APTR, that acts in trans to repress the CDKN1A/p21 promoter independent of p53 to promote cell proliferation. APTR associates with the promoter of CDKN1A/p21 and this association requires a complementary-Alu sequence encoded in APTR. A different module of APTR associates with and recruits the Polycomb repressive complex 2 (PRC2) to epigenetically repress the p21 promoter. A decrease in APTR is necessary for the induction of p21 after heat stress and DNA damage by doxorubicin, and the levels of APTR and p21 are anti-correlated in human glioblastomas. Our data identify a new regulator of the cell-cycle inhibitor CDKN1A/p21 that acts as a proliferative factor in cancer cell lines and in glioblastomas and demonstrate that Alu elements present in lncRNAs can contribute to targeting regulatory lncRNAs to promoters.Long noncoding RNAs (lncRNAs) have emerged as a major regulator of cell physiology, but many of which have no known function. CDKN1A/p21 is an important inhibitor of the cell-cycle, regulator of the DNA damage response and effector of the tumor suppressor p53, playing a crucial role in tumor development and prevention. In order to identify a regulator for tumor progression, we performed an siRNA screen of human lncRNAs required for cell proliferation, and identified a novel lncRNA,  Xist is a cis-acting RNA responsible for the initiation and spreading of inactivation of the very X chromosome that it is transcribed from, by interacting with and recruiting the Polycomb repressive complex 2 (PRC2) Xist-PRC2 complex is recruited to the X-chromosome inactivation center by DNA binding protein YY1 trans-acting lncRNAs, like HOTAIR, regulate gene expression at sites away from where they are transcribed, by recruiting to these sites chromatin-modifying complexes such as PRC2, LSD1 and CoREST/REST trans-acting lncRNAs are recruited to their target gene locations.Long noncoding RNAs (lncRNAs), transcribed from outside (intergenic) or within protein coding regions (intragenic), are >200 nucleotides in length but do not code for proteins. Thousands of lncRNAs have been identified in mammalian cells, many with expression patterns specifically restricted by cell or tissue-type and development stage p21 is expressed ubiquitously and associates with and inhibits kinases important for G1/S transition such as cyclin D/CDK4, Cyclin D/CDK6 and Cyclin E/CDK2 The cyclin-dependent kinase (CDK) inhibitor Alu-mediated p21 transcriptional regulator (APTR) necessary for cell proliferation. APTR represses p21 transcription by recruiting the PRC2 complex to the p21 promoter. The complementary Alu (c-Alu) element embedded in APTR is required for the localization of APTR to the p21 promoter, suggesting that embedded Alu elements in lncRNAs can contribute to the functions of lncRNAs. Cellular stresses that induce p21 such as heat shock and doxorubicin treatment down-regulate APTR, and this downregulation is important for the induction of p21 independent of whether p53 is active or not. A survey of gliomas suggests that APTR and p21 levels are anti-correlated. Our results identify a new regulator of p21, an lncRNA APTR, that silences p21 epigenetically by recruiting PRC2 to the p21 promoter.In this study, we identify a novel lncRNA Ten fresh frozen primary glioblastoma multiforme specimens and two normal brain tissue samples were obtained from patients undergoing surgical treatment at the University of Virginia Hospital following written informed consent and in accordance with a protocol approved by the University of Virginia's Institutional Review Board for Health Sciences Research. These tumors have been studied in two previous publications APTR was obtained from the FLJ cDNA library. For MS2 pulldown assays, plasmid pUC-MS2 coat protein (MS2BP) fused to YFP (Addgene plasmid 27122) and plasmid pUC-24MS2 were used. The 24 copies of MS2 stem loops were amplified from the 24MS2 stem loop cassette (Addgene 45162) by PCR and inserted at the 3′ end of APTR in the pcDNA3-APTR plasmid (Invitrogen). For luciferase assays, various lengths  of the p21 promoter with/without the Alu-containing region (−3389 to −2595 from the TSS) were cloned by PCR using 293T cell genomic DNA and inserted into the HindIII sites of the pGL4.20 plasmid (Promega).The full length nucleotide sequence of In all the figures, Student's t-test was used for testing whether the differences were statistically significant, and analyzed using GraphPad Prism . Statistical significance was defined at P<0.05.TM2000 or RNAiMAX (Invitrogen) according to the manufacturer's protocols. The siRNA sequences are provided in GL2, APTR#1 or #2. After 24 hrs of siRNA transfection, cells were treated with nocodazole (0.1 µg/ml) for 16 hrs and then harvested and analyzed by FACScaliber (BD biosciences) for DNA content.MCF10A, PC3, 293T and U87 cells were grown in DMEM, glioma cell line A172 and HCT116 cells were grown in McCoy's 5a Medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. MCF10A, PC3, 293T, HCT116, A172 and U87 were purchased from the American Type Culture Collection . HCT116 and p53−/−, p21−/− derivatives were supplied by Dr. B. Vogelstein 2SO4 to stop the reaction. For data analysis, each assay plate contained four wells of negative control  and two wells of positive control (ORC2) siRNAs for normalization. To normalize values of BrdU incorporation, we calculated the inhibition index (%) using the following equation: Inhibition index of gene X (%)  =  (GL2av−X)/(GL2av−ORC2av) ×100, where X, GL2av, and ORC2av represent BrdU incorporation (absorbance at 450 nm) of gene X and average of GL2 and ORC2, respectively. Genes with a SD of inhibition indices  greater than a cutoff value were eliminated to select technically reproducible data.Three different siRNAs were designed against each of the 286 candidate lncRNAs to avoid off-target effects, and transfected into PC3 and MCF10A cells using three 96-well plates with three technical replicates. Two to three days after transfection, cells were incubated with 10 µM BrdU for 15 min to 1 hr, and fixed with FixDenat  for 30 min. Cells were blocked with 3% BSA in PBS for 1 hr, then incubated with HRP-coupled anti-BrdU antibody (Roche) diluted in 3% BSA in PBS for 1 hr. After washing three times with PBS containing 0.1% TX-100, cells were incubated with TMB substrate  for 5–10 min and analyzed by the absorbance at 450 nm following the addition of 1 M H18 or Random hexamer by the Superscript III First Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's instructions. Quantitative RT-PCR was performed by iCycler iQ™ Real time PCR Detection System (BioRad) with SYBR green Master Mix (BioRad). The Ct values were determined with the default threshold setting. Relative expressions for the target RNAs were determined by the comparative CT (2−ΔΔCt) method after normalization to GAPDH.Total RNA was extracted from cells using Trizol total RNA isolation reagent (Invitrogen). cDNA was synthesized with oligo (dT)APTR deletion mutant-encoding plasmids. Total RNA was analyzed by RT-PCR or Q-RT-PCR after 72 rs of siRNA transfection. The knockdown efficiency (more than 80% reduction) of endogenous APTR in each experiment was confirmed by Q-RT-PCR using the primer set 1 and 2 , and then replated at 1×106 cells/well. All experiments were performed on three biological replicates. P values were calculated by two-way ANNOVA analysis using GraphPad Prism .For cell proliferation assays, HCT116 32P]-dCTP-labeled DNA oligonucleotides were made by Rediprimer II DNA labeling system . The probes were generated from a 300 nt fragment of APTR cDNA (651–950), amplified using the following primer sets:Total RNA was extracted from 293T cells using TRIzol reagent (Invitrogen). Poly(A) RNA was purified from total RNA using a PolyA TRACT kit (Promega) according to the manufacturer's protocol. Northern blot analysis was performed by standard protocol.  followed by sonication, and then subjected to RNA immunoprecipitation by the following antibodies; anti-EZH2 , SUZ12  and rabbit polyclonal IgG antibody. For biotinylated RNA pulldown, biotinylated sense/antisense wild type or deletion mutants of APTR were generated by MEGAscript T7 kit (Life technologies) according to the manufacturer's protocol. Whole cell extracts derived from 293T cells were pulled down by Streptavidin sepharose beads-Biotinylated APTR complex after 12 hr incubation at 4°C. The precipitated RNA was subject to cDNA synthesis and analyzed by RT-PCR. The primer sets are provided in For RNA immunoprecipitation and biotinylated RNA pulldown, 293T cells (5×10p21 promoter (200 ng), control Renilla luciferase reporter (5 ng) and either empty vector or vector expressing wild type or deletion mutants of APTR (2 ug). After 48 hrs of transfection, the lysates were analyzed by Dual-Luciferase reporter assay system (Promega) according to the manufacturer's protocol. Firefly luciferase activity of each reporter was normalized to the Renilla luciferase activity.293T cells were seeded on 6-well plates and co-transfected by Firefly luciferase reporter fused to various lengths of the p53-wild type, non-transformed breast epithelial cells). BrdU incorporation was measured in each well after 72 hrs of siRNA transfection and compared to that in wells transfected with si-GL2 (negative control) and si-ORC2 . Knockdown of 74 lncRNAs inhibited BrdU incorporation to at least 90% of the level of inhibition seen with si-ORC2  screen for genes required for cell proliferation, where we performed a manual, moderate-throughput siRNA transfection against a targeted gene set and measured effects on cell proliferation or viability by a quantitative comparison of BrdU incorporation in wells transfected with different siRNAs  si-ORC2 . Among tAPTR is a 2303-nucleotides lncRNA expressed from the opposite strand of the intergenic region between PTPN12 and RSBN1L in chromosome 7q21 (http://www.repeatmasker.org) found that APTR possesses two sequences complementary to SINE/Alu elements (c-Alu) as well as one sequence complementary to LINE/L2 element  cells to the same extent as knockdown of ORC2  tailed and spliced lncRNA expressed mainly in nucleoplasm  for 16 hrs, and then analyzed by FACS analysis for propidium iodide staining for DNA content. As shown in siGL2 transfected control cells accumulated in M phase, depletion of APTR led to an increase of G1 and S populations. In agreement with the increase in G1 population, depletion of APTR resulted in a decreased RB phosphorylation   , suggestsfection . Notablyll lines , indicatAPTR knockdown? To address this, we observed cell growth in p21−/− HCT116 cell lines APTR. Knockout and siRNA-mediated knockdown of p21 attenuated the cell growth inhibition upon APTR knockdown  is bound by bacteriophage MS2 binding protein (MS2BP) fused to fluorescent protein YFP (MS2BP/YFP). We adapted the MS2 tagging system for MS2-crosslinking and immunoprecipitation (MS2-CLIP) to enrich the chromatin bound to MS2 tagged RNA (APTR binds to multiple DNA sites between −1.6 and −4 kb relative to the transcription start site (TSS) of p21 (Accession: NM_000389)  , indicatp21 promoter and is responsible for stable p21 gene silencing APTR silences the p21 gene, we examined the interaction between APTR and the PRC2 complex in RNA immunoprecipitation assays with antibodies to EZH2 and SUZ12 followed by RT-PCR . An unrelated RNA, GAPDH mRNA was also not precipitated with EZH2 or SUZ12 (in vitro transcribed biotinylated sense APTR associated in vitro with cellular EZH2 and SUZ12 and pulled them down on streptavidin beads (APTR did not do so. Biotinylated APTR with specific deletions (APTR (1311–2303) lacking any Alu elements was sufficient for binding EZH2 and SUZ12, but portions containing only the c-Alu sequences (1–980 or 650–1311) could not bind EZH2 and SUZ12 . Knockdown of endogenous APTR increases p21, but this was reversed by over-expressing exogenous full length APTR (APTR indicated which portions of APTR are required for p21 silencing (APTR are expressed at comparable levels (APTR (650–2303), deleted of the first partial c-Alu element but retaining the second complete c-Alu, silenced p21. APTR that cannot bind PRC2 (1–980 or 650–1311) was unable to silence p21, suggesting that interaction with PRC2 is essential for silencing p21. Interestingly, APTRΔAlu(650–2303) missing all c-Alu elements did not silence the p21 promoter even though it could bind to PRC2 (To perform structure-function studies on ilencing . All thee levels . APTR (6 to PRC2 .p21 promoter (2.7 kb in length from TSS) with a plasmid expressing wt or mutant APTR (APTR(650–2303) repressed the p21 promoter, but deletions that removed the c-Alu elements or the 3′ end of APTR abrogate this repression. These findings suggest that the 3′ terminal portion and the embedded second c-Alu of APTR are both required for p21 silencing.We confirmed these results by co-transfecting a firefly luciferase reporter driven by the ant APTR . Here agAPTR is required for APTR to interact with PRC2. To explore whether the embedded second c-Alu element is required for targeting APTR to the p21 promoter, we assessed the localization of APTRΔAlu(650–2303) in MS2-CLIP assay. As shown in APTRΔAlu(650–2303)/MS2 was not recruited to the p21 promoter. Consistent with this and APTR/MS2 suppressed the p21 promoter luciferase activity (2.7kb in length from TSS), but APTRΔAlu(650–2303)/MS2 failed to do so (data not shown). These findings suggest that the c-Alu element of APTR is important for recruiting the APTR-PRC2 complex to the p21 promoter  up-regulated p21 mRNA  in these GBM samples (APTR is important for determining the expression of p21 in GBM cell lines and tumors.Glioblastoma multiforme (GBM), the most aggressive primary brain cancer in humans, often inactivates the p21 mRNA  and causp21 mRNA . Notablynockdown . In addi samples . TherefoAPTR, which is expressed from a distant site in the genome, but targets the p21 promoter in trans for epigenetic repression. Depletion of APTR resulted in the dissociation of the PRC2 complex throughout the APTR localization sites in the p21 promoter  epigenetically suppresses p21 transcription in cis, when transcribed from the antisense strand and overlapping with the p21 transcript APTR is clearly very different from these known lncRNAs, in that it is encoded elsewhere in the genome, and acts in trans on the p21 promoter itself.The APTR is its ability to interact with PRC2 so that the latter can be recruited to the p21 promoter. More than 20% of lncRNAs are bound by PRC2 in various cells XIST and HOTAIR, interact with local chromatin or DNA binding proteins to recruit the chromatin modifying machinery to specific sites in the target loci APTR similarly appears to recruit PRC2 to the p21 promoter, although not to all PRC2 target loci, as exemplified by HOXD11. Future studies will explore which proteins in PRC2 interact with APTR and what sequence or structural features of APTR are essential for interaction with PRC2. Clearly significant structural studies are necessary to understand exactly how these critical protein:RNA interactions are established and what determines the specificity and affinity of the various PRC2:lncRNA interactions. An interesting question is whether different lncRNAs that interact with PRC2 have similar or different affinities for the protein complex and whether they compete with each other to regulate gene expression by targeting PRC2 to certain sites in the genome and away from other sites. It will also be interesting to know in the future whether certain lncRNAs alter the conformation and enzymatic activity of PRC2 complex, or whether their activity is limited to the targeting of PRC2 to specific sites in the genome.One essential activity of Alu deletion mutant of APTR could not suppress p21 transcription because the mutation prevented APTR recruitment to the p21 promoter. We do not know how APTR interacts with the p21 promoter and how the APTR-embedded Alu element is required for this interaction. For example, is there a specific “receptor” for the c-Alu element, or c-Alu-bound proteins, in the p21 promoter? Or does the c-Alu region of APTR play a structural role so that APTR can fold in the correct conformation before it can interact with the relevant targeting proteins? Regardless of the mechanism this is another example of how Alu elements encoded in RNAs are functionally important.Importantly, the c-APTR localization sites in the p21 promoter (−1.6 kb to −4 from the TSS) contains tandem inverted partial and full Alu elements (Alu element from both strands of this site at very low levels (our unpublished data). Thus, we tested the possibility that Alu: c-Alu base-pairing interactions between cis-lncRNAs from the p21 promoter and APTR tether the latter to the p21 promoter. Luciferase assays on p21 promoter with or without the Alu region , however, clearly showed that APTR suppressed the p21 promoter without the Alu region Click here for additional data file.Figure S2Alignment of sequences from the repetitive elements in APTR with the retrotransposon SINE/Alu or LINE/L2.(EPS)Click here for additional data file.Figure S3Enough overexpressed APTR persists after siAPTR transfection to exceed the amount of endogenous APTR present normally.APTR expression levels normalized to GAPDH in cells transfected either siGL2 or siAPTR#2 as analyzed by Q-RT-PCR. Residual expression level of the siRNA-sensitive APTR after knockdown is higher than that in wild type cells transfected with siGL2 and empty vector. Percentages represent extent of reduction of APTR in siAPTR-transfected cells compared to that in siGL2-transfected cells.(EPS)Click here for additional data file.Figure S4APTR knockdown results in induction of p21 in multiple cancer cells without wild type p53. (A) RT-PCR shows APTR knockdown efficiency by siAPTR#2 in GL2 or siAPTR#2. PC3 and H1299 have mutant, and 293T has inactivated p53.(EPS)Click here for additional data file.Figure S5Knockdown of p21 alleviates cell growth inhibition upon APTR knockdown. (A) Growth suppression after siAPTR is alleviated in p21 knockdown HCT116 cells. The indicated siRNAs were transfected at Day1. Data represents total cell number at Day 0 and Day 4 (after 3 days of transfection) with mean ±s.e.m., n = 3, *:P<0.05. (B) The expression levels of APTR and p21 relative to siGL2-transfected cells were measured by Q-RT-PCR at Day 6 (after 5 days of transfection).(EPS)Click here for additional data file.Figure S6APTR knockdown. Immunoblot of H3K27me3 in 293T cells transfected with siGL2 or siAPTR#2.Expression level of Histone H3K27me3 does not change after (EPS)Click here for additional data file.Figure S7Expression of exogenous APTR RNA in APTR knockdown-APTR rescue assays. (A) All cells express the expected exogenous APTR RNA in APTR WT and mutants (1–980) after transfection of siAPTR#2. Percentages represent reduction rate of APTR in siAPTR-transfected cells compared to that in siGL2-transfected cells. The residual level of APTR1-980 after siAPTR is still significantly higher than endogenous APTR levels.(EPS)Click here for additional data file.Figure S8Knockdown of p21 alleviates cell growth inhibition in APTR knockdown glioma cells. (A) MTT assays (after 48 hrs of siRNA transfection) show that growth suppression after siAPTR is alleviated in p21 knockdown U87 glioma cells. Data represents mean ±s.e.m., n = 3, **:P<0.005. (B) Q-RT-PCR shows that the expression levels of APTR and p21 relative to siGL2-transfected cells in (EPS)Click here for additional data file.Figure S9Anti-correlation between APTR and p21 RNAs in human glioma cells. The two technical replicates of the experiment in (EPS)Click here for additional data file.Figure S10The tandem inverted Alu elements in the APTR regulatory p21 promoter is not involved in the APTR-mediating p21 silencing. (A) Schema represents firefly luciferase plasmid construction with various length of p21 promoter. (B) Firefly luciferase activity of various p21 promoters in cells transfected with empty vector or vector expressing wild-type APTR. Firefly luciferase activities were normalized to Renilla luciferase from co-transfected plasmid .(EPS)Click here for additional data file.Table S1Sequences of primers and siRNAs used for RT-PCR, Q-RT-PCR, ChIP/CLIP assays and siRNA interference in this study.(EPS)Click here for additional data file.Table S2siRNA based lncRNA functional screening in MCF10A cells. F-BRAMY2034329_1 was renamed as APTR. The positive control siORC2 and negative control siGL2 were transfected in six to eight wells in each biological replicate. The inhibition of BrdU incorporation by each siRNA is expressed as a % of the extent of inhibition by siORC2. For each lncRNA, we showed the % inhibition for each of three siRNAs and the average and standard deviation of the inhibition. Each siRNA was tested in triplicate. The average inhibition was calculated from all three siRNAs, unless the SD>0.2 x mean, in which case the outlier siRNA was discarded when calculating the average.(XLSX)Click here for additional data file.Method S1Supplementary Method.(DOC)Click here for additional data file.


Optical methods provide exceedingly powerful tools in science and technology for measuring, analyzing and manipulating, from optical microscopy and spectroscopy to the characterization of ultrafast processes by femtosecond pulses and the modification of materials by intense laser radiation. However, when it comes to applications in the nanometer-regime, the conventional optical techniques suffer from the resolution limit – formulated by Ernst Abbe one and a half centuries ago – that light cannot be focused to a diameter much less than half its wavelength. This seems like a serious drawback for the use of optics in nanoscience and nanotechnology, since many of the nanostructures of interest are distinctly smaller than this limit of a few hundred nanometers. Fortunately, this restriction can be overcome, if one does not rely on focusing with lenses and mirrors in the optical far-field, but rather exploits the optical near-field in the vicinity of nanostructures. In this Thematic Series, various examples for the use of optical near-fields and near-field optics are presented.Metallic nanostructures, especially noble metals such as gold and silver, are efficient for nano-focusing and controlling light on the nanoscale, because they support surface plasmons, i.e., collective excitations of the electron gas, which couple strongly to light. As a result, the optical near-field around such plasmonic structures can be enhanced by orders of magnitude compared to the incident light intensity, and can be localized in “hot spots” with a length of a few nanometers. This effect of strong near-field enhancement around sharp structures of noble metals has been known from Surface Enhanced Raman Scattering (SERS) for a long time . Yet, thA challenging question, investigated in this series by Esmann et al. , is how The interaction of plasmonic structures with their surroundings can be employed to tune their optical properties, e.g., by using a dielectric phase change material like GeSbTe as a substrate, whose refractive index can be externally adjusted in various ways. As shown by Hong et al. , this isThe mechanical effects of the optical near-fields can be substantial. Examples are specially shaped nano-holes, studied by Rosa et al. , which cIn spite of the high efficiency of plasmonic metal nanostructures, the near-field enhancement of dielectric structures is preferable for some applications. Walhorn et al.  have devThe examples given above illustrate the broad spectrum of applications of optical near-fields and near-field optics. The goal of this Thematic Series is not to provide a comprehensive review of all the aspects of this theme, but rather to highlight several facets in a rapidly developing research field. We sincerely thank all colleagues who contributed to this Thematic Series with their interesting and exciting results.Alfred J. Meixner and Paul LeidererTübingen, Konstanz, January 2014


Alcohol drinking and tobacco smoking are assumed to have significant independent and joint effects on oral cancer (OC) development. This assumption is based on consistent reports from observational studies, which, however, overestimated the independent effects of smoking and drinking, because they did not account for the interaction effect in multivariable analyses. This case-control study sought to investigate the independent and the joint effects of smoking and drinking on OC in a homogeneous sample of adults. Case patients  were affected by invasive oral/oropharyngeal squamous cell carcinoma confirmed histologically, diagnosed between 1998 and 2008 in four hospitals of São Paulo (Brazil). Control patients  were not affected by drinking-, smoking-associated diseases, cancers, upper aero-digestive tract diseases. Cumulative tobacco and alcohol consumptions were assessed anamnestically. Patients were categorized into never/ever users and never/level-1/level-2 users, according to the median consumption level in controls. The effects of smoking and drinking on OC adjusted for age, gender, schooling level were assessed using logistic regression analysis; Model-1 did not account for the smoking-drinking interaction; Model-2 accounted for this interaction and included the resultant interaction terms. The models were compared using the likelihood ratio test. According to Model-1, the adjusted odds ratios (ORs) for smoking, drinking, smoking-drinking were 3.50 , 3.60 , 12.60 , respectively. According to Model-2 these figures were 1.41 , 0.78 , 8.16 . Analogous results were obtained using three levels of exposure to smoking and drinking. Model-2 showed statistically significant better goodness-of-fit statistics than Model-1. Drinking was not independently associated with OC, while the independent effect of smoking was lower than expected, suggesting that observational studies should be revised adequately accounting for the smoking-drinking interaction. OC control policies should focus on addictive behaviours rather than on single lifestyle risk factors. Tobacco smoking and alcohol drinking are lifestyle risk factors which play an etiological role in oral cancer development with sufficient evidence. Such a high level of evidence is corroborated by a multitude of consistent observational studies published since the 70′s, which reported that these lifestyle risk factors were significantly associated with oral cancer These data demonstrated that the smoking-drinking interaction has a statistically and clinically significant effect on oral cancer. Therefore, observational studies designed to investigate the effects of smoking and drinking on oral cancer must necessarily account for this significant joint effect, because the absent account of the interaction term leads to overestimate the independent effects of smoking and drinking on oral cancer, in addition to insufficient adjustment for distal covariates in studies assessing other risk factors. The simplest method to account for such a joint effect in regression analysis is the interaction term approach, which implies that an additional variable -the interaction term- is included in the model Astonishingly, no observational study published during the last twenty-five years has accounted for the smoking-drinking interaction .From November 1998 to December 2008 we enrolled patients undergoing treatment for oral and oropharyngeal cancer in four main hospitals of the city of São Paulo, Brazil  to participate in case-control studies assessing different aetiological hypotheses. One of these four studies th revision) and oropharyngeal (C09–C10) squamous cell carcinoma, confirmed histologically. More specifically, cancers of tongue , gum (C03), floor of mouth (C04), palate (C05), other unspecified parts of mouth (C06), tonsil (C09) and oropharynx (C10) were included, while cancers of lip (C00), nasopharynx (C11), hypopharynx (C13) and other sites of lip, oral cavity and pharynx (C14) were excluded. Stage-0 cancers, corresponding to carcinoma in situ, were excluded because they were not necessarily invasive.The current sample was gathered by merging the databases from the four hospitals. The case group was exclusively composed by newly diagnosed patients with invasive oral . Controls were not to be affected by diseases potentially related with drinking and smoking exposures. In addition, subjects with current or past experience of cancer or aero-digestive tract diseases were not eligible.The data from the four study centres, merged in this study, followed the matching procedures for gender and age. As one of the study centres Specifically trained examiners interviewed participants immediately after their clinical consultation in a separate room; hospital files were consulted to register information comprised in the medical record of patients. All cases and controls underwent identical personal interviews regarding gender, age, schooling level, tobacco smoking and alcohol drinking.The assessment of smoking and drinking habits followed the methods endorsed by IARC Patients reporting having never consumed at least one drink at a regular monthly basis were considered non-drinkers. Alcohol drinking was measured by grams of ethanol, considering that one litre of ethanol weighs 798 g and that beer contains 5% ethanol in volume; wine 12%; liqueurs 30% and distilled spirits 41% The differences between cases and controls regarding age, gender and schooling level were assessed through logistic regression analysis using controls as reference group. Age was categorized into <45, 45–49, 50–54, 55–59, 60–64, 65–69, >69 years, while schooling level into <5, 5–8, >8 years of formal education. Variables which provided odds ratios (ORs) for oral cancer which were statistically significant at 95% level were included in the statistical models as confounders.β = {√ N [r/(r+1)] (p1–p2)2 [1/p(1-p)]} - Zα”, where “Zβ” is the value of the standard normal distribution corresponding to the value of β; “N” is the sample size (cases+controls); “r” is the ratio of controls to cases; “p1” and “p2” are the proportions of exposed to drinking in cases and controls; “p” is the average proportion of exposed to drinking “Zα” is the value of the standard normal distribution corresponding to the value of α = 0.05 using a two-sided test .In order to explore the level of beta error, the power of the study, corresponding to “1-β”, was assessed with the formula, “ZThe effect of drinking and smoking on oral cancer risk was explored through unadjusted ORs with 95% confidence intervals (95% CI), which were assessed by unconditional logistic regression Unconditional logistic regression analysis was also used to assess the adjusted individual and joint effects of smoking and drinking on oral cancer risk. Age, gender and schooling level were considered as potential confounders if they resulted significantly associated with oral cancer. Two analyses were run. In the first analysis, the exposure variables drinking and smoking were treated as binary . In the second analysis, they were categorized into three levels . Two models were designed:Model-1 was the simplest model, conventionally used by observational studies investigating the effects of lifestyle risk factors on cancer. This model accounted exclusively for confounding and assumed that: (1) smoking and drinking exerted individual effects on oral cancer risk; (2) they were reciprocally associated; (3) they did not exert interaction effect.According to this model, the OR for the “ever smoking, ever drinking” category was obtained by the inverse of the logarithm of the sum of the coefficients of smoking and drinking, as provided by logistic regression.Model-2 was the model proposed here to investigate the effects of lifestyle risk factors. This model accounted for confounding and interaction and assumed that: (1) smoking and drinking exerted individual effects on oral cancer risk; (2) they were reciprocally associated; (3) they exerted interaction effect.In order to account for the interaction effect, the interaction term approach was preferred to other methods, such as the stratified analysis with joint categories, because it was the most widely applicable and practical When exposure variables were categorized into three levels, four interaction terms were generated, one for every type of joint exposure, namely, “level-1 smoking, level-1 drinking”, “level-1 smoking, level-2 drinking”, “level-2 smoking, level-1 drinking”, “level-2 smoking, level-2 drinking”.In order to check whether the OR estimates were artificially inflated or overestimated by excess collinearity and multicollinearity, before running the regression analyses, the explanatory variables were tested by means of pairwise Pearson’s correlation coefficient (r) and variance inflation factor (VIF). The highest acceptable values for r and VIF were set at 0.6 and 10, respectively. The robustness of the OR estimates was also investigated through validation analysis. Namely, the case and the control groups were split into two halves: two random variables were generated, one for cases, one for controls; the case and the control groups were ordered according to these variables; the first half of cases and the first half of controls were grouped in sub-sample 1, the remainder in sub-sample 2. Coefficients for smoking, drinking and interaction terms and goodness of fit of regression models were re-estimated in both sub-samples. According to the validation analysis, coefficients, compared using the 95% CIs, must not differ between sub-samples, while p-values of −2log likelihood must be similar 2 . The goodness-of-fit of Model-1 and Model-2 was statistically compared using the likelihood ratio test, with approximately χ2 distribution. The smoking-drinking interaction effect was considered statistically significant if the goodness-of-fit was significantly better in Model-2 than in Model-1.The goodness of fit of the regression models was assessed using the −2log likelihood  and Pseudo-RAll analyses were performed using Stata 12.0 .The study included 1,144 cases and 1,661 controls Table 1.The distribution of cases and controls by gender, age group and schooling level is displayed in β were 20.71 and 21.74 for smoking and drinking, respectively, which resulted in power levels higher than 99.9% for both smoking and drinking (data not in Table). In the multivariable assessment Table 3,An analogous result was obtained when both exposures were classified using three categories Table 3.The values of “r” and VIF were below the limits of 0.6 and 10, suggesting that the coefficient estimates were substantially not inflated or overestimated. According to the validation analysis, the coefficient estimates and goodness of fit of regression models did not change substantially, thus corroborating the robustness of the various risk estimates (data not in Table).The most important findings of this study were that (1)the independent effect of drinking substantially decreased and was no longer associated with oral and oropharyngeal cancer accounting for the smoking-drinking interaction term; (2)the independent effect of smoking also considerably decreased, although it remained significantly associated with the disease; (3)the smoking-drinking joint effect remained significantly associated with oral cancer; (4)regression models accounting for the smoking-drinking interaction term had a significantly better fit than those exclusively assessing the individual effects of behavioural exposures.We gathered an exceptionally high number of participants for a single case-control study on oral cancer risk factors (see previous observational studies reviewed by Being hospital-based, this study was potentially subjected to criticism regarding selection bias, since the present sample was not representative of all residents in the city of São Paulo. Indeed, factors which bring people to public hospitals, such as financial standings, area of residence, ethnicity, religious affiliation, are not distributed uniformly within the underlying study population, thus making it difficult to define the population from which our case patients arose. In order to minimize the consequences of selection bias on the consistency of results, we decided to select hospital-based controls who followed the same referral route as cases. In these circumstances, the underlying study population was the same in the two groups and was identifiable in those residents in the city of São Paulo who sought for care in public hospitals Information bias regarding lifestyle variables, which are generally underreported by heavy users is another important form of bias Recall bias and interviewer bias, two specific forms of information bias, are also relevant limitations of case-control studies Finally, it is possible that other variables, strongly associated with both drinking and smoking, also were associated with oral and oropharyngeal cancer risk. Indeed, smoking and drinking are not only associated with each other, they are also associated with other behavioural risk factors for cancer and other degenerative diseases, such as unsafe sex, use of other addictive substances, unhealthy diet, low physical exercise, etc. The data of the present study were partly corroborated. Indeed, two multi-centre studies from IARC, one based on seventeen centres from Europe and America This study may have implications in the design of effective oral cancer control policy. Indeed, policies based on the control of a single risk factor are fated to fail in the long term, as already demonstrated for alcohol drinking In conclusion, the present data suggest that in this large-sized and homogeneous sample from São Paulo, drinking was not independently associated with oral/oropharyngeal cancer, while the independent effect of smoking was lower than expected. Instead, the joint effect of drinking and smoking was significantly associated with this condition. We strongly recommend that observational studies from other countries are designed accounting for such an important interaction, thus allowing investigating whether this result may be generalized to populations with different lifestyles, and with various drinking and smoking modalities. According to these findings, oral cancer control policies should focus primarily on the addictive behaviour which induces people to adopt several unsafe lifestyles.


Presbyopia is the age-related decline in accommodation that diminishes the ability of the eye to focus on near objects. Presbyopia is common and easy to correct; however, many communities lack access to basic eye care. The purpose of this project was to assess the burden of uncorrected presbyopia in a rural Filipino population and to pilot an intervention aimed at increasing access to reading glasses in the community.Individuals above the age of 40 who presented to a health outreach in the Philippines were invited to undergo a near vision exam to detect the presence of functional presbyopia and be fitted with ready-made, single-vision glasses. The change in stereoacuity was used as a surrogate measure of functional improvement after near vision correction. A questionnaire was administered to assess this population’s perceived barriers and benefits to correcting near vision.The average age of the participants was 57 ± 11 years, with 87.6% of participants having an uncorrected near visual acuity of <20/50. Reading glasses improved near vision to 20/40 or better in 77.7% of participants having near-vision impairment . Over 75% of participants also showed improvement in stereoacuity. Cost, rather than availability, was perceived to be the greater barrier to the procurement of glasses, and 84% of participants reported that the glasses dispensed would greatly improve their ability to earn a living.Dispensing ready-made, single-vision glasses is a simple and cost-effective intervention to improve near vision and enhance depth perception. A greater understanding of the barriers and benefits to correcting near vision will inform the design and execution of a sustainable program to correct presbyopia in developing countries. Presbyopia is the age-related decline in accommodation that diminishes the ability of the eye to focus on near objects . This prPresbyopia is common and easy to correct, even when it occurs together with underlying refractive error. Whereas a modest amount of uncorrected myopia may be somewhat protective of the effects of presbyopia, a mild to moderate amount of uncorrected hyperopia tends to accelerate the age at which a decline in near vision is first perceived. While glasses for near vision, commonly referred to as “reading glasses”, provide obvious benefits in a contemporary, literate society, the difficulties caused by uncorrected presbyopia may nonetheless be quite burdensome in areas with lower literacy rates because near visual acuity is required for many activities of daily living besides reading, such as cooking, sewing, and close family interactions . Even thTo this end, studies have been performed in developing countries to assess the impact of uncorrected presbyopia ,5-9. TheThe current investigation is the first to examine presbyopia in the Philippine Islands, focusing on a rural population in the province of Nueva Vizcaya. This study aims to measure the extent to which the application of single vision reading glasses alone can improve near vision. A simple and cost-effective intervention strategy was developed to rapidly dispense reading glasses to achieve the best corrected, binocular, near visual acuity through a successful collaboration between two international health care organizations. In the process, a pilot survey of the perceived barriers and benefits of correcting near visual acuity, as well as access to the correction of presbyopia, was conducted. A better understanding of these factors will help in the design and execution of a sustainable program to improve near vision in developing countries.Members of the Global Medical Foundation, U.S.A., and Saint Mary’s University School of Health Sciences  undertook the present project in order to correct presbyopia in an aged Filipino population (>40 years old) in the rural province of Nueva Vizcaya in the Philippine Islands. The villages of Dupax Sur and Bambang were chosen because of their relatively central location and accessibility by passable motorways. A majority of the participants served by the outreach activities are members of several indigenous people groups in the Philippines, protected and legally recognized minorities. In order to comply with the Indigenous Peoples Rights Act of 1997 (Republic Act No. 8371), no ethnographically identifying data was collected out of sensitivity to these indigenous cultural communities. The local provincial authorities were responsible for announcing the availability of the health outreach, which offered a variety of medical and dental services for all ages. Services were also offered to residents of neighboring municipalities who journeyed to the mission sites. One of the services was a mass-screening program aimed at identifying individuals with uncorrected presbyopia and fitting them with reading glasses to correct near vision. A cross-sectional, pilot survey of the perceived barriers and benefits of correcting near visual acuity was administered.Persons above the age of 40 without bilateral visual impairment or blindness, as determined by both history and a screening exam with a pen light to exclude gross abnormalities, who presented to the health outreach were invited to undergo a near visual acuity exam and be fitted with reading glasses . The near visual acuity exams were conducted by senior nursing students from St. Mary’s University of Health Sciences, who were members of the eye care team, at a standard distance of 40 cm in outdoor illumination, binocularly, using either the Rosenbaum or LEA near vision card, depending on patient preference and/or literacy level. The distance of approximately 40 cm was maintained by a string attached to the bottom of the near visual acuity chart at one end with the other end held taught against the person’s chin. Near visual acuity exams were initially conducted unaided. Persons were then furnished with a pair of reading glasses based on their age, ranging from +1.00 D to +3.50 D ,12, withFunctional improvement upon application of reading glasses was assessed by comparing the subject’s score on the RANDOT® stereoacuity test  with and without the near vision correction provided by reading glasses at a distance of 40 cm. This test was administered by the lead investigator as well as by senior nursing students specifically trained by the lead investigator. This test incorporates simple shapes at two levels of disparity (500 and 250 arc-seconds), pictures of animals arranged at three levels of disparity , and finally, a series of ten panels that provide a graded disparity between 400 and 20 arc-seconds. Participants who did not demonstrate measurable stereoacuity on a particular portion of the test  were assigned a value of 1000 arc-seconds (twice the limit of measurable stereoacuity in this study) for the purposes of analysis.Following the completion of this medical intervention, individuals were invited to participate in a questionnaire. Of note, the intervention was offered regardless of willingness to participate in the survey. Verbal informed consent was obtained and communications took place in Ilocano, a local Filipino dialect prominent in the province of Nueva Vizcaya, or Tagalog or English, both official languages of the Philippines, depending on the participant’s preference and comprehension. Senior nursing students fluent in these languages administered the questionnaire. Participants were not required to answer every question. Some participants may have not understood every question secondary to education level, having a different dialect as their primary language, or not being applicable to their current situation. The questionnaire was based in part on the National Eye Institute’s Visual Function Questionnaire (NEI-VFQ)  but modiAll data were analyzed using Microsoft Excel  version 12.2.7. All tests of association  were considered to be statistically significant at p <0.05.More than 600 people voluntarily underwent near visual acuity testing and were fit with reading glasses. Among these, 142 people (24%) agreed to take part in the near vision questionnaire that assessed their perceived barriers and benefits to the treatment of presbyopia. The low response rate may be due in part to the fact that participants preferred to partake in the other services at the health outreach rather than take the questionnaire. The mean age of the participants was 57 ± 11 years, 75.3% were female, and 52.6% had secondary or higher education  of the participants responded they were able to read to some degree. Yet, approximately two thirds of the participants stated they had at least a moderate degree of difficulty reading small print, such as that in newspapers. Additionally, when asked about the cost of reading glasses and the availability of an eye doctor as barriers to obtaining reading glasses, participants perceived that cost greatly  had a previous pair of reading glasses. Furthermore, 46.2% (36/78) of those who had purchased their previous pair of reading glasses spent >10 USD. The main reason cited for not having a previous pair or reading glasses was lack of money.The participants who had a previous pair of reading glasses were asked how that pair impacted certain daily tasks Table . Not surFinally, the participants were questioned about their perceived value of the present intervention. A bimodal distribution was observed in the participants’ responses when asked about their willingness to pay for the pair of reading glasses they received at the health outreach: 28.4% stated they would be willing to pay >4 USD while 27.7% stated that they would be unable to pay. Considering the progressive nature of presbyopia, the final question posed to the participants dealt with their willingness to participate in a program where their current pair of reading glasses would be exchanged for a new pair in 3–5 years. Over 80% of the participants responded that they would be very likely to participate in such a program. An unexpected finding was that 84.3% (118/140) of the participants stated the reading glasses they received at the health outreach would greatly improve their ability to earn a living.This study piloted a simple and cost-effective intervention aimed at increasing access to reading glasses in a rural, indigenous, Filipino population. At the same time, the intervention served to increase awareness of presbyopia and assessed the perceived barriers and benefits to accessing treatment for the age-related decrease in the ability of the eye to focus on near objects in this population.Clinical evidence suggests that the prevalence of uncorrected presbyopia can approach 100% among the elderly population and begins at earlier ages in the setting of developing countries ,15. The With regards to our expedited intervention, which did not take into account best corrected distance vision, a study examining presbyopia in Zanzibar, Africa  showed tA recent study assessing presbyopia in a rural, Chinese population  also utilized a functional definition of presbyopia ,17 and dIn conjunction with the improvement in near visual acuity, we wanted to ascertain that our pilot intervention was providing functional improvement as well. The study in Zanzibar, Africa  assessedStereopsis is the binocular perception of depth , which iAmong those participants that demonstrated stereopsis, more than two-thirds showed an improvement in arc-seconds of resolution of stereoacuity upon the application of reading glasses. Subsequently, it may be suggested that our simple and cost-effective intervention provided functional improvement as well as improvement in near visual acuity. However, ten participants did fail to demonstrate measurable stereoacuity pre- and post-correction and another 16 participants actually showed a small decline in stereoacuity upon the application of reading glasses Table . AlthougWith regards to those who actually showed a decline in the best-measured stereoacuity post-correction, more than half of these individuals actually showed improvement on some portions of the test but not others. Specifically, they showed improvement on the simple shapes and animal portions of the stereotest that contain two and three levels of gross disparity, respectively, but exhibited a decline in measured stereoacuity when tested on the finely graded ten panel sequence (see Methods). Considering stereoacuity was measured immediately after the participants received their reading glasses, relaxation of the accommodation reflex with the addition of plus lenses may be a possible cause for the decline in measured stereoacuity in a small subset of patient with underlying hyperopia. Giving participants time to adapt to the reading glasses could have potentially overcome this effect.Beyond correcting near visual acuity and providing functional improvement with ready-made near spectacles, we also sought to understand the perceived barriers and benefits to accessing treatment to presbyopia in a predominantly indigenous population. Two different barriers were the focus of our investigation: cost of so-called “reading glasses” and the availability of an eye doctor. Not surprisingly, more participants perceived that the cost of reading glasses greatly prevented them from obtaining access as compared to the availability of an eye doctor Table . FurtherThe final aspect of the questionnaire focused on the participants’ perceived value of the present intervention. 28.4% of the participants were willing to pay >4 USD for the current pair of reading glasses, approximately eight times the actual cost of the spectacles provided (0.50 USD), which may be depicting the value the participants placed on the current intervention. However, in agreement with cost as a major barrier to accessing treatment for presbyopia, >40% of the participants stated they could pay <1 USD or nothing at all for the current pair of reading glasses. Hence, a community distribution scheme is needed that is amenable to both subsets of participants. For that reason, a cross-subsidization protocol may not be financially sustainable. Further information, such as the least amount that would be possible for those unable to pay, is necessary before such a protocol can be enacted. Therefore, we proposed a glasses trade-in/recycle program to the participants. In this program, an individual would have the opportunity to trade-in his or her reading glasses for a different pair every three to five years , if upon examination, they were deemed to need a different, stronger power. If in useable condition, the spectacles could then be recycled to another individual.While this pilot study is the first to investigate presbyopia in a rural Filipino population and highlights the possible benefits that can be achieved by the correction of near vision by simple ready-made glasses, it does have its limitations. First, we had a limited sample size (142 participants) in comparison to other studies that have been performed in the developing world ,7,16,17.Uncorrected presbyopia is a significant cause of age-related visual impairment in this indigenous Filipino community, and dispensing ready-made single-vision glasses is a simple and cost-effective intervention to improve near vision and enhance depth perception. Furthermore, understanding of the barriers and benefits to correcting near visual acuity will help in the design and execution of sustainable programs to improve access to ready-made spectacles in developing countries. Further research and resources should be directed toward conducting comprehensive population-based intervention studies aimed at developing sustainable programs capable of relieving the unmet global burden of presbyopia.D: Diopter; ANOVA: Analysis of variance; USD: United States Dollar.The authors declare that they have no competing interests.TJW participated in data collection, data analysis, and drafted the article. CMG played a predominant role in the design of the study. MS participated in the data collection and questionnaire administration. GSW participated in the design of the questionnaire. GPG participated in data collection. DJR played a major role in the design of the study, data analysis, and conceptualization of the article. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2415/14/9/prepub


Previous studies have shown that the sliding velocity of class-5 myosins obeys modulation by free Mg2+-ions. Free Mg2+-ions affect ADP release kinetics and the dwell time of actin-attached states. The latter determines the maximal velocity of actin translocation in the sliding filament assay. We measured the temperature dependence of sliding velocity in the range from 5 to 55°C at two limiting free Mg2+-ion concentrations. Arrhenius plots demonstrated non-linear behavior. Based on this observation we propose a kinetic model, which explains both sensitivity towards free Mg2+-ions and non-linearity of the temperature dependence of sliding velocity. According to this model, velocity is represented as a simple analytical function of temperature and free Mg2+-ion concentrations. This function has been applied to global non-linear fit analysis of three data sets including temperature and magnesium (at 20°C) dependence of sliding velocity. As a result we obtain thermodynamic parameters (ΔHMg and ΔSMg) of a fast equilibrium between magnesium free (AM·D) and magnesium bound acto-myosin-ADP (AM· Mg2+D) states and the corresponding enthalpic barriers associated with ADP release (ΔH1‡ and ΔH2‡). The herein presented integrative approach of data analysis based on global fitting can be applied to the remaining steps of the acto-myosin ATPase cycle facilitating the determination of energetic parameters and thermodynamics of acto-myosin interactions.Kinetic and thermodynamic studies of the mechanochemical cycle of myosin motors are essential for understanding the mechanism of energy conversion. Here, we report our investigation of temperature and free Mg Despite the high degree of structural conservation of the myosin motor domains 2+ from the nucleotide binding pocket i precedes the fast dissociation of Mg2+ADP ·D) and magnesium bound acto-myosin-ADP (AM·Mg2+D) states One important parameter that varies between the myosins is the fraction of time the motors spend in the strongly actin attached states during the ATPase cycle in vitro motility assays over an extended temperature range with a high duty ratio class-5 myosin from Dictyostelium. Myosin-5b is a processive motor like myosin-5a and possesses motor properties that can be modulated by changes in the concentration of free Mg2+-ions 2+-sensitive ADP release steps in the ATPase cycle of myosin-5b. Our results provide important insights on the critical role of the Previous thermodynamic investigations have addressed temperature dependences of individual myosins in terms of their ATPases, motile activities, or ADP-release and nucleotide induced acto-myosin dissociation kinetics 2 purchased from Sigma was used for the adjustment of free Mg2+-ion concentrations in the experimental buffers. D. discoideum myosin-5b motor domain fused to an artificial lever arm consisting of two α-actinin repeats (2R) and C-terminal octa-His tag was purified according to the previously described procedures Standard chemicals, anti-His antibodies, trichloromethyl-silane (TCMS), and tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin were purchased from Sigma; rabbit skeletal muscle actin was purified from acetone powder and labeled with TRITC-phalloidin. A titrated 3.9 M stock solution of MgCl2 was adjusted accordingly to provide the desirable free Mg2+-ion concentration using the Maxchelator software (http://maxchelator.stanford.edu). Free Mg2+-ion concentrations are assigned as [Mg2+]. The affinity of Mg2+ for ATP is temperature dependent in aqueous solution and changes in the studied temperature range from approximately 140 µM (5°C) to 50 µM (55°C). We ignored this temperature effect for the calculation of free Mg2+-ions concentrations in the assay buffer, since we used excess ATP concentrations making the error of free Mg2+-ions concentrations negligibly small in the temperature range studied. Saturating concentrations of myosin molecules were used to obtain maximum sliding velocities. The myosin molecules were immobilized via anti-penta-His antibodies (concentration 0.025 mg/ml) on silanated glass surfaces of a flow-cell. TCMS coated cover slips were prepared according to All experiments were performed in assay buffer (AB) containing 25 mM imidazole pH 7.4, 25 mM KCl, 1 mM EGTA, 10 mM DTT. Preparation of actin and labelling with TRITC phalloidin was done according to v1, v2, ΔH1‡, ΔH2‡, ΔHMg, ΔSMg), which are assumed to be temperature independent. The function was written in the script form of the software to allow conditional reassignment of the independent variables, either as reciprocal temperature or concentration of free Mg2+-ions. Additionally, errors of experimental data were included to the fit as statistical weighting parameters. The global fit analysis was performed several times with different initial guesses of parameters to avoid solutions which could correspond to local minima of fit.Actin filament tracking was performed with DiaTrack 3.01 . Data analysis and graphical representation of results were done with Origin 7.0 . The non-linear least square fitter of Origin 7.0 was used for global data analysis. 2+-ion concentrations [Mg2+] over a temperature range of 50°C using in vitro motility assays. 2+] dependence of the sliding velocity at 20°C. The latter data have been reported earlier in the context of the kinetic characterization of the myosin-5b 2+] inhibited actin filament sliding velocity 2.5-fold with an apparent dissociation constant (KMg) of 0.4 mM. Thus, saturating free Mg2+-ion concentrations do not fully inhibit the motile activity of myosin-5b. Previous kinetic investigations with myosin-5a propose a sequential model of product dissociation in which Mg2+ is released prior to ADP 2+] inhibit the sliding velocity of the motor 2+] do not completely inhibit sliding velocity. In order to address this problem, we performed temperature-dependent motility assays at two limiting [Mg2+] of 0.05 mM and 4.5 mM representative for the high and low limits in sliding velocity at 20°C. Over the entire temperature range velocities at low 0.05 mM [Mg2+] (upper trace) were higher than at 4.5 mM [Mg2+] (lower trace). For both experiments a non-linear Arrhenius dependence was observed. This indicates that the underlying kinetics, which determine sliding velocity cannot be considered as a single elementary step in the reaction pathway. A similar observation was reported for the sliding velocity driven by muscle myosin-2 2+ inhibition should include a fast and temperature sensitive equilibrium between Mg2+-bound and Mg2+-unbound acto-myosin-ADP (AM·D)-states that precede the subsequent and rate limiting steps of the myosin-5b ATPase cycle  between Mg2+-bound and Mg2+-unbound AM·D states.The motile activity of myosin-5b was studied at two defined free Mgse cycle . Accordi2+-concentration dependence) to a global non-linear least square fitting analysis using function 1. Results of the fit are shown as solid lines in 2+-ions occurring in biological systems 2+-unbound and Mg2+-bound AM·D states. At low temperatures the Mg2+-unbound state AM·D dominates. The corresponding activation enthalpy ΔH2‡ of the ADP release is 64 kJ/mol and can be seen from the steepness of the Arrhenius plot in the low temperature range  towards the temperature gives a plausible explanation for the convex non-linear behavior of myosin-5b motility in the Arrhenius plots. The pronounced difference in activation enthalpies can be explained as a result of neutralization of the electrostatic charge of ADP by Mg2+ and therefore weakening of binding energy to the protein. The 3-fold reduction of activation enthalpy upon binding of Mg2+ is in line with an increase in the enthalpy (ΔHMg) of the Mg2+-bound state of the equilibrium. The equilibrium shift towards the AM·Mg2+D at higher temperatures implies that this state is entropically favored (ΔSMg = +234 J/molK). Moreover, since the AM·D state has a lower entropy than the AM·Mg2+D state, we can assume that the height of the entropic barrier for the release of Mg2+-ADP is dictated by the entropy of the AM·Mg2+D state. As a consequence, the rate constant for Mg2+-ADP release becomes slower. From the v1 and v2 pre-exponential factors we can estimate the difference of entropic activation barriers between the two pathways: 1 and ν2 . The high enthalpy difference ΔHMg of 66.5 kJ/mol accounts for the large change in KMg. Note, in aqueous solution (pH 7.0) the increase in enthalpy for Mg2+ binding to ADP is approximately 10.5 kJ/mol, as calculated from the temperature dependence of the dissociation constant of the Mg2+-ADP complex according to NIST database parameters of the Maxchelator program. This calculation indicates that the process of Mg2+-ion coordination in the nucleotide binding site of myosin-5 is accompanied by a significant change in the energy of interactions by the protein surrounding. On the other hand the entropy driven mechanism of binding does not principally change (according to NIST database the Mg2+-ADP complex at pH 7.0 in aqueous solution has a 95 J/molK higher standard entropy than the dissociated form). The fraction of the AM·Mg2+D state depends on the concentration of free Mg2+-ions. This is shown exemplary for 20°C in 2+-ion concentration from approximately 0.1 to 2 mM. Interestingly, this concentration range matches the physiological range of free Mg2+-ions found in cells 2+-ions might be of importance for regulation of myosin motor function in vivo. 2+-regulated part of the enzymatic relaxational pathway obtained from our data analysis. The diagram illustrates the temperature independent enthalpic difference of the Mg2+-equilibrium and two enthalpic barriers of the rate limiting steps of the reaction. Obviously, the actual rates of transitions and population of states within the equilibrium are defined by the Gibbs free energy values (2+D and AM·D states. This barrier is drawn low enough to be consistent with the model assumption of a fast equilibrium.We applied our three data sets (two T-dependences and one free Mgre range . At highd ν2 see . This es2+-dependence of motile activity and regulation of this motor by a simple two-state kinetic model. The herein reported methodological approach of data analysis by global fitting can be useful to interpret kinetic and thermodynamic data of other experimentally accessible reaction steps in the acto-myosin ATPase cycle, thus providing a consistent description of the energetics and equilibria of acto-myosin interactions. Moreover, our results underline the significant contribution of entropic terms to the free energies for acto-myosin interactions. At least four additional myosins including Dictyostelium myosin-1D and -1E, human nonmuscle myosin-2c, and human myosin-7a show similar variations in their kinetics as reported for myosin-5a and myosin-5b, with the ADP release step being the rate limiting and Mg2+-ion sensitive step in their ATPase cycle Our kinetic and thermodynamic analysis of myosin-5b allowed us to describe the Mg


Occasionally the thymus may extend in the neck, from its normal location in the anterior mediastinum. The cervical extension, especially when the native thymus is hyperplastic, can mimic a mass. We describe the detection of cervical extension of the hyperplastic thymus, presenting as a suspicious recurrent soft tissue mass in the neck in a patient with Ewing’s sarcoma. Cervical extension of the thymus is often mistaken for a soft tissue mass in the neck, particularly in children and young adults. The problem in differentiating thymic tissue from a mass is compounded when the thymus is hyperplastic and shows increased fluorodeoxyglucose uptake. We describe the case of a child on follow-up for Ewing sarcoma in whom we detected an avid thymic extension in the neck, which mimicked recurrent disease both anatomically and functionally.An 8-year-old child presented in 2009 with a swelling on the left side of the back in the scapular region. Clinical examination revealed a lobulated nontender soft tissue mass. Biopsy was suggestive of Ewing sarcoma. A staging positron emission tomography/computed tomography (PET/CT) study performed at that time revealed increased tracer accumulation in the soft tissue mass associated with the left scapula . The resThe focus of uptake in the neck [arrowhead in A dedicated contrast-enhanced CT was then performed to characterize the suspicious lesion reported on the PET/CT study. Axial CT image confirmed the presence of a hypoenhancing lobulated soft tissue lesion between the trachea and the jugular vein . Closer The thymus gland develops from the third pharyngeal pouch on each side. In the eighth week of intrauterine life the primordial thymus from both sides fuses in the midline and migrates inferiorly to its normal retrosternal location in the superior mediastinum. The migrIt has been shown that cervical extension of the thymus, generally, is anterior to the brachiocephalic vein; has a density identical to that of the thymus, with no intervening fat planes; and does not produce any mass effect on the surrounding structures. All of tIn summary, a soft tissue mass in the lower neck near the midline, particularly in pediatric patients with a history of chemotherapy, should be viewed with caution. Careful scrutiny of the multiplanar CT reformations and knowledge of the imaging attributes of cervical extension of the thymus can, as shown in our case, thus help the nuclear medicine physician to differentiate it from an actual mass or an enlarged node.


Sight loss has wide ranging implications for an individual in terms of education, employment, mobility and mental health. Therefore there is a need for information and support to be provided in eye clinics at the point of diagnosis of sight threatening conditions, but these aspects of care are often missing from clinics. To meet these needs, some clinics employ an Eye Clinic Liaison Officer (ECLO) but the position has yet to be widely implemented. The aims of this study were:(1) To evaluate the forms of advice and emotional support in eye clinics provided by ECLOs.(2) To determine the cost of the ECLO service per patient.Micro-costing was carried out using interviews, a survey and administrative data. The survey was completed by 18 of the 49 accredited ECLOs in the UK (37%) and provided information on the activities performed by ECLOs, numbers of patients seen per day, training costs incurred and the salary of the ECLOs.ECLOs provided information about the services in eye clinics and the community, referral to social services, emotional support to patients and also other advice. The cost of an ECLO per patient per contact was £17.94 based on an average annual ECLO salary of £23,349.60 per year, reviewing on average 9.1 patients per day, in a 42 week year.This study provides the first costing of support services in hospital eye clinics, providing a range of estimates to suit the circumstances of different clinics. The information can be used by local decision makers to estimate the cost of implementing an ECLO service. There is a growing recognition that better support mechanisms are needed for patients in eye clinics, particularly at the time of sight loss. Previous research conducted in the UK suggests that eye clinic patients with sight loss do not always receive emotional support ,2 or infDespite the growing awareness about the need for timely support, the ECLO service has not been widely implemented. Other countries have implemented a similar support/co-ordinator role such as the USA, Australia, Sweden and Poland, but in some countries the role is only available within visual impairment organisations in the community rather than in eye clinics . The utiOne of the reasons why the ECLO service has is not been widely implemented may be due to financial concerns and a lack of cost estimates available in the literature. In a recent report of the key cost drivers for low vision services in the UK, concern was raised over the sustainability of the ECLO role due to cost factors, despite the general feeling that these individuals played a valuable role in low vision services . To provThe aims of this study were to determine (1) whether ECLOs are providing the services previously mentioned as missing in eye clinics, and (2) the cost of an ECLO per patient per contact and whether sufficient facilities have been provided for ECLOs. Cost per patient per contact is the cost of one consultation with an ECLO.An invitation was sent to ECLOs known to the researchers to take part in semi-structured interviews to inform items for use in the questionnaire  and only the results of accredited ECLOs are described. Most ECLOs had the job title of Eye Care/Clinic Liaison Officer (n=13), other titles included Low Vision Co-ordinator/Support Worker (n=3), Information Officer (n=1) and Independent Living Advisor (n=1). Funding for the ECLO position was provided by local authorities (n=5), NHS (n=5), jointly by the local authority and NHS (n=2), local charities (n=2) and national charities (n=4).The information and support activities provided by ECLOs are shown in Table Table Table Table Using the proportions of people registered as severely sight impaired at each age group  we estimECLOs were asked what facilities were provided for their role in the hospital. 72.2% had a private room, 66.7% had a telephone and 44.4% had a computer (n=18). When asked if they were satisfied with the facilities provided, only 44.4% answered yes. ECLOs who provided additional comments about facilities often mentioned that a lack of space or sharing the space with other staff made it difficult to discuss patient’s private situations and to provide emotional support.ECLOs provide information about low vision clinics, social services and signposting to other agencies. Emotional support to patients and family members is widely provided. Just over 10% do not provide information about low vision aids, and most do not provide training in their use, however, this is provided in the community  and may After the first year of set-up costs, the full economic cost of employing a full time ECLO is £34,290 per annum, and £17.94 per patient per contact. This increases the estimated cost of a hospital based low vision service  by 10.8%The sensitivity analysis showed that cost per patient could range from £32.66 to £10.89 if 5 or 15 patients were seen per day respectively. The variations in cost highlight the importance of effectively utilising the service once implemented. However, some variation is inevitable due to the geographical distribution of the elderly population, for example, there are greater proportions of elderly people in coastal areas in the UK due to migration . AlthougThe average lifetime cost of an ECLO per patient ranged from £260.18 for someone first seeing an ECLO at age 8, and £24.60 for someone first meeting an ECLO at age 84, assuming an ECLO is met once every 5 years. Although meetings with ECLOs are not meant to be provided regularly to patients, as they refer patients on to service providers, the changing social needs of patients as a result of progressing visual impairment and/or the changing demands put on an individual at each age may require more regular meetings for advice about education, employment, childcare, welfare rights, benefits and other developing conditions and this must definitely be borne in mind when looking at the cost of the service. Culham et al.  suggest Although ECLOs are providing the services reported as previously missing in eye clinics , earlierECLOs were not always provided with facilities to carry out their role effectively. Computers and telephones, useful for making referrals and obtaining information for patients, were only available to 44.4% and 66.7% respectively. Although a private room was the resource most ECLOs had access to (72.2%), those without this commented it caused many problems as there was no place to discuss a patient’s situation and feelings. In addition most ECLOs were unsatisfied with the resources available to carry out their role.There are certain limitations to our study which may have resulted in an underestimation of costs. The cost per contact was based on self-report of the average number of patients (9.1), and a smaller sample diary study of ECLOs reported an average number of 6 patients per day . HoweverOur response rate was 37% of all accredited ECLOs. One potential reason for the low response rate could be due to the sensitive nature of some questions, in particular those regarding salary. In addition, only 44.4% of ECLOs reported they had access to a computer. Thus, many may have completed the survey at home/elsewhere, and perhaps some ECLOs did not want to answer the questionnaire outside their working hours.In conclusion, ECLOs are providing previously missing information and support to patients and their families in eye clinics. However, ECLOs may not always be able to provide these services effectively due to a lack of facilities. Implementing an ECLO service may increase the cost of a low vision clinic by approximately 10.8%, but Hodge et al.  suggest ECLO: Eye Clinic Liaison Officer; LVA: Low Vision Aid; RNIB: Royal National Institute of Blind People.The authors declare that they have no competing interests.AS and MC conceived of the study and HGG acquired the data. All authors contributed to the design of the study, analysis and interpretation of the data, the drafting and revision of the manuscript and final approval of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/13/482/prepubQuestions.pdf contains a list of questions used in the survey to ECLOs.Click here for file