Systematic search for genes whose gain-of-function by exogenous expression confers an advantage in cell-based selective screenings is a powerful method for unbiased functional exploration of the genome, and has the potential to disclose new targets for cancer therapy. A major limit of this approach resides in the labor-intensive cloning of resistant cells, identification of the integrated genes and validation of their ability to confer a selective advantage. Moreover, the selection has to be drastic and genes conferring a limited advantage are typically missed.We developed a new functional screening strategy based on transduction of mammalian cells of a given species with an expression library from another species, followed by one-shot quantitative tracing with DNA microarrays of all library-derived transcripts before and after selection. In this way, exogenous transcripts enriched after selection, and therefore likely to confer resistance, are readily detected. We transduced a retroviral cDNA expression library from mouse testis into human and canine cells, and optimized the use of commercial murine gene expression arrays for species-specific detection of library-derived transcripts. We then conducted a functional screening by growing library-transduced canine MDCK cells in suspension, to enrich for cDNAs conferring anchorage independence. Notably, these cells show partial resistance to loss of anchorage, and the selection can be of limited stringency, compromising approaches based on clonal selection or anyway requiring high stringency. Microarray analysis revealed reproducible enrichment after three weeks of growth on polyhema for seven genes, among which the Hras proto-oncogene and Sox5. When individually transduced into MDCK cells, Sox5 specifically promoted anchorage-independent growth, thereby confirming the validity and specificity of the approach.The procedure described here brings substantial advantages to the field of expression cloning, being faster, more systematic and more sensitive. Indeed, this strategy allowed identification and validation of genes promoting anchorage-independent growth of epithelial cells under selection conditions not amenable to conventional expression cloning. Different strategies have been developed to overcome at least in part these limitations, such as vector mobilization and re-screening [Functional screenings based on the gain-of-function approach proved extremely valuable in the identification of novel genes involved in key processes related to cancer onset and progression, such as neoplastic transformation, resistance to apoptosis, or escape from senescence -3. Identcreening , or genecreening . These aHere we introduce a novel approach, that we named "xenoarray analysis" Figure , in whicTo assess whether commercial expression arrays can be used for species-specific detection of exogenous transcripts within a background of endogenous transcripts, we analyzed the 46133 50-mer probes of Mouse-6_V1 expression arrays from Illumina. We noticed that 18019 out of 32826 annotated probes (55%) hybridize with at least 25 bases to the 3'-UTR or 5'-UTR regions of the corresponding mouse transcripts. Given the higher level of interspecies sequence variation in the UTR regions , such prTherefore, the commercial arrays analyzed can in principle be used for Xenoarray analysis, without the need of a specific design. To facilitate identification and filtering of probes giving cross-hybridization signals, and subsequent assessment of exogenous transcript enrichment after the selection, we implemented a software named Xenoarray Analysis Studio (XAS). XAS enables reading and displaying gene expression data from wild-type and transduced cells, before and after selection, filtering non-specific probes, and comparing selected and unselected cells to identify enriched exogenous transcripts.To set-up the procedure, we transduced human HeLa cells with a retroviral mouse testis expression library at the estimated multiplicities of infection (MOIs) of ~1.25, ~2.5 and ~5 . The Scatter Plot in Figure To evaluate whether a higher MOI could increase detection of library-derived genes, Xenoarray analysis was performed also on the populations transduced at MOI ~2.5 and ~5, using the standard or the T7-pFB primer for cDNA synthesis. The analysis confirmed that use of the T7-pFB primer increased detection of library-derived transcripts also at higher MOIs Figure . NotablyHeLa cells are already transformed, highly proliferative and anchorage independent . TherefoThe four populations were also assayed for proliferation in adherence conditions and in soft agar Figure . The LIB2 ratio between the signals in selected and unselected cells for each transcript in each selection, and compared the results of the two selections. Over 70 percent of the genes enriched more than 2-fold in selection A were also enriched over 2-fold in selection B. To validate the 2-fold threshold for significant enrichment, we compared Xenoarray data obtained from the two LIB-UNS populations. Only two probes gave a fold-change higher than 2 (2.08 and 2.04) in this control comparison.Interestingly, no library-derived transcripts are less abundant after selection. This indicates a limited stringency of the selection, allowing even rare populations to remain represented. In fact, we noticed that after an initial crisis in the first two days of culture in suspension, MDCK cells adapted to the condition by forming aggregates and reducing the proliferation rate, without massive cell death. More drastic selection procedures, such as crossing a transwell membrane and "diving" to the bottom of the well, induced loss of the majority of the library-derived transcripts (data not shown). To validate reproducibility of the selection within the same transduced population, a second selection had been conducted in parallel on the library-transduced cells described above ("Selection B"). Similarly, also the LIB-UNS population was split in two for replicate analysis (LIB-UNS A and LIB-UNS B). Also in Selection B, Xenoarray analysis highlighted enriched transcripts [see Additional file Hras1, Mos and Rad-9b, known to be capable of transforming cells, conferring anchorage independence and/or promoting cell proliferation [The 29 genes (detected by 34 probes) reproducibly enriched over 2-fold in both selections are listed in Table feration -19.To verify the specificity of the selection hits, we carried out two alternative selection procedures, one based on serum withdrawal, and therefore not involving anchorage independence, and the other based on the crossing of a transwell membrane and "diving" to the bottom of the plate, more directly linked to anchorage independence. Then we compared the enrichment of the 34 probes emerged from polyhema selection with the enrichments observed in these two selections [see Additional file Reproducible enrichment of exogenous cDNAs by parallel selection in the same transduced population does not rule out two main possible artefacts: (i) enrichment may derive from deregulation of an endogenous gene by insertional mutagenesis; (ii) a very small subpopulation of resistant cells may exist from the beginning, and a cDNA transduced in these cells may get reproducibly enriched by carryover.Hras1 enrichment was confirmed, albeit less evident, and the Sox5 and Sox6 transcription factors displayed the highest concordant enrichment.Therefore, to further validate the screening hits, we performed a second, independent transduction of MDCK cells with the mouse testis library ("Infection 2"), using a lower MOI (~1). It should be noticed that double or triple infection of the same cell is not a problem for the xenoarray approach, because in the one-shot analysis the selection drivers will be present in all selected cells, while the bystanders will be diluted out. The lower MOI was chosen to reduce the possibility of phenotypes deriving from insertional mutagenesis. Again, after expansion, the cells were split to generate unselected and selected populations. Xenoarray on LIB-SEL vs LIB-UNS populations from this second infection was then compared with the results of the first infection Figure . Even wiRepetition of the transduction and selection procedure led to a limited validation of the hits identified in the first screening. This result corroborates the idea that process streamlining allowed by the xenoarray approach should be exploited for performing multiple independent screenings, thereby allowing identification of more consistent hits. However, it should be noted that genes enriched in only one transduction-selection experiment are not necessarily false hits. Indeed, the complexity of the integrated cDNA repertoire can vary across independent transduction experiments. This is particularly true for rare transcripts, which may not be represented in one of the two transduced populations, or may integrate in unfavourable regions of the host genome. Moreover, rare transcripts need to be highly enriched to emerge from the microarray background, and even in that case the differential signal may not necessarily be greater than 2-fold.Sox5 mRNA enrichment in selected MDCK cells by PCR . The main disadvantage of using libraries is that some genes may fail to be represented and others may be overrepresented. In this view, Xenoarray analysis could also be applied to ORF collections, which would allow a much tighter control on the composition and relative abundance of the cDNAs used for the screening, as well as focused exploration of gene subsets. In this case, however, it should be noted that the majority of the probes of commercial expression arrays fall outside of the transcripts' ORFs. Therefore, custom species-specific expression arrays should be designed to cover areas of non-homology within the ORF regions. As an advantage, libraries provide a more comprehensive repertoire of all the transcripts and of their isoforms expressed in the tissue of origin, thereby being more explorative. Moreover, when expression libraries are derived from cancerous tissues or cells, this approach can be combined with resequencing of the hits to highlight mutated genes, potentially exploitable as therapeutical targets. It therefore provides a powerful tool for the dissection of the mechanisms of cancer onset and progression and of resistance to anti-neoplastic treatments.The Xenoarray technology described here provides a new, efficient approach to expression cloning and functional genomics. It takes full advantage of genome-wide expression profiling to identify genes that confer resistance to a specific selective stress, thereby establishing a cause-effect relationship. Being more sensitive and systematic, the procedure does not require extreme selection stringency and isolation of resistant individual clones. In this way, also genes conferring a partial advantage can be identified and further explored for their possible reciprocal cooperation.4 cells onto 35 mm tissue culture plates. The following day, 1 ml of dilutions from 10-1 to 10-5 of the pFB-hrGFP retroviral supernatant in growth medium supplemented with 10 μg/ml DEAE-dextran (Amersham Bioscence) were added to each well. After 3 hours, an additional 1 ml of growth medium was added to each well. GFP expression analysis was performed after 48 hours by flow cytometry: cells were trypsinized, diluted in a 1% paraformalhdeide-2% FBS solution and analyzed on a FACS Calibur flow cytometer (Becton Dickinson). The titer of the library, expressed as GFP transduction units (TU) per ml, was calculated as 105 (the number of infected cells) times the fraction of green cells times the dilution factor. HeLa transduction experiments were performed by plating 5*104 cells in 35 mm wells. After one day, 1 ml of library supernatant dilutions prepared as above were added. Medium with no virus was added to generate an uninfected control. The plates were returned to 37° for 3 hours, then 1 ml of growth medium was added to each well. After 48 hours infected and control cells were expanded and used for microarray analysis. MDCK transduction was performed as above, except that 5*105 cells were infected with about 1*106 TU of viral supernatant in 60 mm dishes (MOI = 2). The pFB-hrGFP retroviral supernatant was used at the same MOI as a control. After 48 hours library- and GFP-infected cells were expanded for the functional screening.Madin-Darby canine kidney (MDCK) cells and HeLa cells were from ATCC. They were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% FBS (Sigma) in a humidified atmosphere of 5% CO2. The mouse testis retroviral expression library, packaged in the VSV envelope was purchased from Stratagene . To titer the library viral supernatant, we used a GFP retroviral supernatant provided by the manufacturer at the same titer, seeding 5*10Probes cross-hybridization analysis was performed by blasting all the probes from the Illumina Mouse-6_V1 chip against the transcriptome databases from Ensembl  and resuspending the pellet in fresh medium every 2–3 days. Cells were then allowed to recover on normal dishes for 24 hours, after which the selection was repeated for a total of 3 cycles. Selected cells were expanded on regular plates for one week before being used for microarray analysis and functional assays.Polyhema-coated 100 mm Petri dishes were prepared by applying 4 ml of a 12 mg/ml solution of poly-hydroxy-ethyl-methacrylate  in ethanol, drying under tissue culture hood, repeating the application once and incubating the plates overnight at 37°C. For the selection, after trypsinization, 1.5 × 103 cells of each cell line were seeded in triplicate in 96-well plates, one for each time of the growth curve assay, both on plastic and on Polyhema coated plates. The day after, a tetrazolium salt-based reagent  was added to each well according to the instructions provided by the manufacturer. After an incubation of 2 h, absorbance was read at 490 nm on a DTX 880 plate reader . For the soft agar growth assay, 104 cells were resuspended in 1 ml of 0.5% top agar  in growth medium and seeded in 6-well plates previously coated with 2 ml of 1% basal agar in growth medium. The assay was performed in duplicate. After 2 weeks, phase-contrast pictures were captured and analyzed with a BD Pathway Workstation.For cell viability assays, 10RNA was extracted using the TRIzol reagent (Invitrogen), according to the manufacturer's protocol, and then further purified using the RNeasy Mini kit from Qiagen. The quantification and quality analysis of RNA was performed on a Bioanalyzer 2100 (Agilent). Synthesis of cDNA and biotinylated cRNA was performed using the Illumina TotalPrep RNA Amplification Kit (Ambion Cat. n. IL1791), according to the manufacturer's protocol, with the following variations to optimize xenoarray analysis: (i) standard cDNA synthesis with the T7-dT(24) primer: 1 μg of total RNA was used, with the doubling of the reaction volume and of all reagents; (ii) library-specific cDNA synthesis: 20 μg of total RNA were used with 4 pm of a pFB-specific primer  with standard reaction conditions. The T7-pFB primer contains the T7 promoter (for cRNA synthesis) followed by a vector-specific sequence, which is present in the 3' region of all transcripts derived from the library. Quality assessment and quantification of cRNAs were performed on Bioanalyzer 2100.Hybridization of HeLa-derived cRNAs was carried out for 18 hours on Illumina Mouse_Ref-8_V1 arrays (Cat. N. BD-26-201) according to the manufacturer's protocol, using 15 μg of T7-dT(24)-derived cRNA or 1.5 μg of T7-pFB-derived cRNA. These arrays contain circa 24000 probes exploring the transcripts contained in the Refseq database, and therefore more reliable. Hybridization of MDCK-derived cRNAs was carried out on Illumina Mouse-6_V1 arrays (Cat. N. BD-26-101), using 1.5 μg of T7-pFB-derived cRNA. These arrays contain all the RefSeq probes present in the Mouse_Ref_8_V1 arrays, plus additional 24 k probes exploring less characterized transcripts, additional Unigene clusters and singletones, to allow a more complete coverage of the transcriptome. Array washing was performed using Illumina High-stringency wash buffer for 30 min at 55°C, and followed by staining and scanning according to standard Illumina protocols. Probe intensity and detection data were obtained using the Illumina BeadStudio software, and further processed with the XAS software. Microarray data are available at GEO, dataset GSE11721.2 of the ratio between the signals before and after selection. If multiple selection experiments are conducted, the software can compare them to identify reproducibly enriched exogenous cDNAs. The software and user guide are available on Sourceforge [The Xenoarray analysis pipeline was implemented in the XAS software (C++ with QT library from Trolltech ) and subrceforge .For PCR, cDNA was synthesized using the RT high capacity cDNA kit (Applied Biosystems), and the mouse-specific Sox-5 primers: 5'-GATATTGGGATCTCGCTGGA-3'; 5'- AAGTACTGCCGCATTTCCTG-3', and the following cycle program: 5' at 94°, followed by 25 cycles (2' at 94°- 30" at 55°- 30" at 72°), and 10' at 72°. For Western blot, anti-human Sox5 antibody was purchased from Abcam . Total cellular proteins were extracted by solubilizing the cells in boiling Laemmli buffer followed by sonication. 100 μg of lysates were run on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes . Nitrocellulose-bound antibodies were detected by the ECL system .The full length human SOX5 cDNA was purchased from RZPD German Resource Center for Genome Research and the full coding sequence was cloned into the pFB retroviral vector (Stratagene). Retroviral supernatant was produced by transfecting pFB-SOX5 or pFB-hrGFP with the pVPack-GP  and the pVPack-VSV-G (env-expressing vector (Stratagene) in 293T packaging cells. The pFB-hrGFP retroviral supernatant was used as control of infection efficiency. MDCK transduction with the supernatants was performed as above.UTR: Untranslated Regions; XAS: Xenoarray Analysis Studio; MOI: Multiplicity of Infection; SAGE: Serial Analysis of Gene Expression; PPM: Part per Million; MDCK: Madin-Darby Canine Kidney; GFP: Green Fluorescent Protein; P-POD: Princeton Protein Orthology Database; ORF: Open Reading Frame.EM conceived the experimental strategy. MLM and EM designed the experiments, analyzed the data and wrote the manuscript. CI performed computational analyses. AM designed and performed experiments for Sox5 expression and functional assays. LF wrote the XAS software. CI and LF contributed to the writing of the manuscript. DC performed experiments. All authors read and approved the final manuscript.representation of mouse testis transcripts according to SAGE analysis. Transcripts are ordered by decreasing representation, measured as PPM (Parts per Million).Click here for filexenoarray analysis on library-transduced MDCK cells before and after a replicate selection performed on the first infection.Click here for filedot plots of xenoarray analysis on the 34 probes reported in Table Click here for file


Poor adherence with prescribed asthma medication is a major barrier to positive treatment outcomes. This study was designed to determine the effect of a once-daily administration of mometasone furoate administered via a dry powder inhaler (MF-DPI) on treatment adherence compared with a twice-daily administration.This was a 12-week open-label study designed to mimic an actual clinical setting in patients ≥12 years old with mild-to-moderate persistent asthma. Patients were randomized to receive MF-DPI 400 μg once-daily in the evening or MF-DPI 200 μg twice-daily. Adherence was assessed primarily using the number of actual administered doses reported from the device counter divided by the number of scheduled doses. Self-reports were also used to determine adherence. Health-related quality of life, healthcare resource utilization, and days missed from work or school were also reported.P < 0.001) with MF-DPI 400 μg once-daily in the evening (93.3%) than with MF-DPI 200 μg twice-daily (89.5%). Mean adherence rates based on self-reports were also significantly better (P < 0.001) with MF-DPI 400 μg QD PM (97.2%) than with MF-DPI 200 μg twice-daily (95.3%). Adherence rates were lower in adolescents (12-17 years old). Health-related quality of life improved by 20% in patients using MF-DPI once-daily in the evening and by 14% in patients using MF-DPI twice-daily. Very few (<8%) patients missed work/school.1233 patients were randomized. The mean adherence rates, as measured by the automatic dose counter, were significantly better (Mean adherence rates were greater with a once-daily dosing regimen of MF-DPI than with a twice-daily dosing regimen.This trial was completed prior to the ISMJE requirements for trial registration. It is well established that inhaled corticosteroid (ICS) therapy is the most effective treatment for patients with persistent asthma,2. UnforAlthough further investigation on the effects of once-daily dosing on ICS treatment adherence under more controlled settings are needed, better adherence has been observed in asthma and otheMometasone furoate administered via a dry powder inhaler (MF-DPI) has been shown to be effective in treating asthma, as shown in comparative studies with other ICSs -18. AlthThe primary objective of this study was to determine the effect of once-daily versus twice-daily dosing of MF-DPI on treatment adherence in a study designed to mimic the actual clinical setting.This multicenter study (143 sites in the United Kingdom) enrolled adolescents and adults aged 12 years or older with mild-to-moderate persistent asthma, and a diagnosis of asthma for at least 1 year. The study had institutional review board approval at all sites, and all patients provided informed consent before any study procedures were conducted. The first visit date was February 5, 2003, and the latest visit/contact date was February 26, 2004. Eligible patients had been treated with either beclomethasone dipropionate (BDP) hydrofluoroalkane (≤500 μg/d) or BDP chlorofluorocarbon (≤1000 μg/d) for ≥12 weeks, and had a stable BDP dosing regimen for ≥4 weeks immediately before study entry. The inclusion of patients who used BDP as their prior ICS therapy was justified because BDP was the ICS prescribed most commonly in the UK when the study was conducted, thereby providing a patient population as large and homogeneously-treated as possible. Eligible patients had no clinically significant disease that would interfere with study evaluations, and female patients of childbearing potential were required to use medically accepted birth control. Patients who required ventilator support for respiratory failure due to asthma within the previous 5 years or were hospitalized within the previous 3 months because of asthma were ineligible.This was a 12-week, open-label study designed to mimic an actual clinical setting. Visits took place every 4 weeks. Patients were randomized to receive either MF-DPI 400 μg once-daily in the evening or MF DPI 200 μg twice-daily from inhalers measuring 220 μg/actuation and delivering 200 μg/inhalation. Assessments were made at baseline, and weeks 4, 8, and 12. Patients were instructed in inhaler use and peak flow measurement to demonstrate proficiency and received salbutamol for rescue medication. Optional diary cards were provided to record adverse events (AEs), use of rescue medication, and changes in concomitant medication between visits. Completed diary comment cards were reviewed at all subsequent visits. Following increases in asthma symptoms from baseline, patients used the peak flow meter to obtain an objective measure of asthma worsening. Daily use of the peak flow meter was not required to more closely mimic real-life practice. Patients were also instructed to follow an asthma action plan based on their personal best peak flow at the first visit. If peak flow was <75% personal best for 2 consecutive days, the patient was required to consult the investigator. Recommended clinical practice guidelines were also provided to investigators.Adherence was calculated as the number of administered doses (as determined by device counter number) times 100 divided by the number of scheduled doses. Data were not included for analysis if invalid . Examples of gross misuse of the device included failure to twist the cap when opening/closing the inhaler, or removing and replacing the inhaler cap more than once without taking a dose. If a device was not returned, it was assumed to have been unused.Secondary evaluations included measurements of adherence based on the patients' or their guardians' reports of adherence, regardless of the actual counter readings. The physician's assessment of a therapeutic response was documented at all visits. Health-related quality of life (HRQOL) was assessed using the Integrated Therapeutics Group Asthma Short Form (ITG-ASF), a brief and reliable disease-specific questionnaire. For thiSafety assessments included reporting of AEs at all visits. An abbreviated physical exam and vital signs  were performed on visits 1 and 4. An evaluation of asthma worsening was performed at all visits, with asthma worsening being defined as an increased use of rescue medication , a decrease in peak flow of >25% on 2 consecutive days, or clinical asthma exacerbations .A total of 1300 patients, or 650 per treatment group, were targeted for inclusion in this study, allowing a 90% power to detect a difference of at least 10% between distributions of adherence rates. The study was not powered to test differences in mean adherence. A sample of this size also allowed a sub-group analysis, with a 90% power to detect positive correlations of more than 0.15 between the level of adherence and the total ITG-ASF within each group (0.05 significance level). The Kolmogorov-Smirnov test was used to determine the significance of observed differences in the distribution of adherence rates between the 2 treatment groups. The Pearson correlation coefficient was used to analyze the correlation between adherence and changes in the ITG-ASF score. The chi-square test was used to analyze health care utilization and number of missed days of school/work. An analysis of variance (ANOVA) was used to extract sources of variation by treatment and center.A total of 1233 patients were randomized to treatment; 611 patients in the MF-DPI 400 μg once-daily group and 622 patients in the MF-DPI 200 μg twice-daily group . Adherence was numerically lower for 12-18 year olds (once-daily group = 80.8% [95% CI: 64.1-97.5]), twice-daily group = 87.3% [95% CI: 81.2-92.4]); these patients comprised only 3% of the population, and statistical analysis was not done because there were very few patients in this subgroup. The rates of adherence for female and male patients were similar to each other and to the overall population.The primary endpoint of adherence as measured by device counter was 93.3% (95% CI: 91.6-94.9) for the once-daily group and 89.5% (95% CI: 88.1-90.8) for the twice-daily group; the maximum difference in distribution of adherence rates results was significant at 1 Figure . As meas1 Figure . In the P = not significant; Figure P = 0.08). Regarding healthcare utilization, few patients had missed days of work or school  or unscheduled health visits . No significant difference was seen between treatment groups (P ≥ 0.48).Using 2-way ANOVA with treatment and site effects, 52% of individual patients showed improvement in the physician's evaluation of therapeutic response on a scale of 1-5 from baseline, with 1 representing much improved and 5 representing much worse . The adherence rates in this study are much higher than the rates previously reported in the literature (30% to 70%),,6 and maThis study was designed to prevent some of these issues. Patients were not informed that device dose counts were checked at each visit. The study also tried to decrease aspects of a clinical study that would promote adherence and tried to mimic asthma management in a typical clinical practice. Doctor visits were limited to once a month, and patients were not required to keep daily records of symptoms or peak flow. In addition, in accordance with clinical practice guidelines,,2 patienA limitation of this study may be its duration. Treatment adherence in clinical trial settings does not typically reflect normal rates of adherence until a more prolonged period has passed. One lonP < 0.0001), during double-blind treatment and 71.4% and 63.9%, respectively (P = 0.001), during open-label treatment. Although adherence rates in the study were less than prescribed, it was found that adherence with once-daily treatment was superior to adherence with twice-daily treatment. This difference could be related to the fact that montelukast is an oral medication and fluticasone is an inhaled medication.The literature is lacking clear documentation on the impact of once- vs twice-daily dosing. However, reliable measurements of medication use were obtained in a study that evaluated adherence by patients with mild asthma randomized to treatment with oral montelukast 10 mg once-daily (n = 189) or inhaled fluticasone propionate 88 μg twice-daily (n = 191). The monDespite limitations of the present study, the observed efficacy results suggest that patients were taking their medication and that their asthma was more adequately treated than it had been prior to randomization. Both treatment groups showed improved HRQOL, and over half of all patients in both treatment groups were rated as having an improved therapeutic response from baseline. Very few patients had unscheduled office or home visits or reported missed days from work or school. The differences in adherence between the once-daily and twice-daily groups were statistically significant, yet they were small and not correlated with improvements in HRQOL. Moreover, once-daily dosing in adolescents was associated with numerically lower adherence than twice-daily dosing, although this difference was not statistically significant.Asthma therapy is complex, and treatment dosing frequency is one of many factors that affect adherence. Both groups in the present study had high rates of adherence, which suggests that factors including the physician-patient relationship, medical costs, and patient beliefs regarding illness and medication strongly influence adherence in clinical practice,34. AccoOne indication for prescribing once-daily asthma therapy is for patients who are likely to experience difficulties with twice-daily therapy. It is clear that most patients randomized to twice-daily treatment in the present study did not find this regimen to be a barrier to adherence, although overall adherence rates in clinical practice appear to be significantly lower than adherence rates in this trial. Further studies are needed to determine the degree to which twice-daily dosing might erode adherence over time, and to measure the effects of switching to a once-daily regimen for those patients who find that twice-daily therapy is a barrier to adherence. Although for some patients it is also possible that dosing frequency may have an impact on treatment efficacy, given that a once-daily medication has only one trough per day, whereas a twice-daily medication has two troughs per day. In the present study, however, the once- and twice-daily doses of MF-DPI had similar efficacy.Overall adherence to treatment with mometasone furoate dry powder inhaler was very high in this study, with a statistically greater mean adherence rate using a once-daily dosing regimen versus a twice-daily dosing regimen.AE: adverse event; ANOVA: analysis of variance; BDP: beclomethasone dipropionate; BID: twice daily; HRQOL: health-related quality of life; ICS: inhaled corticosteroid; ITG-ASF: Integrated Therapeutics Group Asthma Short Form; MF-DPI: mometasone furoate administered via a dry powder inhaler; QD PM: once-daily in the evening.Dr. Price or his team have received grants and research support from UK National Health Service, Altana Pharma, AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Merck, Sharpe and Dohme, Novartis, Pfizer, Schering Corp., a division of Merck & Co., and Teva; has consultant arrangements with Boehringer Ingelheim, GlaxoSmithKline, Merck generics, Merck, Sharpe and Dohme, Novartis, Schering Corp., a division of Merck & Co., and Teva; and has spoken for Altana Pharma, Boehringer Ingelheim, GlaxoSmithKline, Merck, Sharpe and Dohme, Pfizer, and Schering Corp., a division of Merck & Co., Dr. Robertson declares no competing interests. Dr. Bullen declares no competing interests. Dr. Rand has served on the Schering-Plough Respiratory Leadership Council and on the Merck Childhood Asthma Network advisory board. Dr. Horne has received grants from Gilead Life Sciences and Hayward Medical Communications/Shire Pharmaceuticals, and has consulted for various pharmaceutical companies. He is an officer of the University of London, School of Pharmacy. Dr. Staudinger is an employee and has stock and stock options for Schering Corp., a division of Merck & Co.Drs. DP, AR, KB, CR, and RH contributed to the acquisition, analysis, and interpretation of the data. Drs. DP, CR, and RH contributed to the design of the study. Dr. HS contributed to the design of the study and interpretation of the results. All authors critically revised the manuscript for intellectual content and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/10/1/prepub


Rates of surgically treated carpal tunnel syndrome (CTS) among blue- and white-collar workers and housewives in the general population were compared.Surgically treated cases of idiopathic CTS were investigated among 25–59-year-old residents of Tuscany, Italy, during 1997–2000, based on obligatory discharge records from all Italian public/private hospitals, archived according to residence on Tuscany’s regional database. Population data were extracted from the 2001 census.After excluding repeat admissions, 8801 eligible cases were identified. Age-standardised rates (per 100 000 person-years) of surgical CTS were: “blue-collar women”, 367.8; “white-collar women”, 88.1; “housewives”, 334.5; “blue-collar men”, 73.5; and “white-collar men”, 15.3. Compared with reference categories (same-sex white-collar workers): female blue-collar workers experienced a 4.2-fold higher standardised rate; housewives, a 3.8-fold excess; and male blue-collar workers, a 4.8-fold excess . Male and female blue-collar workers showed approximately three to sevenfold higher age-specific rates compared to their white-collar counterparts . Housewives’ rates were similar to those of blue-collar female workers up to 40–44 years of age, after which they were significantly lower (p<0.002). At all ages, housewives’ rates were much higher (p<0.001) than those of white-collar women.Surgically treated CTS was three to seven times more common (depending on age/gender) in blue-collar than in white-collar workers, which is difficult to explain by differences in body weight or other individual factors. Thus, occupational risk factors seem relevant throughout working life. The high rates for full-time housewives suggest that domestic chores should be investigated as a possible risk factor for CTS. Carpal tunnel syndrome (CTS) is a disabling condition6A survey of the general population in southern Sweden found that blue-collar workers had approximately double the risk of symptomatic CTS  compared to white-collar workers.Surgery is the treatment of choice for cases of severe chronic CTS refractory to conservative approaches.Using hospital discharge records and census data, we evaluated age- and sex-specific incidence rates of surgically treated CTS among blue- and white-collar workers and full-time housewives in the general population of Tuscany (3.5 million inhabitants), Italy in 1997–2000. In Italy during this period, diagnosis of CTS supported by median nerve conduction studies was considered a prerequisite for surgical treatment, and out-of-hospital CTS surgery was almost completely absent due to reimbursement regulations. In Italy, all public/private hospitals are obliged to provide individual, codified discharge records, even after day treatment. These discharge records are stored in databases of the patients’ region of residence, irrespective of hospital location. The discharge records of hospitals within the administrative Region of Tuscany (Regione Toscana) from the period also contain codified information on the generic occupational categories listed in –To restrict the study to idiopathic CTS, we excluded cases with secondary  diagnoses of conditions thought to be associated with a increased risk of CTS,16Population data for the age groups of interest in the study area were extracted from the most recent national census, which was conducted in 2001; of note, we are not aware of any major workforce changes in Tuscany between 1997 and 2001. To facilitate white-/blue-collar classification we chose to use census data that had been tabulated based on the ISTAT-2001 Classification of Occupations.19We calculated age-sex specific incidence rates (per 100 000 person-years) and standardised rates (age adjusted by the Standard European Population proposed by WHO)The process used to identify surgically treated cases of CTS meeting the study eligibility criteria is described in Overall age-standardised incidence rates of surgically treated CTS (per 100 000 person-years) were 255.4 (95% CI 249.6 to 261.2) for women and 46.4 (95% CI 43.8 to 49.0) for men. Among women, age-standardised rates were 367.8 (95% CI 355.1 to 380.5) for blue-collar workers, 334.4 (95% CI 322.0 to 346.9) for housewives, and 88.1 (95% CI 81.9 to 94.2) for white-collar workers. Thus, with respect to their white-collar counterparts, female blue-collar workers had a 4.2-fold higher rate of surgically treated CTS, and housewives had a 3.8-fold excess. Among men, the age-standardised rates were 73.5 (95% CI 68.9 to 78.0) for blue-collar workers and 15.3 (95% CI 13.1 to 17.5) for white-collar workers. Thus, male blue-collar workers experienced a 4.8-fold higher rate of surgically treated CTS with respect to their white-collar counterparts. This study indicates that surgically treated idiopathic CTS may be as much as three to seven times more common (depending on age and gender) in blue-collar workers than in their white-collar counterparts. It must be stressed that we were unable to adjust for factors other than age and sex. Nevertheless, we believe that such high excess risks among male and female blue-collar workers of various ages would be difficult to attribute to only moderate differencesUnsurprisingly,29The primary explanation for the greatly increased risk of surgically treated CTS recorded among male and female blue-collar workers at all ages is likely to be exposure to manual work which involves taxing hand-wrist activities, such as prolonged, highly repetitive wrist flexion/extension, forceful grip in awkward postures, and use of hand-held vibratory tools.It might be argued that blue-collar workers are more likely to access surgical treatment due to their greater need to conserve manual fitness. However, in a comparison of surgically treated and electromyographically diagnosed CTS patients in the Tuscan town of Siena,The characteristics of the Tuscan coding system also allowed us to evaluate the risks associated with unemployed housewife status. The remarkably similar patterns of surgically treated CTS among Tuscan housewives and female blue-collar workers highlights the possibly aetiological relevance of domestic work. In a case–control study of determinants of surgically treated CTS, we found that full-time housewives again appeared to have an approximately fourfold excess risk in comparison with white-collar workers (unpublished data). Furthermore, in an evaluation of associations between marital status and surgically treated CTS in several regions of Italy,33The study design allowed estimation of age-related risks associated with blue-collar employment and housewife status in the general population. We are confident that use of the hospital discharge records which every Italian public/private hospital is obliged to supply to local administrations allowed us to identify the vast majority of surgically treated cases of idiopathic CTS which occurred among residents of Tuscany between 1997 and 2000, when out-of-hospital CTS surgery was almost completely absent in Italy and going abroad for treatment was presumably not a widely considered option for CTS. Nevertheless, subsequent exclusion of retired subjects from the main analysis due to lack of information on occupational history limits the validity of the results around retirement age. Furthermore, our attempt to restrict the numerators to cases of “idiopathic” CTS may have been affected by under-reporting of concomitant conditions in the discharge records, perhaps especially in the context of patients with lower educational status. This factor might have led to slight overestimates of (i) the overall rates of surgically treated idiopathic CTS, and (ii) the rate ratios for blue-collar workers and housewives. The routine data on which the analysis was based did not permit adjustment for likely confounding factors other than age and gender. The results regarding housewives may have been particularly vulnerable to unadjusted confounding from factors such as BMI, parity and past working history, which could have contributed to an overestimate of risk. Thus, we think that our findings regarding housewives should primarily be considered a stimulus for further study. Clearly, investigation of possible aetiological relationships between manual work and body weightPossible discrepancies in blue-/white-collar/housewife classification between cases and the general population must be considered. After exclusion of military personnel and subjects with unknown active occupational status, the occupational groupings used for the census data and the hospital discharge records were readily classifiable into blue-/mixed collar and white-collar status. However, it is likely that both the hospital discharge records and census data contain some incorrectly coded information . Of note, we do not think that differential classification of housewives should be a major concern since both the hospital discharge records and the census data provide specific categories for full-time housewives.Manual work-related risk factors for surgical treatment of idiopathic carpal tunnel syndrome (CTS) appear to be relevant throughout working life for both men and women.As an occupational category, full-time housewives seem to have a raised risk of surgically treated CTS, possibly related to domestic work.In aetiological studies of carpal tunnel syndrome, attention should be paid to domestic chores as a potentially relevant risk factor.Although we were able to exclude repeated admissions with a principal diagnosis of CTS during the study period, we had no reliable way of identifying previous admissions before the study period (an issue likely to mainly effect the early part of the 4-year study period): thus the rates recorded are likely to be a slight overestimate. Of note, a sensitivity analysis based on the last 2 years of the study period did not show signs of distortion due to the inevitable inclusion of some prevalent cases. As regards the external validity of the findings, it is noteworthy that the overall rates of surgical treatment were broadly in line with those reported in other population-based surveys.In conclusion, blue-collar workers appear to have much higher rates of surgically treated idiopathic CTS than white-collar workers, irrespective of gender and age group. It is not easy to attribute such discrepancies exclusively to confounding factors such as differential body weight. Thus, the present work seems to underline the relevance of occupational risk factors for surgically treated CTS. Moreover, the remarkably similar patterns recorded for full-time housewives suggest that the role of domestic chores deserves more investigation as a possible risk factor for CTS (not only in full-time housewives). In conjunction with analysis of the possible risks associated with domestic duties, study of the biomechanical profiles of manual domestic chores could also be informative.


C. elegans model is highly suitable for testing hypotheses regarding miRNA impact on aging biology: the lifespan of the animal is approximately three weeks, there exist a wealth of genetic mutations that alter lifespan through characterized pathways, biomarkers that report strong healthspan have been defined, and many miRNA genes have been identified, expression-profiled, and knocked out. 50/114 C. elegans miRNAs change in abundance during adult life, suggesting significant potential to modulate healthspan and lifespan. Indeed, miRNA lin-4 has been elegantly shown to influence lifespan and healthspan via its lin-14 mRNA target and the insulin signaling pathway. 27 of the C. elegans age-regulated miRNAs have sequence similarity with both fly and human miRNAs. We review current understanding of a field poised to reveal major insights into potentially conserved miRNA-regulated networks that modulate aging.The last decade has witnessed a revolution in our appreciation of the extensive regulatory gene expression networks modulated by small untranslated RNAs. microRNAs (miRNAs), ~22 nt RNAs that bind imperfectly to partially homologous sites on target mRNAs to regulate transcript expression, are now known to influence a broad range of biological processes germane to development, homeostatic regulation and disease. It has been proposed that miRNAs ensure biological robustness, and aging has been described as a progressive loss of system and cellular robustness, but relatively little work to date has addressed roles of miRNAs in longevity and healthspan (the period of youthful vigor and disease resistance that precedes debilitating decline in basic functions). The  This small (1mm long) transparent animal develops in ~ 3 days through a reproducible set of fully documented cell divisions to generate a sexually mature adult of 959 somatic cells. C. elegans somatic cells do not undergo any regenerative cell divisions and developmental cell divisions are completed during larval development. Thus, the aging biology of this animal best models the situation in mammalian tissues that do not divide to replenish or repair cells. The average C. elegans lifespan is about 21 days under standard lab conditions , although lifespan can be dramatically changed by environmental factors such as low temperature growth or food limitation (dietary restriction). Notably, there is a considerable degree of variation in mean and maximum lifespan within a genetically uniform population reared under the same conditions, indicating that stochastic factors have a significant impact on healthspan and lifespan [The nematode lifespan .C. elegans life can be described as three phases: 1) a period of development to sexual maturity ; 2) the reproductive period ; and 3) in life . For exarcopenia , 10. Pumrcopenia , 12. As rcopenia , 14. TheC. elegans have been reported to affect lifespan [ C. elegans lifespan and healthspan is a conserved insulin/IGF-like signal transduction pathway that was first characterized for its role in formation of dauer larvae under adverse growth conditions. Reduction-of-function mutations in the daf-2 insulin/IGF-1 receptor and downstream signaling kinases can confer longevity [C. elegans lifespan [age-1 PI3 kinase [C. elegans model appears optimally poised to be exploited to address roles of miRNAs on lifespan and healthspan.More than 300 genetic manipulations in lifespan , a clearongevity -19 by reongevity -23. In alifespan . Mutatio3 kinase , but sta3 kinase , 11, 26,3 kinase , 14, pha3 kinase , and eve3 kinase , now mak C. elegans miRNA genes that encode sequences with characteristic miRNA precursor features , there are 154 identifiedre miRNA ). Given s animal , 30, it  in ref. ), or in C. elegans miRNAs in miRBase release 10.1 [C. elegans miRNAs share significant sequence similarities with distinct miRNAs encoded elsewhere within the nematode genome. These sequence relationships suggest considerable potential for functional redundancy within families, although evaluation of physiological redundancy requires addressing the question of whether related miRNAs are co-expressed in the same cell types at the same time, a project well underway [mir genes may not suffice to reveal the biological function of a given family. Indeed, elegantly studied examples of miRNA functional redundancy have been published for the role of the let-7 family members in regulating timing of developmental processes [What does a genome-wide overview of miRNAs reveal with regard to numbers of distinct miRNA families and their potential for functional redundancy? We evaluated all sequence relationships of mature ase 10.1 . We clasase 10.1 -36. In gunderway . One improcesses , 36. Therocesses . Thus, sC. elegans miRNAs are related to Drosophila miRNAs and ~55% of C. elegans miRNAs are related to human miRNAs—more than half of C. elegans miRNAs have common sequences in both flies and humans [let-7 family regulation of RAS oncogene expression. C. elegans let-7 miRNA regulates the timing of fate differentiation of multipotent seam cells during C. elegans development [let-7 and miR-84 have been shown to negatively regulate C. elegans RAS gene let-60, which contributes to developmental signaling [let-7 family mir genes are poorly expressed in a range of malignancies including lung cancer [In vitro, human let-7 has been shown to down-regulate expression of RAS protein and over-expression of let-7 inhibits the growth of human lung cancer cells [let-7 in vivo [Another important question germane to use of genetic models to inform on miRNA functions in the biology of aging regards how well miRNA sequences and functions might be conserved in evolution. miRNAs pose interesting problems in evolutionary biology because sequences need to be maintained in both the miRNA as well as in the critical target transcripts; miRNAs standardly modulate expression of multiple targets, adding to potential constraints . This bed humans . This exelopment , and seqignaling . In humag cancer , 42. In er cells , 42. Rec in vivo , a resulC. elegans model, with the plethora of molecular and genetic manipulations that can be executed, the comprehensive identification of conserved miRNAs that has been accomplished [Overall, however, the mplished , the weamplished , recordsmplished , 44-47, mplished  within amplished  ,C. elegans life [C. elegans adults and detected expression levels of the 114 C. elegans miRNAs registered in miRBase 5.0 via microarray hybridization [C. elegans strain used for this analysis was rendered sterile by a temperature-sensitive fertility mutation so that age-synchronized cultures could be easily maintained. Fig. (1A) depicts sampling time points relative to the lifespan curve. For each age, we normalized miRNA levels to levels in day4 first-day adult animals and plotted values over time. Fig. (1B) gives an example of how conserved miRNAs let-7, lin-4 and miR-1 change over adult C. elegans life.How miRNAs might regulate healthspan and lifespan is a question that is just coming into focus and is likely to remain at the forefront of aging research for some time. As a first step toward understanding how miRNAs might impact aging, we documented how miRNAs change in abundance during adult ans life , the firdization . The C. mir genes appear to have similar expression patterns, but in many they do not.Of the 114 miRNAs screened, we found that 50 change in expression during adulthood with a 90% confidence statistic--we refer to these 50 as age-regulated miRNAs. 34 of these age-regulated miRNAs fell within a 95% confidence level. Nearly two thirds (31) varied more than twofold between maximum and minimum expression levels over adult life. In terms of expression patterns, we did not note a clear correlation in the expression of age-regulated miR family members or in that of age-regulated miRNAs clustered in the genome—in some cases family members or clustered C. elegans study provides an indication of how whole-body levels of individual miRNAs vary on average, but that miRNAs that change in only a few cells, or miRNAs that change reciprocally in different tissues could be missed in this analysis. Thus, there is likely even more extensive modulation of miRNA levels during aging than appears from this first general survey. Nevertheless, one certain conclusion emerges: there are substantial changes in levels of many miRNAs during adult C. elegans life. As such, these age-regulated miRNAs are candidate modulators of aging and longevity.It is probably worth emphasizing that this C. elegans adulthood, 27 share sequence conservation with human miRNAs  have been investigated for roles in biology apart from aging. Which age-regulated miRNAs influence aging in nematodes, flies and/or humans remains mostly to be experimentally determined. Given that conserved mechanisms such as insulin signaling, dietary restriction and oxidative stress responses impact both lifespan and healthspan across phyla, it is plausible that functional studies of conserved miRNAs in facile invertebrate models could identify a novel set of small molecule modulators of universal components of aging.Interestingly, of the 50 miRNAs that significantly change in expression during refer to  for detaC. elegans adulthood. Interestingly, a considerable number of age-regulated miRNAs diminish in expression over adult life. This trend is most striking when considering expression changes during the reproductive phase of early adulthood (days 6-8) or from early to mid adult life (days 6-11). Expression of about two thirds of the age-related miRNAs  diminishes during the reproductive stage (d6-8) or during the reproductive to mid-life stage (d6-11). This downregulation is not likely to be solely attributed to germ cell production since germ cell proliferation would increase miRNA abundance, and because many of the miRNAs that change with age do not appear to be germline expressed in published studies. Expression of most of these age-regulated miRNAs is similar in wild-type and young glp-4(bn2) mutant adults that lack most germline cells [spe-9 mutant (our study used mutations to block fertilization so embryonic development cannot contribute). That there appears to be a general down-regulation of miRNAs suggests that a relaxation in precise transcriptional/translational controls might be a general feature of advancing adult life in C. elegans. It is conceivable that a modest dysregulation of protein synthesis could initiate declines in multiple systems during adulthood. This hypothesis remains to be tested, beginning with whole-genome analysis of proteomic consequences of miRNA decline.We found that miRNA expression levels varied in a range of pattern types during ne cells , indicatE and Suppl. Fig. S2 in [C. elegans equivalent of centenarians. If any of the late-changing miRNAs identified are causative in end-stage aging biology, these two models might be experimentally addressed by testing consequences of deletion or over-expression of miRNAs that change the most at the day 15 timepoint. Finally, one might wonder about the miRNAs with greatest changes in relative abundance over adult life. One miRNA, conserved miR-231 , stands out as the most abundant age-regulated C. elegans miRNA (highest levels at day 15) with the largest net increase (~11-fold) and greatest variation over adulthood . miR-231, and other miRNAs that change with age, might impact expression of gene batteries that modulate lifespan or the quality of aging, a general hypothesis that can be tested with the powerful research tools that can be applied in this model. We list age-regulated C. elegans miRNAs exhibiting largest changes in expression in (i) early adulthood, (ii) late adulthood or (iii) over adult life in Table 2.One can speculate that specific patterns of expression might identify miRNAs that exert specific influences on the biology of aging. For example, miRNAs that decline early in adult life might be associated with, or possibly even causal in, “mid-life” crises in metabolism such as the transition to increased rates of age pigment/lipofuscin accumulation or the onset of sarcopenia , 14. Theg. S2 in  for a lilin-4 impact on aging [A key question is to what extent miRNAs influence the biology of healthy aging and lifespan in a given organism and across species. It has been proposed that miRNAs function in biology to insure developmental robustness and maintain homeostasis . Since aon aging  is the fC. elegans lin-4 binds to 3’ UTR sequences of the lin-14 mRNA to downregulate expression of the transcription factor LIN-14, enabling specification of hypodermal cell fate transition from the first to the second larval stage [2A). With regard to adult phenotypes, lin-4 loss-of-function (lf) shortens lifespan, whereas over-expression of lin-4 lengthens lifespan . Long life is associated with lowered levels of age pigment accumulation and increased heat stress resistance, suggesting that not just lifespan, but also healthspan is modulated by the lin-4 miRNA. In support of a normal role for lin-4 in healthspan promotion, we have also observed accelerated locomotory decline in middle-age lin-4(lf) mutants . lin-4 requires the presence of target lin-14 to exert its effects in adult life, with lin-14(gf) (a mutant that lacks the lin-4 binding sites in the 3’UTR) mimicking the deleterious effects of lin-4(lf) [lin-4 function in adult life limits pro-aging effects of lin-14. Since lin-4 is one of the miRNAs that decline in abundance with age, we could speculate that as lin-4 levels diminish during adult life, elevated expression of lin-14 occurs in aging animals with deleterious consequences for the quality of aging . This may be a good example of antagonistic pleiotropy in which a gene that confers developmental benefit is actually deleterious later in life [During development, al stage   . Togethe in life .lin-14 promote age-associated decline? lin-14(lf) does not extend lifespan without the critical downstream transcription factors of insulin signaling FOXO/DAF-16 or HSF-1, suggesting that lin-14 modulates the level of insulin signaling, a well characterized mechanism for modulation of lifespan .How does C. elegans mir genes have been knocked out, including 87 deleted in one massive screen [mir deletion mutants did not exhibit lethality or major phenotypic changes in developmental timing, mobility, gross morphology and reproduction. One possible explanation is that there could be a considerable amount of functional redundancy among sequence-related or other miRNAs. As noted above, there is strong experimental evidence for functional redundancy for the let-7 family [C. elegans miRNAs share potential for functional redundancy [lin-4 investigations on lifespan [C. elegans miRNAs on lifespan and healthspan. Although much carefully controlled work remains to be done, a first pass look at age-associated phenotypes in several mir mutants suggests that multiple miRNAs may prove to promote healthspan , consistent with the hypothesis that this gene class may contribute to robustness required for maintenance of healthspan. If this proves to be the case, the C. elegans model will be an important proving ground for genetic miRNA “therapies” that might promote healthy aging in transgenic studies.Many e screen . Interes7 family , 36, anddundancy . It is alifespan , there aCentral to the understanding of miRNA impact on aging will be the identification of physiologically relevant targets. The expectation is that a highly complex web of miRNA interactions must exist, given that individual transcripts can be targeted by multiple miRNAs and that individual miRNAs can interact with multiple transcripts. In the absence of quantitative, cell-specific biochemical identification of miRNA-targeted transcripts, computational target predictions have been generated to suggest the arrays of mRNA transcripts that might be subjected to regulation by specific miRNAs e.g. , 59, 60), 60 59, C. elegans transcripts for 3’ UTR target sites of the 50 age-regulated miRNAs  to idenle S3 of  for totae lin-14 , 57, 64)Computational strategies such as these can highlight specific miRNAs for experimental investigations of tissue-specific age-related declines. For example, with an interest in identifying miRNAs that might impact muscle aging, we searched a list of genes expressed preferentially in muscle  for 3’ UGigantic strides have been accomplished in the understanding of miRNA biogenesis and in deciphering mechanisms of miRNA regulation of cellular processes during development, health and pathology. Mammalian miRNA profiles in the areas of diseases of aging such as cancer  and neurC. elegans. However, intriguing potential connections have begun to appear. For example, it is tantalizing that in mammalian cells, miR-34 elevation is associated with p53-induced cell senescence [C. elegans miR-34 is one of the few miRNAs that increase in abundance with age [C. elegans could easily be tested given reagents available in this facile model. In the future, key advances in cataloging age-regulated miRNAs in mammalian tissues should be compared with studies of roles of C. elegans miRNAs in analogous tissues to begin to address whether conserved miRNA-regulated processes relevant to aging biology might exist.At present, it is probably premature to compare mammalian tissue-specific studies to whole animal surveys in nescence , 73 and with age . HypotheC. elegans converge with extensive lifespan and healthspan studies conducted in this model to date to set up a powerful system for the experimental evaluation of roles of miRNAs in the biology of aging of a single organism. We know that there are significant changes in miRNA abundance during adult life that occur in various “patterns” (with nearly 50% of 114 tested miRNAs modulated) and the elegant lin-4 study from Bohem and Slack [mir mutants and in lines that over-express individual mir genes. It will be particularly important to determine whether there a few key “node” miRNAs that regulate large networks of genes that impact aging, such as oxidative stress response networks, metabolic pathways, and insulin signaling pathways. Defining such node miRNA regulators of aging biology will hold particular significance if they exert conserved roles in mammalian healthspan.In this review we have emphasized how basic characterization of miRNAs in nd Slack  serves aC. elegans [vs those that age poorly might indicate expression patterns correlated with strong healthspan. Given the suggestion that miRNA changes in midlife might underlie some of the early pre-clinical changes that ultimately manifest as age-associated decline [It is also possible that specific miRNA levels in adult life have potential to be used as diagnostics for scoring “physiological”  age as is the case for age pigment levels in  elegans . Compari decline , it shouC. elegans model might also be used to address how well genetic interventions using miRNAs can be used to extend healthy life phases. Genetic engineering or direct miRNA administration that modifies miRNA expression time or level can be conducted to address basic principles regarding small molecular therapeutics that promote healthy aging. Our initial impression is that whole life over-expression of individual mir genes may not be easily tolerated by the animal —however, late life expression/administration, more relevant to potential clinical application, remains to be evaluated. Success will support conjecture that miRNA modulation might have therapeutic value [Finally, the ic value , 76 in m


Caenorhabditis elegans, male mating behavior, which provided a model for understanding motor behaviors at the genetic, molecular as well as circuit level. C. elegans male mating behavior consists of a series of six sub-steps: response to contact, backing, turning, vulva location, spicule insertion, and sperm transfer. The male tail contains most of the sensory structures required for mating, in addition to the copulatory structures, and thus to carry out the steps of mating behavior, the male must keep his tail in contact with the hermaphrodite. However, because the hermaphrodite does not play an active role in mating and continues moving, the male must modify his tail posture to maintain contact. We provide a better understanding of the molecular and neuro-muscular pathways that regulate male tail posture during mating.To survive and reproduce, animals must be able to modify their motor behavior in response to changes in the environment. We studied a complex behavior of Genetic and laser ablation analysis, in conjunction with behavioral assays were used to determine neurotransmitters, receptors, neurons and muscles required for the regulation of male tail posture. We showed that proper male tail posture is maintained by the coordinated activity of opposing muscle groups that curl the tail ventrally and dorsally. Specifically, acetylcholine regulates both ventral and dorsal curling of the male tail, partially through anthelmintic levamisole-sensitive, nicotinic receptor subunits. Male-specific muscles are required for acetylcholine-driven ventral curling of the male tail but dorsal curling requires the dorsal body wall muscles shared by males and hermaphrodites. Gamma-aminobutyric acid activity is required for both dorsal and ventral acetylcholine-induced curling of the male tail and an inhibitory gamma-aminobutyric acid receptor, UNC-49, prevents over-curling of the male tail during mating, suggesting that cross-inhibition of muscle groups helps maintain proper tail posture.Our results demonstrated that coordination of opposing sex-specific and core muscle groups, through the activity of multiple neurotransmitters, is required for regulation of male tail posture during mating. We have provided a simple model for regulation of male tail posture that provides a foundation for studies of how genes, molecular pathways, and neural circuits contribute to sensory regulation of this motor behavior. Caenorhabditis elegans. Arguably the most complex motor behavior displayed by this organism, male mating can be separated into at least six sub-steps: response, backing, turning, vulva location, spicule insertion, and sperm transfer . Do. Dotph-1lin-39 mutant males to respond to aldicarb. In lin-39 mutant males, CP neurons 1–4 die or are necrotic, while CP neurons 5 and 6 take on a more posterior fate, and lin-39 mutant males have a turning defect similar to that of males in which the CP neurons have been ablated [lin-39 (n1760) mutant males in response to aldicarb was lower, but not significantly so , UNC-49 ,36. To taldicarb . Mutatiog Figure  and 10b.unc-30, a homeodomain containing transcription factor that is required for the terminal differentiation of the D-type motor neurons [unc-30 mutant males showed a significant decrease in dorsal tail curling, indicating that acetylcholine may act in part through the D-type neurons to promote dorsal tail curling  mutation in the background of the wild-type N2 strain in the following strains: unc-29(e1072), unc-38(sy576), tph-1(mg280), lin-39(n1760), unc-64(e246), and unc-30(e191). To generate males in the following strains, hermaphrodites were heat shocked at 29°C for approximately 10 hours and then the resulting male and hermaphrodite progeny were crossed to generate males: unc-63(e384), unc-25(e156), and unc-49(e407).All strains were cultured using standard methods . To incrAs there is day-to-day variation in behavioral assays, in all assays, control animals were assayed on the same day as mutant or ablated males for comparison.unc-31(e169) hermaphrodites. Response behavior was recorded as follows: the total amount of time it took males to respond to a hermaphrodite was noted . Also, the length of time males spent responding to a hermaphrodite before leaving or finding the vulva was noted (continued response). Backing behavior was assayed as follows: males were observed for their ability to back along the hermaphrodite. In addition to noting the ability to back along the hermaphrodite, it was also noted if males instead of backing along the dorsal and ventral sides of the hermaphrodite, backed along the lateral sides of the hermaphrodite. Turning behavior was assayed as follows: the number of bad turns was noted. Turns were categorized as missed, sloppy, tip, or stutter as described in Liu et al. [Mating behavior was observed using a Wild M5A microscope at ×25 and ×50 magnification. Males were isolated from hermaphrodites as L4s and used the following day. Males were only assayed one time and then destroyed. An exception to this was that M cell-ablated and sham-ablated control males were used 16 to 32 hours after being isolated as L4s. Also, these males were assayed two times with at least a 1-hour recovery period between assays. Males were assayed for their ability to mate with partially paralyzed u et al. . In addiAldicarb was purchased from Chem. Service . For assays, aldicarb was diluted from a 5 mM, or for the assays comparing males and hermaphrodites, a 25 mM, frozen stock solution. Levamisole was purchased from Sigma-Aldrich. Levamisole was diluted from a 50 mM frozen stock solution. All drugs were diluted in ddH20. L4 males were isolated the day before assays. An exception to this is M cell, dorsal body wall, and sham-ablated males, which were first assayed for mating behavior and then after a recovery period were assayed for tail curling in response to drugs. To assay males for tail curling in response to drugs, 500 μl of drug or ddH20 control solution were put in Becton Dickinson 24-well tissue culture plates. Three to six males were assayed at one time and the solution used was discarded after each assay. Males were assayed immediately, and then at 2.5-minute intervals for the first 10 minutes and then at 5-minute intervals. At each time point, males were examined for 20 seconds. Males were considered positive for each behavior if they curled their tail for >5 seconds. If both types of tail curling were seen during that period only the first response seen was recorded.Laser ablation was done using a VSL-337 nitrogen laser and a Zeiss axioskop using standard protocols . Sham-abunc-29::dsRed construct was made by amplifying a fragment containing 1529 base pairs upstream of the start of unc-29 and having PstI sites designed at the ends of both primers. This was then first sub-cloned into the T-easy vector and then digested with PstI. The PstI fragment was then sub-cloned into the vector PSX77, containing dsRed 2 followed by the unc-54 3' UTR (courtesy of S. Xu), and injected into pha-1; him-5 males along with a pha-1 rescuing plasmid. Three stable lines were examined for expression of unc-29::dsRed and all had similar expression patterns.The All statistical tests were done using the GraphPad InStat software. Appropriate tests were chosen based on the recommendations of the program.GABA: gamma-aminobutyric acid; GABAR: gamma-aminobutyric acid receptor.AJW and PWS conceived the study and designed the experiments. AJW carried out the experiments. AJW wrote the manuscript and PWS provided input and revised the manuscript.


We have reviewed the pivotal presentations rcelated to colorectal cancer (CRC) and other gastrointestinal malignancies from 2008 annual meeting of the American Society of Clinical Oncology (ASCO). We have discussed the scientific findings and the impact on practice guidelines and ongoing clinical trials. The report on KRAS status in patients with metastatic CRC receiving epidermal growth factor receptor (EGFR) targeted antibody treatment has led to a change in National Comprehensive Cancer Network guideline that recommends only patients with wild-type KRAS tumor should receive this treatment. The results of double biologics (bevacizumab and anti-EGFR antibody) plus chemotherapy as first-line treatment in patients with metastatic CRC has shown a worse outcome than bevacizumab-based regimen. Microsatellite Instability has again been confirmed to be an important predictor in patients with stage II colon cancer receiving adjuvant treatment.Adjuvant gemcitabine therapy for pancreatic cancer was investigated by the CONKO-001 study; this resulted in superior survival as compared with observation and can be regarded as an acceptable option, without the addition of radiotherapy. The addition of bevacizumab to gemcitabine and erlotinib was not supior to gemcitabine and erlotinib for advanced disease. Second-line therapy for advanced pancreatic cancer with 5-fluorouracil and oxaliplatin resulted in a survival benefit. Irinotecan plus cisplatin and paclitaxel plus cisplatin result in similar survival when combined with radiotherapy for esophageal cancer. The novel fluoropyrimidine S1 appears to be active in gastric cancer, as a single agent or as combination therapy. Adjuvant intraperitoneal mitomycin-C may decrease the incidence of peritoneal recurrence of gastric cancer. Sorafenib is an effective agent in Asian patients with hepatocellular carcinoma secondary to hepatitis B; its utility in child's B cirrhosis remains to be proven. Sunitinib is also an active agent in hepatocellular carcinoma, and may represent an alterative to sorafenib for advanced disease. These and other important presentations from the 2008 ASCO annual meeting are discussed in this article. Colorectal cancer (CRC) is among the top three most common malignancies and cancer-related death in Western world including United States . In 2008Cetuximab and panitumumab are epidermal growth factor receptor (EGFR) targeted antibodies approved for clinical use in patients with metastatic CRC. Ligand binding of the EGFR activates the RAS/RAF/MAPK, STAT, and PI3K/AKT signaling pathways, which modulate cellular proliferation, angiogenesis, and survival. However, the level of EGFR expression as measured by immunohistochemistry does not predict clinical benefit .KRAS, the human homolog of the Kirsten rat sarcoma-2 virus oncogene, encodes a small GTP-binding protein, and acts as signal transducer in response to ligand binding of growth factor receptor, including EGFR . KRAS caThe CRYSTAL study is a phase III study comparing first-line chemotherapy with a regimen of 5-fluorouracil (5-FU), leucovorin (LV) and irinotecan, known as FOLFIRI, with or without cetuximab. At 2007 ASCO annual meeting, data from the CRYSTAL study was first presented, which showed that addition of cetuximab to FOLFIRI increased response rate (RR) by 8% and prolonged progression-free survival (PFS) by 0.9 months  fluoro-2-deoxy-D-glucose (FDG) and positron emission tomography (PET) is thought to be better than CT scan, however the evidence of improved clinical management and outcome is lacking. Wiering et al. reported a randomized controlled study enrolling 150 CRC patients with liver metastasis planning for surgical resection . PatientChemotherapy is frequently administered after complete resection of liver metastases from CRC, but the optimal regimen is yet to be established. Dr. Marc Ychou presented preliminary finding from CPT-GMA-301 study, which was conducted to compare 6-month of adjuvant chemotherapy with 5-FU/LV vs. FOLFIRI in this setting with DFS as the primary endpoint . This raAllegra et al. reported the initial safety report of NSABP C-08, a phase III randomized study enrolling 2,710 patients to compare a modified FOLFOX regimen known as mFOLOX6 (every 2 weeks for 12 cycles) vs. bevacizumab and mFOLFOX6  as adjuvant therapy in patients with stage II/III colon cancer after surgery . A signiWolmark and colleagues presented an update on the previously reported NSABP C-07 trial . The triThe benefit of adjuvant chemotherapy is still debatable for stage II colon cancer. The United Kingdom QUASAR study has shown chemotherapy with 5-FU and LV in patients with stage II CRC can provide a small improvement (~4%) in rates of recurrence and OS compared to patients on observation . HoweverIn 2008 ASCO annual meeting, Sargent et al. presented an analysis of 491 patients from five clinical trials randomizing patients with stage II and III colon cancer to either 5-FU based adjuvant chemotherapy or no post-operative treatment . Those wThe sensory neuropathy associated with cumulative oxaliplatin treatment frequently interrupts with the administration of oxaliplatin-based regimen for CRC. Two studies presented at the 2008 ASCO annual meeting examined the strategy of using calcium and magnesium, and one study also examined intermittent administration of oxaliplatin in minimizing neurotoxicity from oxaliplatin.The Combined Oxaliplatin Neurotoxicity Prevention Trial (CONcePT) randomized patients receiving first-line therapy for metastatic CRC to either continuous or intermittent FOLFOX plus bevacizumab. The intermittent arm differed from the continuous arm in that oxaliplatin was stopped after 8 cycles in patients who had at least stable disease, then was re-started after another 8 cycles of maintenance therapy with bevacizumab and infusional 5-FU/LV or tumor progression during maintenance treatment. Patients in both arms were also randomized to receive intravenous calcium gluconate and magnesium sulfate (CaMg) before and after oxaliplatin treatment. This study was discontinued prematurely due to interim analysis suggesting there were significantly lower RR in patients receiving CaMg. However, a subsequent independent radiology review did not find any evidence of detrimental effect from CaMg on the activity of FOLFOX plus bevacizumab. An analysis of the 139 patients who received treatment per protocol was performed and presented at 2008 ASCO annual meeting . Time toN04C7 was designed as a placebo-controlled phase III study to prospectively evaluate the activity of CaMg as neuroprotectant against cumulative oxaliplatin-related peripheral sensory neurotoxicity . PatientNon-colorectal gastrointestinal cancers have a significant burden world-wide. Modern multimodal approaches that integrate surgery, radiation and systemic therapy, the development of new cytotoxic agents along with anti-EGFR and anti-VEGF therapies have led to a significant survival improvement for colorectal cancer patients. Advances have been limited however, in the case of non-colorectal gastrointestinal cancers. The research presented at the 2008 ASCO annual meeting indicates that progress in these cancers is forthcoming. Abstracts from the annual meeting are grouped below into categories based on disease sites: pancreatic, esophagogastric, and hepatobiliary.Surgical resection remains the only potential curative therapy for pancreatic cancer patients. However, 5-year survival for surgically resected patients is 30% only and most patients die of disseminated disease. Therefore, effective adjuvant strategies are needed. The Gastrointestinal Tumor Study Group (GITSG) 9173 trial indicated that post-operative 5-FU and radiotherapy extended the median overall survival to 20 months, as compared with 12 months with observation alone. Similar results were reported subsequently by the Johns Hopkins and European Organization for Research and Treatment of Cancer (EORTC) and 5-FU -based chemoradiation became the standard-of-care for nearly two decades . The EurTogether, the CONKO-1 and the ESPAC-1 studies argue against the use of standard, post-operative, adjuvant radiotherapy for pancreatic cancer. Preoperative chemoradiation strategies that decrease margin-positive resections and pre-select patients with better cancer biology for resection deserve further exploration.The addition of erlotinib to gemcitabine improved OS as compared with gemcitabine alone, in the PA.3 study, although the median survival increase was very modest (5.9 to 6.4 months with the addition of erlotinib) . The addThere was no significant prolongation of survival with the addition of bevacizumab, although DFS was significantly improved (from 3.6 to 4.6 months). Bevacizumab was reported to be safe in this combination, despite an increase in the incidence of epistaxis, hypertension and proteinuria. Interestingly, there was no reported increase in thrombotic events with bevacizumab. The AVITA study suggests that antiangiogenic strategies may have merit in the treatment of advanced pancreatic cancer, although the margin of benefit with bevacizumab is modest. Therefore, it is imperative that we identify patient subgroups that may benefit from such an approach.Kindler et al. investigated a multi-targeted strategy against both the EGFR and VEGF in a randomized phase II study. Pancreatic cancer patients (n = 139) received gemcitabine, bevacizumab and erlotinib or gemcitabine, bevacizumab and cetuximab . They noGemcitabine has become the standard therapy for advanced pancreatic cancer, since its approval almost a decade ago. Subsequent investigational strategies have included the addition of other, targeted agents to gemcitabine. There have been very few attempts to address the role of alternative cytotoxic agents (which may represent better platforms for the addition of targeted therapies) other than gemcitabine in the first-line setting. The FFCD group randomized 202 patients with advanced, untreated pancreatic cancer to gemcitabine or 5-FU plus cisplatin . PatientThere are no standard second-line regimens for advanced pancreatic cancer, after gemcitabine failure. However, capecitabine, capecitabine and oxaliplatin (CAPOX), and FOLFOX are commonly used. The CONKO-3 study randomized 168 patients who had gemcitabine-refractory pancreatic cancer to 5-FU, LV and oxaliplatin (OFF) or 5-FU and LV (FF) . The stuNanoparticle albumin-bound (Nab)-paclitaxel is a novel paclitaxel formulation which is currently approved for the treatment of breast cancer. Due to the nano-size and presence of albumin, which attracts the particle to tumor sites, the penetration of cytotoxic agents bound to the nanoparticle is very high, possibly leading to a better anti-tumor response. Preclinical data indicated that increased expression of Secreted Protein and Rich in Cysteine (SPARC) within tumors resulted in improved anti-tumor response to Nab-paclitaxel. Pancreatic cancer overexpresses SPARC protein and impressive partial responses (PRs) were seen in a phase I pancreatic cancer study of Nab-paclitaxel + gemcitabine . Nine PRThe CALGB presented the results of a single-arm phase II study of sunitinib for patients with advanced pancreatic cancer who had previously been treated with gemcitabine-based therapy. No responses were reported in 77 treated patients, and stable disease resulted in 7 patients . The CalGemcitabine in combination with radiotherapy (50.4 Gy in 28 fractions) was compared with gemcitabine chemotherapy  for locally advanced pancreatic cancer by the Eastern Cooperative Oncology Group (ECOG) 4201 study. Although this study was closed early (74 patients enrolled out of planned size of 316 patients) due to poor enrollment, there was a statistically significant median-survival improvement in the combination-therapy arm (9.2 months with gemcitabine and 11 months with gemcitabine and radiation). Unfortunately, this very modest survival improvement occurred at the expense of 40% grade 4 gastrointestinal/hematological toxicities in patients receiving chemoradiation. Kim et al. reported the results of a phase II study of gemcitabine, oxaliplatin and radiotherapy, followed by gemcitabine chemotherapy in 53 patients with locally advanced pancreatic cancer . PancreaInvestigators at Johns Hopkins presented data on their preclinical model termed PanXenoBank, which uses surgically resected pancreatic adenocarcinoma to generate xenograft in mice .in vivo platform for drug testing and provides data that can be used prospectively to treat patients. Of the 90 patients whose cancers were xenografted, 18 failed to engraft and preliminary results in the rest indicated that a tailored-approach, using unconventional agents such as mitomycin-C was possible. While the data in this regard is premature and the technique is labor-intensive, this strategy holds promise. The MD Anderson group presented data on single nucleotide polymorphisms (SNPs) of genes involved in gemcitabine metabolism and showed that cytidine deaminase and deoxycytidine kinase alleles were associated with an improved survival, albeit at the cost of increased toxicity [This xenograft serves as an toxicity . Collisstoxicity . They coThe Japanese Oncology Group (JCOG) presented updated data from the JCOG 9907 study, which randomized 330 patients with stage II or III esophageal cancer to pre-or post-operative chemotherapy with 5-FU and cisplatin . In the Irinotecan plus cisplatin, paclitaxel plus cisplatin, and 5-FU plus cisplatin are commonly used chemotherapeutic combinations along with radiotherapy for the preoperative treatment of esophageal cancer. The ECOG 1201 was a randomized phase II study for paclitaxel plus cisplatin vs. irinotecan plus cisplatin along with radiotherapy for these patients . All patStahl et al. reported the results of a randomized trial of chemoradiation ± surgery for operable squamous cell carcinoma of the esophagus . In theiPeritoneal relapse occurs frequently in surgically resected gastric cancer. Therefore, adjuvant intraperitoneal chemotherapy merits study. Kang et al. randomized 640 patients with gastric cancer that had involved the serosa  to intraperitoneal cisplatin, early mitomycin-C (administered on day 1) after surgery and an extended schedule of doxifluridine (for 12 months) and cisplatin, vs. delayed mitomycin-C (3–6 weeks post-operative) and a 3 month course of doxifluridine . The resDocetaxel, cisplatin and 5-FU (DCF) is a FDA-approved regimen for gastric cancer, based on the results of the V325 study, which proved the superiority of this regimen as compared with 5-FU and cisplatin . HoweverCetuximab was investigated in second-line setting as a single-agent in patients with metastatic esophageal cancer by SWOG . MarginaSorafenib is a novel inhibitor of Raf kinase, VEGF receptor (VEGFR) and platelet-derived growth factor receptor, and has been recently approved for the treatment of advanced hepatocellular cancer (HCC), based on the results of the SHARP trial . In thisRaoul et al. studied the effect of sorafenib in ECOG performance status (PS) 1 and 2 patients as a subanalysis from the SHARP trial . They noIn Asian countries, the incidence of HCC is higher than in the western nations and is more likely to be HBV-associated. Cheng et al. randomized 226 Asian patients with HCC to sorafenib or placebo. Sorafenib significantly prolonged OS and PFS as compared with placebo . The degAbou-Alfa et al. presented the results of sorafenib therapy in HCC patients with child's B cirrhosis . In a phSunitinib is a multi-targeted tyrosine kinase inhibitor that is directed against VEGFR2 and has activity comparable to that of sorafenib. Zhu et al. presented phase II data of 34 patients with HCC who received sunitinib in a dose of 37.5 mg daily. Median PFS was 4 months and OS 10 months . BiomarkBevacizumab and erlotinib may have promising activity in this chemo-refractory disease. In a preliminary analysis of their multi-center phase II study, Holen et al. reported 4 PRs resulting from this biologic-only combination among 20 assessed patients, and 4 cases with stable disease . CetuximPeriampullary cancers may have a better prognosis as compared with pancreatic and biliary cancers. However, the standard of care in this patient population remains to be defined, due to the relative rarity of this disease. Overman et al. reported the efficacy results of a phase II study of CAPOX in advanced periampullary cancer. An impressive 50% RR and a median survival of 20 months was recorded . Grade 3The authors declare that they have no competing interests.Both authors participated in drafting and editing the manuscript. Both authors read and approved the final manuscript.