2 and AB thymomas  . The perThese tumours show variable clinical behaviour ranging from a tendency to be indolent and non-invasive to highly infiltrative tumours with metastatic spread to the pericardium and pleura and, occasionally, to the lung .Thymic tumours are exceptionally rare in the paediatric age group, accounting for less than 1% of childhood mediastinal tumours . As in aRare Tumours in Paediatric Age]) was launched in Italy in 2000 [With a view to promoting research on, and improving the clinical management of very rare paediatric cancers  a nationwide cooperative initiative called the TREP project .All patients under 18 years of age with a diagnosis of "Guidelines were developed to help paediatric oncologists diagnose thymic tumours. After chest X-ray, computed tomography (CT) and/or magnetic resonance imaging (MRI) were recommended to assess local tumour extent. A bone marrow biopsy was part of the diagnostic work-up to rule out lymphatic neoplasms. Surgical guidelines suggested that primary excision should be attempted via a sternotomy if non-mutilating, and complete resection was considered feasible; if not, a biopsy was to be taken for diagnostic purposes. The criteria adopted for diagnosing thymoma or thymic carcinoma were as stated by the WHO classification. Tumours were staged according to the commonly-used Masaoka staging system  Table 11. The diThe paucity of data in the literature prevented us from establishing a strict protocol, but radiotherapy and chemotherapy were recommended in an attempt to reduce the tumour mass and make delayed surgery feasible. The guidelines stated that the most often used multidrug regimens included cisplatin, doxorubicin, vincristine and cyclophosphamide.The patients' demographic data are shown in Table The tumour masses were generally large at diagnosis, exceeding 5 cm in largest diameter in 7 cases (and more than 10 cm in 4 of them).The thymic carcinomas revealed a very aggressive behaviour with infiltration of adjacent organs (in 4 cases), regional lymph node invasion (in 5) and metastases to the bone (in 3) and liver (in 1). One child with thymoma had distant metastases to both kidneys.According to the Masaoka staging system, 3 patients were classified as stage I, 1 as stage III, 1 as stage IVa and 4 as stage IVb.A complete primary resection was performed at diagnosis in 3 patients with stage I thymoma, involving a sternotomy in 2 cases and a left thoracotomy in one.In one child, the histopathological diagnosis of thymoma was established on autopsy after a rapid death due to respiratory failure resulting from compression of the respiratory tract by an unrecognized mediastinal tumour.Among the 5 patients with carcinoma, a tumour resection was attempted in two (after chemo-radiotherapy in one), but left macroscopic residual disease; a biopsy of the enlarged regional lymph nodes was performed for diagnostic purposes in 3 cases. All received multidrug chemotherapy based on platinum derivatives in association with etoposide (2 cases) or other drugs. A tumour volume reduction was evident in three cases , ranging from 25% to 66%, but the response was short-lived (1 to 3 months).Radiotherapy to the mediastinal region was administered in 4 patients, in doses ranging from 45 to 54 Gy.The disease progressed in all 5 patients with carcinoma, who died from 7 to 17 months after its diagnosis. One child with thymoma also died soon after being admitted to hospital due to compression of the respiratory tract by the tumour mass. The 3 patients with stage I thymoma were alive with no evidence of disease 22, 35 and 93 months after complete tumour excision.Our report confirms that thymic tumours are very uncommon in the paediatric age group.In adults, the clinical behaviour of thymic tumours may vary from an indolent course to a very aggressive one. The WHO classification describes subtypes with a progressively worsening prognosis: thymoma types A, AB, and B1 have a relatively good outcome; B2 and B3 are more aggressive and have intermediate survival rates, while thymic carcinoma carries the worst prognosis [For thymomas, the WHO histological classification and the Masaoka staging system are independent prognostic factors. Based on the Masaoka staging system, the 20-year survival rates are reportedly 89%, 91%, 49%, 0%, and 0% in patients with Stages I, II, III, IVa, and IVb disease, respectively .Surgical resection is the mainstay of treatment for patients presenting with Masaoka Stage I or II disease; complete tumour resection is accompanied by complete thymectomy, the removal of all surrounding mediastinal fat and possibly also the pleura, to increase the chances of ensuring negative surgical margins .Patients achieving a complete resection of a stage I tumour have a 5-year survival rate of 100% and a recurrence rate of 1% ; these pOur limited experience suggests that thymomas in children have a similar behaviour with stage I thymoma having a favourable outcome. No further treatment was necessary in our cases after tumour removal. Conversely, the child with stage IV thymoma died of disease even before any therapy could be attempted.Published experiences of paediatric thymoma are limited, but report similar results: In agreement with our results, Dhall  reportedIn adults, thymic carcinomas have a more aggressive clinical course than thymomas and they are associated with a poor prognosis. They are frequently not amenable to radical resection at diagnosis so multimodal therapies (including neo-adjuvant or adjuvant chemo-radiotherapy) are employed. Overall, the results obtained in adults with thymic carcinoma are unsatisfactory, with a reported 5-year survival rate of around 50% and a mean survival of 2.5 years ,16 ,17. Igawa  conducteStudies describing children with thymic carcinoma are scarce. Yaris  reviewedA recent report from the Polish Rare Tumour group  describeThe cases of thymic carcinoma in our series all had unfavourable features: they all presented with large masses and evidence of local or distant spread. Only a diagnostic biopsy was performed in 3 patients and an attempt at tumour resection in 2 left macroscopic residuals. Despite the administration of chemotherapy and radiotherapy, these tumours remained unresectable and all patients died.Four patients in our series received radiotherapy, but it failed to reduce the tumour bulk. In the series published by the Polish group, 4 children were irradiated and 2 of them (with stage II and stage III disease) were still alive, though one of them unfortunately developed severe neurological sequelae due to radiation-induced spinal damage.Although thymic tumours seem to be sensitive to chemotherapy, the most effective regimen remains to be established. Different drug combinations have been used, generally based on cisplatin, doxorubicin, cyclophosphamide and prednisone .In our experience, we only found evidence of a short-lived tumour response in 3 children treated with cisplatin based chemotherapy. Similar results were obtained by the Polish group, with 4 out of 5 assessable children showing a tumour response after initial chemotherapy. Different regimens were administered, however, making it hard to say which is the most effective combination .More effective drugs are therefore needed and targeted molecular therapies might pave the way to new therapeutic options in patients with thymic carcinoma in advanced stages. Strobel et al.  describeThymic tumours are very rare in the paediatric age group. Like their adult counterparts, children with thymomas that are completely resected at diagnosis have an excellent prognosis. Thymic carcinomas behave very aggressively, however, and the prognosis is poor. The TREP project has demonstrated that cooperative studies are feasible even on exceptionally rare tumours and this approach should be transferred to a more international level in an effort to establish the best treatment for these very rare tumours.The authors declare that they have no competing interests.GB substantially contributed to the conception and design of the study and approved the final version of the manuscript.EC and AI were involved in drafting the manuscript and critically revising its intellectual content.GC contributed to data analysis and interpretation, and critically revised the work.AF, MC, AD, RM made substantial contributions to data acquisition.All authors have read and approved the final manuscript.


The thalamus has been proposed to play a role in sensory modulation via switching between tonic and burst dual firing of individual neurons. Of the two firing modes, altered burst firing has been repeatedly implicated with pathological pain conditions, which suggests that maintaining a certain form of thalamic burst could be crucial for controlling pain. However, specific elements of burst firing that may contribute to pain control have not yet been actively investigated. Utilizing the deep brain stimulation (DBS) technique, we explored the effects of bursting properties in pain control by electrically stimulating the ventrobasal (VB) thalamus in forms of burst patterned to test different aspects of bursts during the formalin induced nociception in mice. Our results demonstrated that electrical stimulations mimicking specific burst firing properties are important in producing an anti-nociceptive effect and found that the ≤3 ms interval between burst pluses  and ≥3 pulses per burst were required to reliably reduce formalin induced nociceptive responses in mice. Periodicity of IntraBI was also suggested to contribute to anti-nociception to a limited extent. Parkinson’s disease to depression [Deep brain stimulation (DBS) has been widely used to treat many different types of pathological conditions ranging from pression . Patholopression –4. This Among the various brain regions that have been targeted for the treatments of different types of pain conditions, the somatosensory thalamus has often been targeted to treat neuropathic pain for its relay function ,3,5–7. TOf the many thalamic nuclei, the ventrobasal (VB) complex, which includes the ventro-posterior lateral (VPL) and the ventro-posterior medial (VPM) nuclei, was targeted for the stimulation, since it is suggested to serve a sensory gating function in rodents.The present study investigated the effect of burst components by stimulating the VB with varying burst protocols to compare the nociceptive responses induced by formalin based on the hypothesis that bursts with different properties would have different impacts on anti-nociception. Consistent with our hypothesis, we found that the number of burst pulses composing a burst, the intra-burst-interval (IntraBI), and the possible influence of periodicity of IntraBI play important roles in reducing nociceptive responses.Implantation of electrodes for DBS may alter the physiological state of the brain through the electrode-brain interface ,24 that st phase (0-5 min) nociceptive responses was higher for the sham surgery group, while the 2nd phase (10-60 min) nociceptive responses of the two groups did not differ. Contrary to the clinical result, implantation of electrodes in the VB did not reduce the degree of formalin induced nociceptive responses in mice, but rather increased nociceptive responses only in the 1st phase. The discrepant results may be due to the different nature of pain—neuropathic vs. nociceptive pain of the 1st phase—or placebo effects in human patients. The results also indicate that the stimulating cord attached to a mouse did not interfere with the expression of nociception related behaviors.Formalin injection  to the hind paw pad of a mouse triggered a biphasic surge of behavioral nociceptive responses, a typical response pattern in the formalin test, in both the sham surgery and ‘no-surgery group’  as delineated in the schematic drawing  and 20-25 min  segments (2+ spike (LTS) burst  yieldednd phase ,29, the nd phase [A previous study has shown that the number of burst spikes had a tendency to increase when nociceptive responses were significantly reduced during the 2nd phase , suggestnd phase while only the 4 burst pulse stimulation significantly reduced nociception in the 1st phase  and 20-25 min  segments under the 5 burst pulse stimulation, and 20-25 min  segment under the 4 and 3 burst pulse stimulations. The 2 burst pulse stimulation showed no anti-nociception during the entire recording period. Results showed that the number of pulses within a burst also plays a critical role in reducing nociceptive responses. Three or more burst pulses were required to significantly reduce the nociceptive responses in the 2st phase  while nociception was significantly reduced in the 2nd phase . The 9 ms IntraBI stimulation, on the other hand, could not reduce nociceptive responses in both phases.During sleep, thalamocortical cells burst rhythmically, and the rhythmical bursting is hypothesized to block sensory information from being transferred to the cortex . Likewisnd phase nociceptive responses while the 5 ms IntraBI was ineffective in reducing them. This suggests that periodicity of IntraBI in multiples of 3 may influence the effect of burst stimulations in reducing nociceptive responses, although it appears to be only effective at a very short range of IntraBI.It is noteworthy to mention that the 6 ms IntraBI stimulation reduced the 2nd phase responses when the IntraBI was relatively short (6 ms).This study reports that certain bursting properties are critical for endowing electrical stimulations with its anti-nociceptive ability. Of the parameters tested, both IntraBI and the number of burst pulses were determined to be critical factors in producing anti-nociceptive effect. IntraBI of ≤3 ms and burst pulses of ≥3 were required to reliably reduce formalin induced nociceptive responses. On the other hand, the periodicity of IntraBI, in a multiple of 3, had limited effects on anti-nociception in the 2The burst stimulation patterns tested in our investigation were modeled based on the previous neuronal recording data of VB neurons during the formalin test to isolate the elements of bursting properties that were required for anti-nociception. Considering the nature of the electrical stimulation method, the temporal patterning of burst stimulation would act at multiple levels of the TC circuitry to produce an anti-nociceptive effect. First of all, the RT, which provides the major source of GABAergic inhibition to the TC neurons to initiate LTS bursting in the thalamus , could hThe cortex, another structure connected with the TC neurons , could aAnti-dromic activation of the spinal cord neurons could also have produced the anti-nociceptive effect. High frequency burst stimulations in the spinal cord have been reported to be more effective in controlling pain with reduced side-effects than the conventionally used continuous high frequency stimulation . HoweverActivation of other somatic sensations and symptoms such as paresthesia may be another possible factor contributing to the reduction of nociceptive responses due to the competition. The VB relays not only nociceptive information, but also other sensations such as touch or temperature . Therefond phase by the 6 ms IntraBI stimulation whereas the 5 ms IntraBI had no anti-nociceptive effect at all. Differences between the 5 and 6 ms IntraBI stimulations suggest that in addition to the RT-TC contribution, another mechanism, such as generation of anti-nociceptive brain rhythms or another pathway, may have also contributed to produce the anti-nociception effect. Brain rhythms of patients with chronic pain were altered, and the altered brain rhythms are postulated to contribute to the abnormal pain experience of these patients [The periodicity within a burst may be another factor contributing to anti-nociception to a certain degree, since slight nociceptive effect was present in 2patients . Direct st and 2nd phases. Only a tendency of nociceptive responses reduction was present in the 1st phase while the 2nd phase nociceptive response was reduced almost completely. This may be due to the different nature of nociceptive pain generation. The 1st phase responses are due to direct activation of nociceptors while the 2nd phase responses are due to the development of inflammation by nociceptive stimuli [nd phase responses rather than the 1st phase responses.Even in the burst protocols that have anti-nociceptive effects, the degree of anti-nociceptive effect differed between the 1 stimuli ,29,38. P stimuli ,39. TherIn general, our study demonstrated that burst stimulation with shorter IntraBI, greater number of burst pulses and relatively long intervals between bursts could successfully reduce formalin induced nociceptive responses. This result is consistent with the previous study reporting that bursts analyzed from recordings of spontaneous firing of neuropathic animals had reduced the number of burst spikes and increased IntraBI compared to the intact control animals. Sometimes increased bursting in the thalamus during the awake state is assumed to be a factor causing neuropathic pain ,19. HoweOverall, this study showed that electrical burst stimulations with specific properties would have different anti-nociceptive effects. Future investigation utilizing optogenetic tools would help dissociate the precise circuitry and action mechanism of burst stimulations on pain control.All experiments were conducted in accordance to the guidelines of the Committee for Research and Ethical Issues of International Association for the Study of Pain  and apprFirst generation of C57BL/6J × 129/SvJae hybrid mice  were used in the experiment. C57BL/6J × 129/SvJae hybrids were bred in the animal facility in Korea Institute of Science and Technology. Both inbred mouse lines, C57BL/6J and 129/SvJae, were obtained from The Jackson Laboratory (USA). Mice were maintained at constant temperature (22±1°C) with free access to food and water under a 12 h light and dark cycle (light cycle beginning at 8: 00 AM).2O) was injected subcutaneously into the plantar surface of the left hind paw with a syringe . Immediately after the injection, behavior was videotaped with a camcorder  for an hour and manually analyzed by two blinded investigators. Nociceptive responses were scored by measuring the total duration of licking, biting, and shaking of the formalin injected paw in 5 min blocks. The scores of the two investigators were averaged.The formalin test was used to induce nociceptive pain. All mice used in the experiments were handled and habituated to the experimental setting for a week for approximately 10 min including the test day. The test chamber was an opaque plastic cylinder  placed on top of a beveled mirror for behavior monitoring. Either saline (0.9%) or 10 µl of 5% formalin  and sufficient level of anesthesia was maintained throughout the surgery. Surgery was done using a stereotaxic instrument . Two bipolar stimulating electrodes aligned to be approximately 0.6 mm apart  were implanted in the right VB region  of the Different burst-stimulation protocol was given to stimulate the VB thalamic neurons during the formalin test. None of the stimulation conditions caused visible aversion, irritation, or aberrant behavior. All stimulating pulses were biphasic square pulses with current amplitude of 100 µA and duration of 100 µs. Stimulating conditions differed in IntraBI intervals , the number of burst pulses , or interval between bursts . For details of the stimulation protocols refer to Repeated measures ANOVA was used for statistical analysis over time between experimental groups followed by Games-Howell post hoc test. To analyze the effect of stimulation conditions at different nociceptive phase, statistical analysis was done for each 5 min time segments. First, Levene’s test of equality of error variance was used to determine equal variance at P<0.05. For time segments with equal variance, one-way ANOVA was used while univariate general linear mean was used for time segments with unequal variance, both followed by Games-Howell post hoc test to compare the effect of each stimulation condition to the sham control. Since all the 0-5 min and 20-25 min segments were determined to have equal variance, one-way ANOVA was used for those segments. Student’s t-test was used to compare differences for each 5 min time segments between the sham and ‘no-surgery group’ because main purpose was to compare the difference between the two groups. P-value of 0.05 was used to determine the significance for all statistical analyses. All statistical analyses were done using SPSS (13.0).2O) for a day and stored in 30% sucrose solution at 4°C in the refrigerator for a week before sectioning. Coronal sections (50 µm) were cut through the entire thalamus with a microtome cryostat . The sections were stained with Cresyl Violet  and examined under a light microscope to identify the stimulation sites.Stimulation sites were verified post mortem. Mice were overdosed with 2% avertin, and perfused transcardially first with saline and then with 10% formalin . Brains were removed and further fixed in 10% formalin (1:10 dilution of 37% formalin solution in ddH