Aspergillus fumigatus is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the E. coli tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for Aspergillus spp.Although A. fumigatus using a simple co-transfection approach. Vectors were generated in which the tetracycline transactivator (tTA) or the reverse tetracycline transactivator (rtTA2s-M2) are controlled by the A. nidulans gpdA promoter. Dominant selectable cassettes were introduced into each plasmid, allowing for selection following gene transfer into A. fumigatus by incorporating phleomycin or hygromycin into the medium. To model an essential gene under tetracycline regulation, the E. coli hygromycin resistance gene, hph, was placed under the control of seven copies of the TetR binding site (tetO7) in a plasmid vector and co-transfected into A. fumigatus protoplasts together with one of the two transactivator plasmids. Since the hph gene is essential to A. fumigatus in the presence of hygromycin, resistance to hygromycin was used as a marker of hph reporter gene expression. Transformants were identified in which the expression of tTA conferred hygromycin resistance by activating expression of the tetO7-hph reporter gene, and the addition of doxycycline to the medium suppressed hygromycin resistance in a dose-dependent manner. Similarly, transformants were identified in which expression of rtTA2s-M2 conferred hygromycin resistance only in the presence of doxycycline. The levels of doxycycline required to regulate expression of the tetO7-hph reporter gene were within non-toxic ranges for this organism, and low-iron medium was shown to reduce the amount of doxycycline required to accomplish regulation.Here we describe the construction of plasmid vectors that can be used to regulate gene expression in A. fumigatusThe vectors described in this report provide a new set of options to experimentally manipulate the level of specific gene products in  Aspergillus fumigatus is a saprophytic filamentous fungus that has become the leading mould pathogen in leukemia treatment centers and transplantation units in developed countries, second only to Candida spp. as a cause of systemic mycosis [Aspergillus case-fatality rate demonstrated that more than 50% of patients die with, or as a result of, aspergillosis, despite having received the reference standard of therapy [ mycosis . Despite therapy . The conA. fumigatus genome is expected to greatly facilitate efforts to determine the contribution of specific gene products to the virulence of this opportunistic pathogen. Unfortunately, the genetic tractability of A. fumigatus has lagged behind some other fungal systems, particularly in the area of conditional expression systems. Inducible promoter systems have proven to be instrumental for the elucidation of gene function in a number of species, most notably with essential genes. Experimental manipulation of gene expression in A. fumigatus is presently accomplished through the use of DNA cassettes that are introduced into the organism as transgenes [alcA promoter from A. nidulans has been successfully used in A. fumigatus [alcA promoter can have significant effects on the metabolism of the organism and thus remain a concern for many applications, particularly for in vivo studies.The completion of the annotated sequence of the ansgenes -5, inseransgenes ,6 or expansgenes . An induumigatus . HoweverE. coli tetracycline-resistance operon, a regulatory unit that detects minute concentrations of tetracycline and mounts an appropriate resistance response. Expression of the operon is controlled by a repressor protein, TetR that binds to operator sequences (tetO) in the promoter/enhancer region of the operon and prevents transcription. In the presence of tetracycline TetR is unable to bind tetO, which releases the repression and allows the operon to be expressed. This system has been adapted for experimental gene regulation in eukaryotes by fusing TetR to the VP16 transcriptional activating domain of herpes simplex virus VP16, thereby creating a synthetic tetracycline-regulatable transcriptional activator protein (tTA) that can be used to regulate a gene that is under the control of a tetracycline-responsive promoter  linked to a 175 bp minimal gpdA promoter from A. nidulans  and one showing moderate hygromycin sensitivity (tTA-1). Conidia from each of these transformants were spotted into the center of a plate of minimal medium containing both doxycycline and hygromycin and the radial growth of the colony was monitored with time. The pH of the medium in this experiment was adjusted to 8 in order to maximize the hygromycin toxicity. As shown in Fig. A. fumigatus .Inducible promoter systems are particularly useful for creating strains that can be inducibly depleted of an essential gene product . To modeulans p48, Fig. 2.tus Fig.  can be uhph RNA than tTA-2  and/or basal expression from one or more integrated copies of the tetO7-hph reporter gene (which would not be affected by doxycycline). Since there was a clear dose-response effect of doxycycline on hph expression and hygromycin resistant growth in this strain  that requires interaction of the transactivator with tetracyclines before 'tet-on' . Unfortunditions , we chosnditions . One paric cells .tetO7-hph reporter . Doxycycline was incorporated into the medium at 100 μg/ml to ensure that the tetO7-hph transgene would be expressed at sufficient levels to protect against hygromycin toxicity. Approximately 15% of 27 hygromycin resistant colonies showed reduced growth when shifted to hygromycin medium without doxycycline, one of which was selected for further analysis. As shown in Fig. tetO promoter in A. fumigatus. A further increase in hygromycin resistance was achieved at 15 μg/ml of doxycycline, but concentrations above 15 μg/ml had no additional effect. Northern blots analysis confirmed that the levels of hph RNA in the rtTA transformant were increased by the addition of doxycycline to the medium .A recent report has shown that iron blocks the accumulation and activity of tetracyclines in bacteria . Since iH20 Fig. . This inS. cerevisiae [Candida albicans and C. glabrata are the only pathogenic fungi in which the system has been successfully applied thus far, however neither of these studies used the tetR-VP16 fusions upon which the tTA and rtTA systems are based [The tetracycline-inducible method of gene regulation has become one of the most popular tools to manipulate gene expression in eukaryotes . The effrevisiae , the sysre based -17.A. fumigatus. Since the hph gene is essential in the presence of toxic levels of hygromycin, the ability to control hygromycin resistance by modulating the levels of hph transcription validates the system as a tool for analysis of essential genes in A. fumigatus. In the tTA system we found that individual transformants varied in the amount of doxycycline that was necessary to regulate expression of the tetO7-hph reporter gene. Since doxycycline prevents the tTA protein from binding to the tetO sequence, this is most likely due to variability in the amount of tTA protein that is expressed in each transformant. A limitation of the tTA approach described here is that the majority of the hygromycin-resistant transformants from the tTA/tetO7-hph co-transfection were not susceptible to regulation by doxycycline. This may be due in part to leaky expression of the tetO7-hph reporter, caused by enhancers in the proximity of the integration site [gpdA promoter used in this study were too high to be removed by non toxic concentrations of doxycycline. Since lower levels of tTA expression are more readily suppressed by doxycycline, it is conceivable that a weaker promoter used to drive tTA would increase the frequency with which doxycycline-regulatable transformants can be isolated. Lower levels of tTA expression could also be accomplished by using a shorter segment of the gpdA promoter used in this study.In this study we show that both the tet-off (tTA) and tet-on (rtTA) systems can be used to regulate the expression of a hygromycin resistance reporter gene in ion site ,25. A setetO7-hph reporter gene was also observed in a strain expressing the reverse transactivator, rtTA. Concentrations of doxycycline from 2 μg/ml to 15 μg/ml gave a graded response of hygromycin resistance, indicating that A. fumigatus is responsive to concentrations of doxycycline that are similarly effective in S. cerevisiae [C. albicans [A. fumigatus. Only 15% of the hygromycin-resistant colonies from an rtTA/tetO7-hph co-transfection showed doxycycline-dependent hygromycin resistance however, suggesting that some of the hygromycin resistance was due to leaky expression of the tetO7-hph gene. Leakage of tetO7-regulated genes has been described in other systems, and is attributed to enhancers located in the proximity of the integration site that increase expression of the tetO-linked gene [tetO7-controlled genes regardless of whether they are integrated randomly in the genome or targeted to specific loci.The ability to quantitatively control expression from the revisiae  and C. aalbicans , raisingked gene ,25. ThisA. fumigatus. A limitation of the system was that only 10–15% of the transformants could be regulated by doxycycline, either when tTA or rtTA were used, emphasizing the need to screen for regulatable transformants. A recent approach to limit the problem of leakiness of a tetO-driven gene is the use of trans-silencer proteins comprised of fusions between tetR and a transcriptional silencing domain [A. fumigatus-derived trans-silencer protein into the co-transfection approach described in this study would improve the efficiency of the system.This report establishes the utility of the tetracycline-regulated system as an approach to regulate gene expression in g domain ,27. It iAll vectors are based on the pBluescript plasmid (Stratagene) and were linearized prior to transfection. PCR amplification of components were performed using standard amplification protocols using PfuTurbo DNA polymerase (Stratagene).tet operator sequence (tetO7) was PCR amplified from pUHD10-3 [aagcttgcgtatcacgaggccctttc and the reverse primer 5'-aagcttctcgacccgggtaccgag (added HindIII cloning sites are underlined) and cloned into the HindIII site of pBluescript. A 1.6 kb fragment containing a minimal gpdA promoter from A. nidulans (-175 relative to the ATG of the hph open reading frame), the hph gene encoding resistance to hygromycin, and the trpC terminator from A. nidulans, was then PCR amplified from pAN7-1 [gagctccccatcttcagtatattcatc (added SstI cloning site underlined) and reverse primer 5'-tctagatcgcgtggagccaagagcgg (added XbaI cloning site underlined) and cloned downstream of tet07 into the SstI and XbaI sites of the plasmid, creating p482. To minimize read-through from flanking sequences into tet07, a 280 bp segment of the terminator region of A. fumigatus cgrA [tet07 PCR to create p500. The cgrA terminator was PCR amplified from genomic DNA of A. fumigatus isolate H237 using the forward primer 5'aagcttacagcagaagaatctctc (added HindIII cloning site underlined) and reverse primer 5'ctcgagatgattcatgacgtatattc (added XhoI cloning site underlined), cloned into pCR2.1-Topo (Invitrogen), excised with HindIII, and inserted upstream of tetO7 in p482 to create p500.A segment containing seven copies of the pUHD10-3  with them pAN7-1  with fortus cgrA  was inseA. nidulans gpdA promoter was amplified from pAN7-1 [hph open reading frame) using the forward primer 5'-aagcttcggagaatatggagctt (added HindIII cloning site underlined) and the reverse primer 5'-gaattcggtgatgtctgctcaag (added EcoRI cloning site underlined) and cloned into pBluescript at the same sites. The tTA gene was then PCR amplified from pUHD15-1 [gaattctggcaatgtctagattagataaaag (added EcoRI cloning site underlined) and reverse primer 5'-atcatgtctggatcctcgcg  and cloned into the EcoRI and BamHI sites downstream of the gpdA (-679) promoter. A 280 bp segment of the terminator region of A. fumigatus cgrA [actagtacagcagaagaatctctc (added SpeI site underlined) and reverse primer 5'-gcggccgcatgattcatgacgtatattc (added NotI site underlined) and inserted into the SpeI and NotI sites downstream of tTA. To introduce phleomycin selection into this construct, a phleomycin resistance cassette containing the A. nidulans gpdA promoter, the Streptoalloteichus hindustanus ble gene encoding resistance to phleomycin, and the S. cerevisiae CYC1 terminator was amplified from pBCphleo  using the forward primer 5'-cctcaggcggagaatatggagcttcatcg and the reverse primer 5'-cctcaggaattaaagccttcgagcgtccc. The PCR product was cloned into pCR-Blunt II-TOPO (Invitrogen), excised with KpnI and XhoI and inserted into the PgpdA-tTA construct to create p444. The phleomycin cassette was excised from p444 with HindIII and re-ligated to create p473. To introduce hygromycin selection into p444, the phleomycin cassette was excised with KpnI and HindIII and replaced with a hygromycin resistance cassette  that was amplified from pAN7-1 [ggtacccggagaatatggagcttc (added KpnI cloning site underlined) and reverse primer 5'-aagcttgcttgagagttcaaggaag (added HindIII cloning site underlined) to make p434.A segment of the m pAN7-1   to create p474. To introduce phleomycin resistance into p474, the phleomycin resistance cassette was excised from p444 with KpnI and HindIII and cloned into the same sites in p474 to create p480. To introduce hygromycin resistance into p474, the hygromycin resistance cassette described in p434 was excised from an unrelated plasmid as a HindIII fragment and cloned into the HindIII site of p474 to make p502.The tTA gene was excised from p473 with A. fumigatus strains used in this study are listed in Table-Aspergillus minimal medium plates [4·4H20. For low-iron minimal medium, the FePO4·4H20 concentration was reduced to 0.45 μM.The m plates . This miA. fumigatus protoplasts as previously described [tetO7-hph reporter construct was co-transfected with 5 μg of the linearized tTA plasmid (p444), or 50 μg of the linearized rtTA plasmid (p474).Plasmids were introduced into escribed . FollowiAspergillus minimal medium agar containing hygromycin and doxycycline at the concentrations specified in the Figure legends. The plates were then incubated at 37°C, and colony diameter was measured with time. Radial growth rates were calculated from the exponential part of the resulting growth curves.For experiments addressing the effects of doxycycline on hygromycin sensitivity, ten thousand conidia were spotted onto the surface of hph gene expression, RNA was isolated from overnight cultures in minimal medium supplemented with the indicated concentrations of doxycycline by crushing in liquid nitrogen and extracting RNA from the crushed mycelium with phenol/chloroform. Twenty micrograms of total RNA were fractionated by formaldehyde gel electrophoresis as previously described [32P-labeled hph DNA probe under stringent conditions in 50% (v/v) formamide/5XSSC /2X Denhardt's solution/10% (w/v) dextran sulfate/1% (w/v) sodium dodecyl sulfate (SDS). The hph probe was an 800 bp EcoRI-BamHI fragment from pAN7-1 [hph open reading frame. Hybridization intensity was quantified with a Phosphorimager (Molecular Dynamics) and normalized for differences in gel loading by quantitating the relative levels of SYBR-green II-stained rRNA .For analysis of escribed , transfem pAN7-1  containitTA tetracycline transactivatorrtTA reverse tetracycline transactivatorTetR tetracycline repressortetO TetR binding sequencehph hygromycin resistance geneble phleomycin resistance geneA. fumigatus, screening of transformants and drafting the manuscript. RB participated in plasmid construction. JCR contributed to the planning of the study. DSA conceived of the project and directed its design and execution. All authors have read and approved the final manuscript.KV participated in vector construction, gene transfer into


Basal Cell Carcinoma (BCC) is the most common carcinoma in humans. It accounts for 20% of carcinomas in men and 10–15% of carcinomas in women. Despite its high incidence, metastatic events are exceedingly rare. The reported frequency of metastatic dissemination is estimated at 0.0028–0.5 percent. Once metastasis is detected, there is a high mortality rate of 50% within 8 months.In this study, we present a case of simultaneous lung and parotid metastases of giant BCC primary located on the right medial canthus of a 62 year old female.Examination of the tumor located on the medial canthus obtained showed "adenoid BCC". Computed tomography (CT) was performed to evaluate parotid region for evaluation of parotid gland and chest. Parotid and lung metastasis were detected in CT. Routine labarotory tests and radiological investigations were done. There was no abnormal finding. We also investigated this patient with a bone scan , abdominal and cranial CT scans .Although metastasis of BCC is a very rare condition, this study reports a case of simultaneous parotid gland and lung metastasis originating from a giant BCC primary that was located on the right inner canthus of a 62 year old female. Basal cell carcinoma (BCC) is the most common carcinoma in humans and accounts for 20% of carcinomas in men and 10–15% of carcinomas in women. Approximately 75–86% of primary BCCs are found on the head or neck. The most common location on the head is the nose, specifically the nasal tip and alae. It constitutes 90% of periorbital malignancies [BCC arising on the medial canthus tends to be deep and invasive and may result in perineural extension and loss of optic nerve function. Pieh et al reported that the highest recurrence rates of BCC following attempted excision, (approximately 60%), was seen with lesions arising from the medial canthus since these lesions tend to be more invasive and difficult to manage . ReclusiA 62-year-old woman was referred to the Plastic and Reconstructive Surgery Department for treatment of a bleeding exophytic tumor located on the right inner canthus. She had had the lesion for approximately 11 years. Initially, the patient was treated with excision and primary closure ten years ago. At this time the tumor had a diameter of 5 cm. The tumor was diagnosed as adenoid BCC microscopically and surgical margins were tumor-positive. The patient was operated on two years later when the diameter of the recurrent tumor was 15 mm. Histological examination of this second specimen revealed an "adenoid BCC" with clear surgical margins.Although the tumor recurred again after the second excision, the patient neglected medical advice and did not undertake any treatment Figure . More reWe also investigated this patient with a bone scan , abdominal and cranial CT scans  and a thoracic CT. Multiple metastatic lesions were seen in the chest CT Figure .Examinations of the cardiovascular, gastrointestinal, neurological, urogenital and hematological systems and other parts of the skin were performed by physical and routine laboratory and radiological techniques. There were no abnormal findings. Biopsy was performed from the tumor located on the inner canthus and revealed "adenoid basal cell carcinoma" Figure . Also, FThe patient did not accept the offer of surgical treatment for the tumor on the inner canthus. She was referred to the Oncology Department and treated with radiotherapy and chemotherapy. The patient received approximately 6000 cGy of external beam radiation over 3 weeks totally. Also, chemotherapy was initiated with cisplatin and 5-fluoruracil. She was followed up with physical examination and CT scans for six months and there were no metastases to other organs. She is still being followed.Spates et al. noted that metastatic BCC was first reported in 1894 . As outl1) the primary tumor must occur in skin containing hair follicles and not the mucous membranes;2) metastasis cannot be by simple extension, but occurring at a site distant from the primary tumor;3) the primary tumor and the metastasis must have similar histologic appearances of basal cell carcinoma; and4) squamous cell features must not be present in the lesions ,8. The cWhile the usual BCC that gives rise to metastases is a large, ulcerated, locally invasive BCC of the head and neck that recurs despite repeated surgical procedures or radiotherapy, these features are not absolute prerequisites for metastasis . ImmunosTumors greater than 3 cm in diameter have a 2 % incidence of metastasis and/or death. This increases to 25% in those lesions more than 5 cm in diameter and to 50% in lesions more than 10 cm in diameter . The proThe author(s) declare that they have no competing interests.EC, conceived the study and coordinated the write-up and submission. AA participated in the writing of the manuscript. All authors read and approved the final manuscript.


Cigarette smoking prevalence among gay men is twice that of population levels. A pilot community-level intervention was developed and evaluated aiming to meet UK Government cessation and cancer prevention targets.Four 7-week withdrawal-oriented treatment groups combined nicotine replacement therapy with peer support. Self-report and carbon monoxide register data were collected at baseline and 7 weeks. N = 98 gay men were recruited through community newspapers and organisations in London UK.At 7 weeks, n = 44 (76%) were confirmed as quit using standard UK Government National Health Service monitoring forms. In multivariate analysis the single significant baseline variable associated with cessation was previous number of attempts at quitting .This tailored community-level intervention successfully recruited a high-prevalence group, and the outcome data compares very favourably to national monitoring data (which reports an average of 53% success). Implications for national targeted services are considered. Analysis of tobacco marketing has demonstrated lesbian and gay youth as an emerging target community , therebyGay men have been disproportionately affected by HIV/AIDS disease in developed countries. HIV risk-taking behaviour is associated with cigarette smoking among HIV-negative gay men , and amoThe National Health Service (NHS) Cancer Plan, a UK Government health strategy, recommends that Primary Care Trusts (PCTs) take a commissioning lead in forming local alliances involving community groups, harnessing community efforts, and dissemination of effective interventions. . In ordeThis innovative pilot study aimed to design, recruit to, and deliver a series of pilot smoking cessation group interventions and to evaluate outcomes using standard UK Government assessment criteria.Setting up and running specialist Smoking Cessation Clinics", part of the Smoking Cessation Training and Research Programme (SCTRP) at St Bartholomew's and Royal London School of Medicine and St George's Hospital Medical School. The programme of withdrawal-oriented treatment combines groupwork, nicotine replacement therapy  and ongoing peer support throughout. An initial information session is followed by 6 closed group sessions, setting a quit data for week 3. This pilot consisted of 4 delivered groups, and each group consisted of 7 closed weekly meetings each of 2 hours.The intervention was developed and delivered by a community-based volunteer-led charity in London UK, with a remit to promote the health of gay men. Potential acceptability and effectiveness were maximised by providing an NHS-approved programme adapted for an appropriate service wholly facilitated and attended by gay men. Seven volunteers experienced in delivering group interventions within the organisation were trained in the 3 day course "The service principle was for a non-judgemental environment where gay men could address socialising and gay social spaces, recreational drug use, sexuality and HIV and the impact of these on their motivations, and ability, to quit smoking. Several specific modifications were made to the taught model. Our intervention modified the SCTRP program's use of "Quit buddies" which promoted partnered support, instead creating "Quit cells" of 3 or 4 participants. This design modification was made in the light of other group interventions delivered by this community organisation in which reliance of a participant on more than one person for support has found to be more reliable. The information on Zyban was expanded to address contraindications with HIV antiretroviral combination therapies. Exercises from assertiveness training courses were imported to assist participants in clearly communicating the intention to remain a non-smoker. In general, group discussion and processes were focussed on culturally-specific contexts to gay men. A detailed intervention programme was written in order to promote consistency across the cycles of intervention delivery.Week 1: information on the course content as well as expectations of quit date are given along with information regarding potential side effects and how to deal with them. Week 2: what to expect when you quit and how to deal with reactions, information on the effects of carbon monoxide, preparation for quit date, personal action plan and the how to use a smoking diary. Week 3: information on how to use nicotine replacements, role play of assertive refusal of cigarettes, selection and formation of quit support cells, and personal statements of cessation. Week 4: group review of challenges of the first week of not smoking with reference to smoking diary and personal action plan, exploration of potential "alternative" support such as meditation and exercise, discussion of the challenges of drug use with respect to smoking cessation. Week 5: group review of previous week's experience, information on health benefits achieved to date and weight gain issues. Week 6: review of previous week's experience, identification of future sources of support. Week 7: review of previous week's experience, information of health benefits to date, elaboration on support sources, small celebration of the group's achievement.Twenty-four recruitment advertisements were placed in free London-wide and national gay press, and accompanying editorial and articles were secured to support the recruitment process.Prior to the initial session, participants were sent the required UK Department of Health self-completion Smoking Cessation Service NHS Client Assessment Form. Carbon monoxide readings were taken at each session from week 2, using the "Smokealyser" calibrated carbon monoxide register, and readings were used in addition to self-report data to confirm smoking cessation at week 7. All intervention attendees were asked to give written permission for data collection purposes and were given guarantees of confidentiality.All data were entered into SPSS for windows V11. In line with NHS monitoring data requirements, the percentage of successful quitters was calculated as those who gave carbon monoxide readings and confirmed they had quit at week 7 as a percentage of those who set a quit date for week 3. Variables were entered individually into univariate binary logistic regressions, with cessation outcome as the dependent variable and participant baseline characteristics, attitudes and behaviour, and nicotine replacement methods as independent variables. Variables with p values below 0.25 were then entered stepwise into a multivariate logistic regression, with 95% confidence intervals (95% CI) reported.Ninety-eight men registered to attend the intervention, and of these 76 attended at least the first session. Sixty-nine of men returned the assessment sheet, and the outcome analysis is of those 69 men.The mean age of participants was 37.1 years , and n = 63 (90%) reported their ethnicity as White. Forty-four men (64%) had been educated to degree level or higher, and n = 52 (75%) were in full time employment with a further 9 (13%) men medically retired, n = 5 (7%) unemployed, n = 2 (3%) in full time education and n = 1 (1%) retired. Seventeen men (25%) were entitled to free prescriptions (i.e. the welfare state pays for their prescribed medications). Sixty-five men (94%) reported that they drink alcohol, consuming a mean of 22.8 units per week .The daily number of cigarettes smoked was as follows: 1–5 ; 5–10 ; 11–20 ; 21–30 ; 31–40 ; 41+ . The first cigarette after waking was smoked during the following number of minutes after waking: 5 minutes ; 6–30 minutes ; 31–60 minutes ; 61+ minutes . Smoking motivations are summarised in Table Participants reported a mean 2.6 of consultations with their primary care General Practitioner (GP) in the previous year . Secondary/hospital consultations in the previous year were reported by n = 35 (52%) men, with a mean of 2.26 consultations for these men . Thirty-four men (51%) had been recommended by their GP to give up cigarette smoking, and n = 26 (38%) men were currently on prescribed medication. Fourteen men (20%) were diagnosed HIV-positive, n = 25 (51%) HIV-negative, n = 16 (23%) untested and n = 4 (6%) refused to answer. The participants rated their health as follows: excellent n = 10 (14.5%); good n = 36 (52%); moderate n = 20 (29%); poor n = 2 (3%); very poor n = 1 (1%).Sixty-one men (90%) had made a previous attempt to quit, and of those who had made an attempt the mean was 2.85 attempts . Previously employed nicotine replacement methods were gum n = 30 (49%), patches n = 30 (49%), nasal spray n = 3 (5%), inhalor n = 12 (20%), microtabs n = 3 (5%), nicotine lozenges n = 4 (7%), and Bupropion (Zyban) n = 12 (20%).Participants described the importance of this current attempt to quit as extremely important ; very important ; quite important ; not at all important (n = 0). Participants rated their chances of quitting for good on this attempt as extremely high ; very high ; quite high ; not very high ; very low .Attendance at sessions was consistently high, of 532 person-sessions 13 sessions were missed. Non-attendance did not apparently cluster around a particular session.At week 3, of the 69 men who gave data, n = 58 men (84%) set a quit date. At week 7 (4 weeks after the quit date) n = 44 men (64%) were confirmed as having quit using the CO monitor, representing 58% of those who attended the first session, 76% of those who set a quit date and 64% of those who gave data at baseline and week 7.A further 3 men reported by telephone that they had quit smoking but did not attend the final session to give clinical data to verify. Nine men (13%) reported not having stopped smoking, n = 6 men (9%) set a quit date at week 3 and did not return to group, n = 7 men (10%) attended the first session only. For the purposes of this analysis, these 25 men were coded as not having quit in the following modelling.This analysis considers those 44 men confirmed as having ceased compared to those 25 categorised as not having quit. Following univariate analysis (see Table This pilot intervention has targeted a hitherto overlooked high smoking prevalence group, and has adapted a Government-approved intervention to meet the specific needs of gay men in an appropriate and acceptable setting. The success rate of 76% of men who had set a quit date being confirmed as having quit at week 7 compares extremely favourably to national monitoring data, which reports a success rate nationally 2001–2002 for smoking cessation services as 53% .Public health targets must consider the needs of high prevalence communities, and this may be achieved through innovative development of existing effective services. However, this study has highlighted the lack of targeted interventions for gay men, and the evidence demonstrates further elevated health needs compared to the general population in the fields of alcohol and drug use  mental hFurther research may identify the factors which contributed to the effectiveness of this pilot complex participative intervention, including offering recruitment and delivery outside of community settings, measuring success rates for gay men in non-gay specific or tailored groups, and the usefulness of "quit cells". Longer-term follow-up data and increasing dosage to include a follow-up session would also provide further useful data. In order to refine the intervention for trial testing, qualitative data regarding the utility, acceptability and preferences for the content of specific sessions would be illuminating. Further, the non-randomised design without comparison group limits presents a limitation to the generalisability of findings, yet still offers cessation outcomes much better than standard national cessation data quoted above which were collected without quasi-experimental design using the same follow-up period. Data were not available on the 29 men who registered for the course but did not attend or complete baseline data, and so it is not possible to compare their demographics or smoking behaviours to those who took up the intervention. Certainly, replication of this first pilot would be necessary in other settings, e.g. non-metropolitan communities, where issues of feasibility and uptake should be addressed. Commissioners may consider the purchase of existing facilitators from cities to deliver in non-metropolitan areas where demand is likely to be lower, as smoking cessation service recommendations state that group leaders need to keep up to date with their skills and to use them on a regular basis .In order to meet the smoking cessation needs of this hitherto overlooked population, and to meet public health policy targets, a rigorous research agenda must be established. While the use of required standard outcome monitoring must be continued, rigorous experimental trials using longer term follow up and commonly reported measures are required. Complex participative interventions must be developed, as in this pilot, from evidence-based interventions with full programme description to ensure replication. The development of appropriate interventions must first pilot services to ensure that they are appropriately adapted to maximise acceptability and uptake among target communities.Lastly, provision of the service by skilled volunteer facilitators has ensured an acceptable, low-cost intervention with a rate of effectiveness in these four pilot groups that compares favourably to national non-targeted interventions outcomes calculated using standard assessment formula. Acceptability of the model appears high with respect to the low number of missed sessions. Voluntary sector provision and delivery should be considered as a low-cost and highly acceptable point of delivery for effective community-level smoking cessation interventions.The authors declare that they have no competing interests.JB and NH managed the pilot study. RH was responsible for data management and analysis and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The Adenomatous polyposis coli (APC) tumour suppressor is found in multiple discrete subcellular locations, which may reflect sites of distinct functions. In Drosophila epithelial cells, the predominant APC relative (E-APC) is concentrated at the apicolateral adherens junctions. Genetic analysis indicates that this junctional association is critical for the function of E-APC in Wnt signalling and in cellular adhesion. Here, we ask whether the junctional association of E-APC is stable, or whether E-APC shuttles between the plasma membrane and the cytoplasm.We generated a Drosophila strain that expresses E-APC (dAPC2) tagged with green fluorescent protein (GFP-E-APC) and we analysed its junctional association with fluorescence recovery after photobleaching (FRAP) experiments in live embryos. This revealed that the junctional association of GFP-E-APC in epithelial cells is highly dynamic, and is far less stable than that of the structural components of the adherens junctions, E-cadherin, α-catenin and Armadillo. The shuttling of GFP-E-APC to and from the plasma membrane is unaltered in mutants of Drosophila glycogen synthase kinase 3 (GSK3), which mimic constitutive Wingless signalling. However, the stability of E-APC is greatly reduced in these mutants, explaining their apparent delocalisation from the plasma membrane as previously observed. Finally, we show that GFP-E-APC forms dynamic patches at the apical plasma membrane of late embryonic epidermal cells that form denticles, and that it shuttles up and down the axons of the optic lobe.We conclude that E-APC is a highly mobile protein that shuttles constitutively between distinct subcellular locations. The Adenomatous polyposis coli (APC) protein is an important tumour suppressor in the colonic epithelium . A key fHowever, APC proteins have additional functions in connection with the actin and microtubule cytoskeletons that appear to be separate from their function in controlling Wnt signalling ,9. One oIn order to explore this mechanism, we asked whether Drosophila E-APC  might have a structural role at AJs. If so, E-APC would be expected to be stably associated with AJs, similarly to the structural components of the adhesive complex. As in mammalian epithelia ,16, the We used the GAL4 system to express GFP-E-APC throughout the embryo, and found that its subcellular distribution is very similar to that of endogenous E-APC in fixed embryos. In particular, GFP-E-APC is concentrated underneath the plasma membrane in apicolateral regions of embryonic epithelial cells Fig. ; Fig. 2bNext, we conducted fluorescence recovery after photobleaching (FRAP) experiments in live embryos, to examine how stably GFP-E-APC is associated with adherens junctions. We bleached the fluorescence in a defined square centred over the junctional region of an epithelial cell with a short laser pulse, and examined the recovery of the fluorescence within this square over time Fig. . This reWe also conducted FRAP experiments with structural AJ components, namely E-cadherin-GFP, Armadillo-GFP and α-catenin-GFP. In these cases, we can only recover a small fraction of the initial fluorescence within the time frame of the experiment Fig. . This alWe conclude that E-APC is significantly more mobile than the structural AJ components. This suggests that E-APC shuttles either within the cortex, along the zonula adherens, or that it shuttles from the cytoplasm to the plasma membrane . IntereIn late embryonic stages, GFP-E-APC forms striking patches underneath the apical plasma membrane of epidermal cells that are in the process of forming denticle extrusions Fig. . These sIt has been reported that E-APC and Armadillo are required for anchoring mitotic spindles in the cortex of dividing blastoderm cells in the early Drosophila embryo . We cannWe also expressed GFP-E-APC in eye imaginal discs, to examine its subcellular distribution within a larval epithelial sheet. We thus noticed striking puncta of green fluorescence within the axons of the optic stalk that connects these discs to the larval brain Fig. . These pshaggy/zeste white3, or sgg, mutants) therefore mimic constitutive and sustained Wingless signalling [sgg mutant embryos [sgg mutant embryos, the levels of membrane-associated GFP-E-APC are also noticeably reduced . Taken together with our results from the sgg mutants, this suggests that the kinetic association of GFP-E-APC with the plasma membrane is unaffected by Wingless signalling.The subcellular distribution of E-APC and its accumulation at the adherens junctions is unchanged in other mutants of the Wingless signalling pathway  were gezw3M11-1 and dshv26 mutant embryos lacking maternal and zygotic gene function were generated as described [sgg mutants (identified with an RFP-marked X chromosome [sgg mutant embryos were hand-picked (from timed egg collections) under the dissecting microscope, and separated into GFP-positive and GFP-negative embryos; unfertilised embryos were discarded.escribed . We did romosome ). For WeAntibody staining of fixed embryos and analysis by confocal microscopy were described previously . The folThe following primary and secondary antibodies were used for Western blotting: rabbit anti-E-APC ; mouse mFor live imaging, embryos were dechorionated in 50% bleach for 1–2 minutes and washed. Embryos were transferred to a moistened black filter (Schleicher and Schüll). Embryos were adhered to coverslips with heptane glue, made by mixing heptane and clear sellotape (Sellotape Ltd). Embryos were mounted in Voltalef oil (10S). For short term imaging (<30 minutes), embryos were mounted on a glass slide with small coverslips as supports. For longer term imaging, e.g. for bleaching of pre-denticle patches, embryos were mounted in oil and placed on Bio-foil gas permeable membrane (Sartorius Ltd) mounted on a perspex frame .FRAP experiments were performed using a Bio-Rad Radiance confocal microscope with a 40× NA 1.3 objective lense. Imaging was performed with a 488 nm argon laser at 5% laser power and the following confocal settings: iris at 4 mm, 50% gain, zoom 10, scan speed 500 lps, box size 512 × 512 pixels. These conditions were found to give minimal photobleaching over the observed time.For each FRAP experiment, a pre-bleach image was recorded by selecting a focal plane and taking a Z-series, consisting of 3 0.5 μm steps either side of the desired focal plane (from -1.5 μm to +1.5 μm). The LaserSharp software was used to define several regions of interest (ROIs) for bleaching. A maximum of one bleach ROI was placed in any cell and several cells were always left unbleached. Typically, 3–5 ROIs were bleached in one field of view on one embryo. These regions were bleached at 100% laser power (scanning at 500 lps). 10 bleach scans were found to produce the best results for all constructs. After bleaching, a Z-series was recorded every 15 seconds for 5 minutes. At the time of these experiments, the LaserSharp software did not contain a function for performing this type of 4D bleaching experiment. This problem was overcome by manually switching between imaging and bleaching settings and manually saving pre bleach images and starting the time course. As a result of this, there was usually a 30–60 second delay between the pre-bleach image and the post-bleach images.Data sets were analysed with the Bio-Rad LaserPix software. For each time point, the total pixel intensity distribution was compared to the pre-bleach image to select the corresponding region. The two images were then compared by eye to confirm that they did correspond to the same focal plane. The coordinates for the bleach ROIs were used to accurately locate the bleach spots on the pre bleach image, and the mean fluorescence intensity for each ROI was calculated. Several equivalent sized ROIs were also placed on unbleached cells to measure any change in fluorescence due to photobleaching or movement.To track movement of the cells, an acetate sheet was placed over the computer monitor and each ROI was marked on it as well as the shapes of the cells surrounding it. By aligning the sheet with the appropriate cell shapes, the ROI could be appropriately positioned for each time point. This process was used to position each ROI on the appropriate image for each time point.Once all ROIs had been placed on the image, the mean fluorescence intensities were calculated for each ROI, and their positions were saved on a copy of the image See Fig . Data waData sets were discarded for any of the following reasons. First, if movement of the embryo in the Z axis took the sample outside the range of the Z-series in any time point. Second, if movement in the X/Y axis was sufficient to move significant numbers of the bleach boxes outside of the observed region. Third, if an ROI ever left the field of view, all data points for that ROI was discarded. Fourth, all data sets were discarded if the intensities of the control ROIs changed dramatically at any point in the experiment, or showed a large general increase or decrease.Pre-denticle structures were bleached in a similar manner to junctional E-APC described above.GFP-E-APC was expressed in eye imaginal discs by the GAL4 system, using the driver line GMR.GAL4 (described in FlyBase). Eye discs and brains were dissected from crawling third instar larvae in PBS. Eye discs were teased away from the brain and inverted to reveal the optic stalk. Whole disc/brains were mounted in a drop of PBS under a cover slip, supported by two smaller cover slips. Each disc was observed for no more than 30 minutes.FRAP experiments were performed using a Bio-Rad Radiance confocal microscope and Bio-Rad LaserSharp software, using the 100× NA 1.4 objective lens. A narrow strip was bleached across the whole field of view by adjusting the size of the scanning area. These experiments were performed before a FRAP program was available for LaserSharp so bleaching was performed manually, leading to somewhat variable intervals between each stage of the experiment. The region was bleached with the 488 nm line of an argon laser for approximately 20 scans. Time courses were recorded after each bleaching experiment for 5 minutes.sgg mutant embryos, including the Western blots; M.B. directed the study, helped with the microscopy and drafted the manuscript. All authors read and approved the final manuscript.A.C. developed and conducted most of the FRAP experiments; J.M. completed the analysis of GFP-E-APC in live and fixed FRAP of GFP-E-APC in early embryonic epithelial cells. Example of a FRAP experiment of GFP-E-APC, as described in Figure 3.Click here for fileFRAP of GFP-E-APC in the larval optical stalk. Example of a FRAP experiment of GFP-E-APC, as described in Figure 6.Click here for file


Are short synthetic peptides the key to developing cancer vaccines? And what are the obstacles in the way? CTLs specifically recognize and lyse targets through the interaction of T cell receptors (TCRs) on the surface of the T lymphocyte with protein fragments (peptides) presented on the surface of target cells, in association with major histocompatibility complex (MHC) class I molecules. When a particular CTL interacts with a target cell, it rapidly divides to form a clonal population of T cells with the identical TCR.CD8Townsend and colleagues first elucidated the molecular basis of target cell recognition by CTLs in 1985 [+ T cell response  that is specific for several of these antigens. The development of such responses, however, requires a large tumor load, occurs late in the disease, and probably does not cause the efficient destruction of the tumor cells [Furthermore, before Boon and colleagues cloned the first antigen recognized by tumor-reactive CTLs in 1991 , it was or cells . Thus, aUnfortunately, some synthetic peptides, including some corresponding to immunodominant epitopes (those which cause the biggest part of the immune response) from tumor antigens, only seem to bind MHC class I molecules with medium to low affinity and/or are recognized by specific T cells with relatively low avidity. These characteristics are the likely cause of the poor immune reaction generated by these peptides . One strSolinger and colleagues were the first to describe antigen variants producing T cell responses that were stronger than those elicited by the parental sequences . Some hePLoS Medicine [In a study by Lee and colleagues in this issue of Medicine , despiteMedicine ,11. In aMedicine , into anIt is increasingly clear that even the smallest alteration in the structure of the MHC peptide complex can result in significant changes in which TCRs are selected after vaccination. Thus, manipulating the immune T cell repertoire in vivo through the use of heteroclitic tumor antigen peptide variants could be harder than anticipated. As the field moves rapidly towards the use of new vaccine adjuvants with high immunogenic potential , reasses


There is a continued need to develop more effective cancer immunotherapy strategies. Exosomes, cell-derived lipid vesicles that express high levels of a narrow spectrum of cell proteins represent a novel platform for delivering high levels of antigen in conjunction with costimulatory molecules. We performed this study to test the safety, feasibility and efficacy of autologous dendritic cell (DC)-derived exosomes (DEX) loaded with the MAGE tumor antigens in patients with non-small cell lung cancer (NSCLC).This Phase I study enrolled HLA A2+ patients with pre-treated Stage IIIb (N = 4) and IV (N = 9) NSCLC with tumor expression of MAGE-A3 or A4. Patients underwent leukapheresis to generate DC from which DEX were produced and loaded with MAGE-A3, -A4, -A10, and MAGE-3DPO4 peptides. Patients received 4 doses of DEX at weekly intervals.Thirteen patients were enrolled and 9 completed therapy. Three formulations of DEX were evaluated; all were well tolerated with only grade 1–2 adverse events related to the use of DEX (injection site reactions (N = 8), flu like illness (N = 1), and peripheral arm pain (N = 1)). The time from the first dose of DEX until disease progression was 30 to 429+ days. Three patients had disease progression before the first DEX dose. Survival of patients after the first DEX dose was 52–665+ days. DTH reactivity against MAGE peptides was detected in 3/9 patients. Immune responses were detected in patients as follows: MAGE-specific T cell responses in 1/3, increased NK lytic activity in 2/4.Production of the DEX vaccine was feasible and DEX therapy was well tolerated in patients with advanced NSCLC. Some patients experienced long term stability of disease and activation of immune effectors Vaccine immunotherapy as an approach to cancer treatment has evolved over the last 10 years as the basic biology of the immune response has been elucidated. Tumor-associated antigens that are capable of eliciting cytotoxic T cell responses have been identified. Among the most frequently expressed across many malignancies are the MAGE antigens, originally described in melanoma, but expressed by other tumors including non-small cell lung cancer (NSCLC) . Immune in vitro and eradicate established murine tumors .Dexosomes have been demonstrated to participate in antigen presentation in the following way ,22. Afteex vivo dendritic cell approach for eradication of advanced cancer. Purified dexosomes were shown to be effective in both suppressing tumor growth and eradicating an established tumor in this model. Furthermore, the effect of the dendritic cell-derived dexosome was greater than that of the dendritic cell from which it was produced [Dexosomes have demonstrated significant antitumor activity in a mouse tumor model, suggesting that the use of dexosomes derived from dendritic cells may result in improved efficacy relative to the produced . TherefoWe performed this study to investigate the safety, feasibility, and efficacy of administering autologous dexosomes loaded with tumor antigens (subsequently referred to as DEX) to patients with advanced NSCLC. We also evaluated the immunologic responses in selected patients and monitored the clinical outcomes.This phase I clinical protocol was approved by the Duke University Medical Center Institutional Review Board and conducted in compliance with the Helsinki Declaration and under an IND from the United States Food and Drug Administration held by Anosys Corporation. All subjects provided written informed consent. Patients were eligible for enrollment if they had histologically confirmed, unresectable Stage III A or B or Stage IV NSCLC, were HLA A*0201 positive, at least 18 years of age, and had adequate organ function and a Karnofsky performance status of at least 80%. Patients were required to have been treated with at least one prior standard chemotherapy regimen and have measurable disease. In addition, patients were required to have tumor expressing MAGE A3 or MAGE A4. To avoid performing repeat biopsies, this was achieved by detecting MAGE A3 or MAGE A4 expression in peripheral blood tumor cells by RT-PCR using established methods.The main exclusion criteria were: prior therapy within 4 weeks of the leukapheresis, CNS disease, history of autoimmune disease, concurrent use of systemic steroids, presence of HIV infection or acute or chronic viral hepatitis B or C. Pregnant or lactating women were also excluded.2 atmosphere in the presence of 50 ng/mL GM-CSF  and 10 ng/mL of IL-4 . On the 7th day of culture, the supernatant of the resulting dendritic cell preparation was harvested, filtered, and concentrated. Dexosomes were then isolated by ultracentrifugation on a D2O/sucrose cushion. As described in table Dexosomes were manufactured from peripheral blood mononuclear cells (PBMCs) as previously described . Briefly13 MHC class II molecules in a volume of 3 mL (divided into twoinjections given at two sites on opposite sides of the body) as a combination of subcutaneous (90% of the volume) and intradermal (10%) injections weekly for 4 weeks. No retreatment was allowed. Vital signs were monitored for 1 hour after each injection. Clinical responses were assessed by RECIST criteria. CT scans of the chest through the upper abdomen were obtained at baseline, 1 month following the last dose of DEX and every 3 months after last dose of DEX for 1 year, but scans to confirm responses were not required in this phase I study. All surviving patients have been followed every 6 months for assessment of vital status.Patients were enrolled into three cohorts  that varied in the method of MHC Class I peptide loading and concentration as described in Table Prior to the initial leukapheresis and 1 week after the last dose of DEX, the following peptides were injected intradermally, in addition to the standard recall antigen panel of Candida, Mumps, and tetanus: MAGE-A3(112–120), MAGE-A4(230–239), MAGE-A10(254–262), and MAGE-A3(247–258), each at 10 μg in 0.1 mL saline. The diameter of the induration and erythema was measured 48 hours following the peptide injection.in vitro stimulation of PBMCs with autologous DCs pulsed with the MAGE-A3(112–120), MAGE-A4(230–239), and MAGE-A10(254–262). The number of spots (interferon-gamma-secreting T cells) per 20,000 responding PBMC was reported. The background number of spots against an irrelevant antigen was subtracted from the number of spots for the experimental conditions.Immune response was evaluated at baseline and 1 week following last dose of DEX with cryopreserved PBMCs obtained by leukapheresis. The ELISPOT assay was performed by ImmunoSite, Inc.  according to previously reported methods  using boNK cells were isolated from cryopreserved PBMCs using an NK Cell Isolation Kit  according to the manufacturer's instruction. NK cell purity was checked by flow cytometry using anti-CD3-FITC, anti-CD45-PerCP, and anti-CD56-APC antibodies . Isolated NK cells and NK cells activated for 40 hours by IL-2  600 Units/ml were incubated at various effector to target rations with chromium-51 labeled K562 cells, an NK target, for 4 hours at 37°C and cytotoxicity was assessed by the amount of radiolabeled chromium released. Cytotoxicity was calculated as follows: percentage of target cell lysis = 100 × (counts per minute (cpm) of experimental release - cpm of spontaneous release) / (cpm of maximum release - cpm of spontaneous release).The primary endpoints of this study were safety and feasibility, with secondary endpoints of clinical and immunologic response rates. The incidence, type and severity of adverse events were recorded during the study treatment through 30 days following the last dose of DEX. Descriptive statistics were used to present the data. Adverse events were coded using MEDDRA version 5.0. Survival and time to progression were measured from the date of the first injection to the date of documented disease progression or death. For patients who progressed, the time to disease progression was determined by the interval from the first injection+ 1 day to the last evaluation of disease staging. For patients who did not progress or die during the two year follow up period, the time of disease progression and survival was determined by the interval between the first dose of DEX and the date of last evaluation of disease staging + 1 day and concatenated with the '+' sign.Thirteen patients,  median age 62 years (range 44–72 years) with unresectable pretreated Stage III or IV NSCLC were enrolled. The median time from original diagnosis to study entry was 9.9 months (range 2–61 months) and the median Karnofsky score of the patient population was 80% (range 80–100). DEX therapy was administered to 9/13  patients. Of the 9 dosed patients, 5 patients had Stage IV and 4 patients had Stage IIIB disease. Six patients had stable disease and 3 patients had progressive disease at study entry. Two patients had squamous cell carcinoma, 4 patients had adenocarcinoma, 2 patients had large cell carcinoma, and in 1 case the histological type was not reported. All patients had received prior chemotherapy , 6/9 patients had received prior radiotherapy and 4/9 patients had prior surgery for cancer treatment. Four patients did not receive DEX for the following reasons: manufacturing failure in 2 cases , one of whom had received chemotherapy 13 days prior to leukapheresis and one of whom (DU39) also had rapid disease progression at the time of leukapheresis; delay in shipment in one instance (DU14); and, rapid disease progression prior to planed dosing with DEX in one case (DU44). The characteristics of all dosed patients are listed in Table 2 (see separate file for Table 2).14 (range 4.1 × 1012 to 9.1 × 1014). This quantity of dexosomes in our advanced NSCLC patients is similar in quantity to that generated from healthy donors .The dose of DEX that was selected corresponded to the maximum dose that could be achieved from healthy donors. We confirmed that this dose could be generated in all but two patients with NSCLC. The mean dexosome generation consisted of a total class II number of 3.14 × 10The DEX immunotherapy was generally well tolerated without evidence of serious toxicity. The most frequently reported adverse events causally related to the use of DEX were mild (Grade 1–2) in severity and included: Injection site reactions  in 8 patients; flu like syndrome (1 patient); and, peripheral edema and pain in the arm (1 patient). There were no significant organ or laboratory toxicities attributable to the vaccine. No autoimmune reactions were observed.All 9 dosed patients underwent DTH testing with individual tumor-associated peptides prior to and following all doses of DEX. There was no DTH response to the specific peptide antigens prior to DEX therapy. Three patients  had a positive response of at least 5 mm erythema or induration in the longest dimension 48 hours after skin testing with one of the MAGE peptides. Specifically, DU06 had 5 mm induration and erythema with MAGE-A4(230–239), DU24 had 6 mm induration and erythema with MAGE-A10(254–262) and DU49 had 5 mm induration and erythema with MAGE-A3(112–120), respectively.The peptide-specific immune response to MAGE and CMV was analyzed using ELISPOT in 5 of 9 dosed patients . One patient (DU49) exhibited detectable increases in T cell precursor frequency to MAGE-A10(254–262) following in-vitro stimulation . Assays for DU50 and DU63 could not be completed because of poor viability. Robust responses to anti-CD3 and to the control peptide CMV pp65 were observed in DU24, DU81, and DU49, but no MAGE-specific responses were detected.Since most patients did not exhibit a significant increase in antigen-specific T cell activity, we hypothesized that regulatory influences such as CD4+CD25+ regulatory T cell populations might inhibit augmentation of the T cell response. In 2/3 patients who had analyzable specimens available, an increase in CD4+CD25+ T cells as a percentage of CD4+ T cells was observed following completion of DEX therapy when compared with baseline values . The small number of samples available for this analysis precludes any conclusions but does suggest that CD4+CD25+ T cell analyses should accompany future studies of DEX immunotherapy.During the study, new data from Escudier et al (manuscript submitted) suggested that the immunologic activity of DEX might be due to activation of NK cells. We therefore explored the hypothesis that NK cells may be activated following DEX therapy. This was not planned as part of the initial analysis and therefore specimens of PBMC were limiting in all but 4 patients . Although there was no consistent change in NK percentage before and after immunization . The possible role of negative regulatory mechanisms was suggested by the presence of elevated levels of CD4+CD25+ regulatory T cells following immunization in some patients.We observed increases in systemic immune responses against MAGE by DTH reactivity in 3/9 patients who had no reactivity to the MAGE peptides prior to immunization and activation of NK cells, but found minimal increases in antigen-specific T cell activity in An intriguing immunologic observation was the increase in NK activity following immunization in 2/4 patients analyzed. Although DEX are intended to activate antigen-specific, MHC-restricted T cell responses, it is possible that cytokines released in response to DEX therapy could cause activation of NK cells or that DEX could directly activate NK cells. DEX therapy may stimulate both innate and adaptive arms of the immune response and thereby provide a rationale for maximizing the anti-tumor effect of this approach, even in cases where tumors have lost Class I antigens, a common finding as cancers become more advanced . Indeed,Despite the small sample size and the fact that 3/9 dosed patients had disease progression at the time of initiation of DEX treatment, we observed prolonged disease stabilization in some patients. Large clinical trials in patients with advanced NSCLC have generally reported median time to progression of 3–5 months in patients with advanced NSCLC treated with systemic chemotherapy regimens -30. The DEX therapy was well. Immune activation and stability of disease was observed in some immunized patients with advanced NSCLC.Michael Morse received funding from NIH 5R21CA89957-02. Additionally, portions of this study were funded by Anosys, Inc.Nancy Valente, Revati Shreeniwas, Mary Ann Sutton, Alain Delcayre, Di-Hwei Hsu, and Jean BernardLe Pecq held stock and were employees in Anosys,H. Kim Lyerly was a consultant for Anosys, Inc.MAM was the principal investigator of the study and oversaw all aspects including protocol development, patient management, data collection and analysis, and manuscript preparation.JG enrolled patients to the study and managed their care and participated in data analysis.Takuya Osada performed the NK assays and analyzed the data.SK enrolled patients to the study and managed their care.AH performed in vitro immunologic assays and analyzed the data.TMC oversaw the immunologic analyses performed at Duke University and analyzed the data.NV, RS, and MAS oversaw development of the protocol, data collection and analysis, and manuscript preparation.AD developed and oversaw the MAGE screening for patient eligibility.D-H H oversaw portions of the immunologic analysis and data analysis.J-B L provided scientific direction regarding generation of the dexosomes, protocol development, and data analysis and manuscript preparationHKL provided consultation on immunologic assay development All authors read and approved the final manuscript.Table 2 (DOC) presents the remainder of clinical and immunological data from all patientsClick here for file


Caenorhabditis elegans is a little less lonely than the rest of us—it is a self-fertile hermaphrodite, which as a larva makes and stores sperm before switching to egg production for the remainder of its lifespan.  A sister species, C. briggsae, is also hermaphroditic, but phylogenetic evidence suggests the last common ancestor of the two species had a female/male mode of reproduction. This raises the question of how the sex determination mechanisms, which must have evolved independently, differ between the two species. In this issue, Sudhir Nayak, Johnathan Goree, and Tim Schedl show that a crucial difference lies in the activities of two genes.The nematode C. elegans, the early period of sperm production is controlled by multiple proteins, two of which are the focus of this study, the RNA-binding protein GLD-1 (encoded by the gene gld-1) and the F-box-containing protein FOG-2 (encoded by the gene fog-2). Together, they repress translation of a gene, tra-2, by binding to its messenger RNA. This allows another gene, fem-3, to transiently masculinize the larval germline to produce sperm.In C. elegans and C. briggsae, Schedl and colleagues found they share 30 out of 31 sex determination genes, but not fog-2. More surprisingly, they found that the role of gld-1 in sex determination is opposite in the two species. When C. elegans is deprived of gld-1, would-be hermaphrodites produce only oocytes. But when C. briggsae is deprived of gld-1, would-be hermaphrodites produce only sperm. Thus, the authors conclude, the control of hermaphrodite spermatogenesis is fundamentally different in the two species.Comparing the genomes of C. elegans genome, the authors showed that fog-2 arose from a gene duplication event after the C. elegans–C. briggsae split, which occurred approximately 100 million years ago. Since then, its final exon, which codes for the C-terminal end of the protein, has undergone rapid evolution. The authors also show that this is the “business end” of the protein for its interaction with GLD-1, suggesting that the divergence of C. elegans and C. briggsae sex determination pathways resulted, in part, from FOG-2's new interaction with GLD-1.By further examining the fog-2 is in C. elegans is still unclear. The authors speculate that it may recruit additional factors onto the gld-1/tra-2 mRNA complex, increasing efficiency of translation repression. Much remains to be discovered about C. briggsae sex determination as well. The authors suggest that additional genetic differences promoting self-fertility are likely to have accumulated since the two species diverged, which may act to strengthen the male–female germline switching signal. Investigation of this possibility may shed more light on how hermaphroditism operates in these two species, and how a developmental pathway controlling sex determination can evolve.Exactly what the role of


Arabidopsis.A novel approach for modeling gene-regulatory networks, based on graphical Gaussian modeling, is used to create a network for the isoprenoid biosynthesis pathway in  Arabidopsis thaliana, we detect modules of closely connected genes and candidate genes for possible cross-talk between the isoprenoid pathways. Genes of downstream pathways also fit well into the network. We evaluate our approach in a simulation study and using the yeast galactose network.We present a novel graphical Gaussian modeling approach for reverse engineering of genetic regulatory networks with many genes and few observations. When applying our approach to infer a gene network for isoprenoid biosynthesis in  The analysis of genetic regulatory networks has received a major impetus from the huge amounts of data made available by high-throughput technologies such as DNA microarrays. The genome-wide, massively parallel monitoring of gene activity will increase the understanding of the molecular basis of disease and facilitate the identification of therapeutic targets.To fully uncover regulatory structures, different analysis tools for transcriptomic and other high-throughput data will have to be used in an integrative or iterative fashion. In simple eukaryotes or prokaryotes, gene-expression data has been combined with two-hybrid data  and phenIn higher organisms, however, little is known about regulatory control mechanisms. As a first step in reverse engineering of genetic regulatory networks, structural relationships between genes can be explored on the basis of their expression profiles. Here, we focus on graphical models ,6 as a pGraphical models are powerful for a small number of genes. As the number of genes increases, however, reliable estimates of conditional dependencies require many more observations than are usually available from gene-expression profiling. Furthermore, because the number of models grows super-exponentially with the number of genes, only a small subset of models can be tested . Most imSome of these problems may be circumvented by restricting the number of possible models or edges ,13 or byAs an alternative approach to modeling genetic networks with many genes, we propose not to condition on all genes at a time. Instead, we apply graphical modeling to small subnetworks of three genes to explore the dependence between two of the genes conditional on the third. These subnetworks are then combined for making inferences on the complete network. This modified graphical modeling approach makes it possible to include many genes in the network while studying dependence patterns in a more complex and exhaustive way than with only pairwise correlation-based relationships.Saccharomyces cerevisiae.For an independent validation of our method, we compare our modified graphical Gaussian modeling (GGM) approach with conventional graphical modeling in a simulation study. We show at the end of the Results section that our approach outperforms the standard method in simulation settings with many genes and few observations. For a further evaluation with real data, we apply our approach to the galactose-utilization data from  to detecArabidopsis thaliana . The grIsoprenoids serve numerous biochemical functions in plants: for example, as components of membranes (sterols), as photosynthetic pigments (carotenoids and chlorophylls) and as hormones (gibberellins). Isoprenoids are synthesized through condensation of the five-carbon intermediates isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In higher plants, two distinct pathways for the formation of IPP and DMAPP exist, one in the cytosol and the other in the chloroplast. The cytosolic pathway, often described as the mevalonate or MVA pathway, starts from acetyl-CoA to form IPP via several steps, including the intermediate mevalonate (MVA). In contrast, the plastidial  pathway involves condensation of pyruvate and glyceraldehyde 3-phosphate via several intermediates to form IPP and DMAPP. Whereas the MVA pathway is responsible for the synthesis of sterols, sesquiterpenes and the side chain of ubiquinone, the MEP pathway is used for the synthesis of isoprenes, carotenoids and the side chains of chlorophyll and plastoquinone. Although both pathways operate independently under normal conditions, interaction between them has been repeatedly reported ,17.Reduced flux through the MVA pathway after treatment with lovastatin can be partially compensated for by the MEP pathway. However, inhibition of the MEP pathway in seedlings leads to reduced levels in carotenoids and chlorophylls, indicating a predominantly unidirectional transport of isoprenoid intermediates from the chloroplast to the cytosol ,18, althTo gain more insight into the cross-talk between both pathways at the transcriptional level, gene-expression patterns were monitored under various experimental conditions using 118 GeneChip (Affymetrix) microarrays . To construct the genetic regulatory network, we focused on 40 genes, 16 of which were assigned to the cytosolic pathway, 19 to the plastidal pathway and five encode proteins located in the mitochondrion. These 40 genes comprise not only genes of known function but also genes whose encoded proteins displayed considerable homology to proteins of known function. For reference, we adopt the notation from  .The genetic-interaction network among these genes was first constructed using GGM with backward selection under the Bayesian information criterion (BIC) . This waet al.  glucose, which revealed that sterols were formed via the MVA pathway, while plastidic isoprenoids  were synthesized using intermediates from the MEP pathway [13C]- and 1-deoxy-D-xylulose into β-carotene, lutein and phytol indicated that the carotenoid and chlorophyll biosynthesis pathways proceed from intermediates obtained via the MEP pathway [On a metabolic level, our results are substantiated by earlier labeling experiments using [1- pathway . Moreove pathway .In contrast, a close connection between the MVA and the MEP pathways could not be detected. This suggests that cross-talk on the transcriptional level may be restricted to single genes in both pathways.DXR, MCT, CMK, GGPPS11, GGPPS12, AACT1, HMGR1 and FPPS1, supporting the hypothesis that AACT1 and HMGR1 are involved in communication between the MEP and MVA pathways.In a further analysis step, we examined which gene pairs the four identified pathways  attached to. Genes from the plastoquinone pathway were predominantly linked to the genes DXPS2, HDS, HDR, GGPPS11, DPPS2 and PPDS2, whereas the chlorophyll biosynthesis appears to be related to DXPS2, DXPS3, DXR, CMK, MCT, HDS, HDR, GGPPS11 and GGPPS12. Genes from the phytosterol pathway attach to FPPS1, HMGS, DPPS2, PPDS1 and PPDS2.Genes from the carotenoid pathway attached to σij|, where σij denotes the pairwise correlation between genes i and j.Incorporating 795 additional genes into the isoprenoid genetic network would not have been feasible with standard GGMs as the graphical model would have had to be newly fitted for each additional gene. Also, hierarchical clustering would not have been an appropriate tool for detecting the similarities in the correlation patterns between the two isoprenoid metabolisms and their downstream pathways. Figure The positions of the MVA pathway genes (labeled 'm') and the non-mevalonate pathway genes (labeled 'n'), respectively, are shown to the right of the figure. The symbol + represents the positions of genes from the downstream pathways identified in Table For an independent comparison between the modified and the conventional GGM approaches, we simulated gene-expression data with 40 genes and 100 observations. This simulation framework corresponds to the data for isoprenoid biosynthesis and is thought to be only exemplary at this point. An extensive simulation study is currently underway and will be presented elsewhere.k edges decays as a power law ∝ kγ-. For metabolic and protein networks, γ is usually estimated to range between 2 and 3, which would result in very sparse networks with fewer edges than nodes in our simulation settings. To allow for denser networks, we generated 100 graphs each for γ = 0.5, 1.5 and 2.5. With 40 nodes, these graphs then comprised 88.3, 49.7 and 30.5 edges on average. For each edge, the conditional dependence of the corresponding gene pairs was modeled with a latent random variable in a structural equation model as described in [N.Following recent findings on the topology of metabolic and protein networks ,30, we sribed in . FurtherThe performance of the graphical modeling approaches was monitored using the rate of true and false positives in receiver operator characteristics (ROC) curves  had GAL4p-binding sites. These genes were also identified in [After incorporating all yeast genes into our network of the nine galactose genes, 13 genes were found to attach significantly well. Among these, ified in . This reified in , we did Analysis of gene expression patterns, for example cluster analysis, often focuses on coexpression and pairwise correlation between genes. Graphical models are based on a more sophisticated measure of conditional dependence among genes. However, with this measure, modeling is restricted to a small number of genes. With a larger set of genes, it is rather difficult to interpret the model and to generate hypotheses on the regulation of genetic networks.In our approaches, in the search for significant co-regulation between two genes all other genes in the model are also taken into account. However, the effect of these genes is examined separately, one gene at a time. Because of this simplification, modeling can include a larger number of genes. Also, each edge has a clear interpretation, representing a pair of significantly correlated genes whose dependence cannot be explained by a third gene in the model. Our frequentist method has a resemblance to the first two steps in the SGS and PC algorithms . By restBy using a Gaussian model, we can only reveal linear dependencies between genes. For handling nonlinearities, gene-expression profiles should be discretized and analyzed in a multinomial framework. In principle, it should be straightforward to adopt our approach to a multinomial model. Because we focused on linear dependencies, we have not addressed this problem so far.A. thaliana, we constructed a genetic network and identified candidate genes for cross-talk between both pathways. Interestingly, both positive and negative correlations were found between the identified candidate genes and the corresponding pathways. AACT1 and HMGR1, key genes of the MVA pathway, were found to be negatively correlated to the module of connected genes in the MEP pathway. This suggests that in the experimental conditions tested, AACT1 and HMGR1 may respond differently (than the MEP pathway genes) to environmental conditions, or that they possess a different organ-specific expression profile. In either case, expression within both groups seems to be mutually exclusive. On the other hand, a positive correlation was identified between IPPI1 and members of the MVA pathway, suggesting that this enzyme controls the steady-state levels of IPP and DMAPP in the plastid when a high level of transfer of intermediates between plastid and cytosol takes place.For the isoprenoid biosynthesis pathways in Although we have considered only metabolic genes in this analysis, the method can be extended to identify genes encoding other types of proteins belonging to the same transcription module. In fact, transcription factors and other regulator proteins, as well as structural proteins such as transporters, are often found in the same expression module . Our resSimilarly, the expression of genes in the phytosterol pathway appears to be influenced by genes from the MVA pathway. For the downstream regulation of plastoquinone biosynthesis, however, genes from both pathways seem to be involved. This finding is in agreement with the dual localization of enzymes from the plastoquinone pathway in either the plastid or the cytosol. The regulation of this pathway may therefore depend on processes happening on the metabolic and regulatory level in both compartments.A. thaliana can only be made on the basis of additional knowledge and biological experiments. At this stage, the use of domain knowledge has provided some means of network validation. As genes from the respective downstream pathways were significantly more often attached to the isoprenoid network than were candidate genes from other pathways, we are quite confident that our method can grasp the modularity in the dependence structure within groups of genes and also between groups of genes. Such modularity would have been difficult to detect by standard graphical modeling or clustering.We have shown in a simulation study that for gene-expression data with many genes and few observations, the modified GGM approaches have performed better in recovering conditional dependence structures than conventional GGM. However, a final evaluation of our inferred network for the isoprenoid biosynthesis pathways in q be the number of genes in the network, and n be the number of observations for each gene. The vector of log-scaled gene-expression values, Y =  is assumed to follow a multivariate normal distribution N with mean μ =  and covariance matrix Σ. The partial correlation coefficients ρij|rest, which measure the correlation between genes i and j conditional on all other genes in the model are calculated asLet ωij, 1, j = 1,...,q are the elements of the precision matrix Ω = Σ-1.where ρij|rest can be estimated and tested against the null hypothesis ρij|rest = 0 [i and j is drawn if the null hypothesis is rejected. Since the estimation of the partial correlation coefficients involves matrix inversion, estimators are very sensitive to the rank of the matrix. If the model comprises many genes, estimates are only reliable for a large number of observations.Using likelihood methods, each partial correlation coefficients rest = 0 . An edgeCommonly, the modeling of the graph is carried out in a stepwise backward manner starting from the full model from which edges are removed consecutively. The process stops when no further improvement can be achieved by removal of an additional edge. The final model is usually evaluated by bootstrapping to exclude spurious edges in the model.i, j be a pair of genes. The sample Pearson's correlation coefficient σij is the commonly used measure for coexpression. For examining possible effects of other genes k on σij, we consider GGMs for all triples of genes i, j, k with k ≠ i, j. For each k, the partial correlation coefficient ρij|k is computed and compared to σij. If the expression level of k is independent of i and j, the partial correlation coefficient would not differ from σij. If on the other hand, the correlation between i and j is caused by k since k co-regulates both genes, one would expect ρij|k to be close to 0. Here, we use the terminology, that k 'explains' the correlation between i and j.Let ρij|k values in a biologically and statistically meaningful way, we define an edge between i and j if ρij|k ≠ 0 for all remaining genes k. In particular, if there is at least one k with ρij|k = 0, no edge between i and j is drawn since the correlation between i and j may be the effect of k. Our approach can be implemented as a frequentist approach in which each edge is tested for presence or absence or alternatively, as a likelihood approach with parameters θij, which describe the probability for an edge between i and j in a latent random graph.In order to combine the different i, j and all remaining genes k, p-values ρij|k are obtained from the likelihood ratio test of the null hypothesis ρij|k = 0. In order to combine the different p-values ρij|k, we simply test whether a third gene k exists that 'explains' the correlation between i and j. For this purpose, we apply the following procedure:For the gene pair i, j form the maximum p-value(1) For each pair pij,max = max{pij|k, k ≠ i, j}.pij,max according to standard multiple testing procedures such as FDR [(2) Adjust each h as FDR .pij,max value is smaller than 0.05, draw an edge between the genes i and j; otherwise omit it.(3) If the adjusted q(q - 1))/2 in the model. Implicitly, multiple testing over all genes k is also involved in step 1. However, because the maximum over all pij|k is considered, a multiple testing correction is not necessary.The correction for multiple testing in step 2 is carried out with respect to the possible number of edges  be a sample of n observations. For estimating θ, we maximize the log-likelihood L(θ) = logPθ(y) via the EM-algorithm [The frequentist approach has the disadvantage that a connection between two genes lgorithm .θt be a current estimate of θ. Further, let g be the unobserved graph encoded as an adjacency matrix with gij ∈ {0,1} depending on whether there is an edge between genes i and j or not. In the E-step of the EM-algorithm, the conditional expectation of the complete data log-likelihood is determined with respect to the conditional distribution p,Let By assuming independence between edges, Equation (1) becomesand further, after replacingand summing out Equation 2 we findP and P at the right side of Equation (3) are approximated by the statistical evidence of edge i, j in GGMs with genes i, j and k. As we only want to estimate the effect of k on the correlation between i and j, we distinguish only the two cases whether k is a common neighbor of i and j, for example, gik = 1 and gjk = 1 or not. When k is a common neighbor, we test ρij|k ≠ 0 versus ρij|k = 0. When k is not a common neighbor of i and j, we test σij ≠ 0 versus σij = 0 for the pairwise correlation coefficients instead. Thus, we obtain and  are p-values of the corresponding likelihood ratio tests. After replacing Equation (4) in Equation (3), the M-step of the EM-algorithm, that is the maximization of Eθ(logPθ(g)|y,θt) with respect to θ, leads to an iterative updating scheme θ t → θt+1 withwhere θ as followsIn summary, we determine the probability parameters i, j, compute P(ρij|k ≠ 0) and P(σij ≠ 0) for all genes k ≠ i, j.(1) For gene pairs θ0, apply iteratively Equation (5) until the error |θt+1 - θt| drops below a prespecified value, for example 10-6.(2) Starting with i, j in step 1 of the analysis, the partial correlation coefficients ρij|k are not only computed and tested for genes k in the model but also for the additional candidate genes. However, the iteration in step 2 is not extended to these candidate genes. In other words, θij is only iteratively updated in Equation (5) if both genes i, j are in the original model. For candidate genes k, θik and θjk are kept fixed at a prespecified value, for example 1, and are not re-estimated in the EM-iteration process.Our latent random graph approach also enables us to fit a large number of additional genes into a constructed genetic network. In this case, for a gene pair θ. If these candidates have an effect on the correlation between i and j, θij will decrease. Thus, by comparing the original network with the network inferred from allowing for additional genes in step 1, we can determine which candidate genes lower the θ-values and, accordingly, fit well into the network.This outline introduces a second level into the modeling process. At the first level, the network between the original genes is constructed. At the second level, we test how additional candidate genes influence the parameters Additional data is available with the online version of this paper. Additional data files The gene expression values of the isoprenoid genesClick here for additional data fileThe gene expression values of the 795 genes from other pathwaysClick here for additional data fileA more detailed description of the microarray data Click here for additional data fileThe correlation pattern of the 40 isoprenoid genes.Click here for additional data file


Carbon dioxide fixation bioprocess in reactors necessitates recycling of D-ribulose1,5-bisphosphate (RuBP) for continuous operation. A radically new close loop of RuBP regenerating reactor design has been proposed that will harbor enzyme-complexes instead of purified enzymes. These reactors will need binders enabling selective capture and release of sugar and intermediate metabolites enabling specific conversions during regeneration. In the current manuscript we describe properties of proteins that will act as potential binders in RuBP regeneration reactors.We demonstrate specific binding of 3-phosphoglycerate (3PGA) and 3-phosphoglyceraldehyde (3PGAL) from sugar mixtures by inactive mutant of yeast enzymes phosphoglycerate mutase and enolase. The reversibility in binding with respect to pH and EDTA has also been shown. No chemical conversion of incubated sugars or sugar intermediate metabolites were found by the inactive enzymatic proteins. The dissociation constants for sugar metabolites are in the micromolar range, both proteins showed lower dissociation constant (Kd) for 3-phosphoglycerate (655–796 μM) compared to 3-phosphoglyceraldehyde (822–966 μM) indicating higher affinity for 3PGA. The proteins did not show binding to glucose, sucrose or fructose within the sensitivity limits of detection. Phosphoglycerate mutase showed slightly lower stability on repeated use than enolase mutants.in-situ removal of sugar intermediate metabolites for forward driving of specific reactions in enzyme-complex reactors.The sugar and their intermediate metabolite binders may have a useful role in RuBP regeneration reactors. The reversibility of binding with respect to changes in physicochemical factors and stability when subjected to repeated changes in these conditions are expected to make the mutant proteins candidates for  Sustained increase of atmospheric CO2 has already initiated a chain of events with unintended ecological consequences  and allowed to run for 5 hours. The chromatograms were removed from the solvent system and subjected to staining. Three different staining techniques were used to detect sugars, ammonium molybdate, silver nitrate and alpha-naphthol staining ,24. For The dissociation constants for protein-sugar binding was estimated by measurements of area in chromatograms. For this purpose covalently immobilized protein A sepharose beads  was used. The proteins were immobilized on protein A using Amino link kit . The known concentration of protein was incubated at room temperature (25°C) with varying concentration of sugar in the range of 1 μM to 1 mM in a 100 μl fixed volume. At the end of incubation (10 min), the mixture was centrifuged at 10000 × g and an aliquot of supernatant was spotted and chromatogram (TLC) was developed. A similar mixture but with BSA coupled beads served as control. Area calibration using varying concentration of sugar with a fixed aliquot spot volume was recorded under identical conditions. From the measurement of area in control and experimental set the free sugar was calculated such bound sugar is control minus sugar left in experimental set. The experimental data was used to draw a Scatchard type plot from where dissociation constant was calculated, represented by P as free protein, L as ligand and PL as the ligand-bound-protein, the dissociation constant is defined as Kd = [Pfree] [Lfree]/ [PL]. The dissociation constant Kd values for PGDM and S39A enolase for 3PGA and 3PGAL were calculated using experimental data using MS excel program.DB and DB carried out purification of enolase mutants, MK and SM carried out the binding assays. MTS, SC, AG and VG participated in design of the study and performing analyses, MTS also helped to draft the manuscript. SKB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.