Brugia malayi termed abundant larval transcript , expression of which reaches ~5% of total transcript at the time parasites enter the human host.Parasites exploit sophisticated strategies to evade host immunity that require both adaptation of existing genes and evolution of new gene families. We have addressed this question by testing the immunological function of novel genes from helminth parasites, in which conventional transgenesis is not yet possible. We investigated two such novel genes from Leishmania mexicana. We then followed the course of infection in vitro in macrophages and in vivo in mice. Expression of ALT proteins, but not a truncated mutant, conferred greater infectivity of macrophages in vitro, reaching 3-fold higher parasite densities. alt-transfected parasites also caused accelerated disease in vivo, and fewer mice were able to clear infection of organisms expressing ALT. alt-transfected parasites were more resistant to IFN-γ-induced killing by macrophages. Expression profiling of macrophages infected with transgenic L. mexicana revealed consistently higher levels of GATA-3 and SOCS-1 transcripts, both associated with the Th2-type response observed in in vivo filarial infection.To test the hypothesis that ALT proteins modulate host immunity, we adopted an alternative transfection strategy to express these products in the protozoan parasite Leishmania transfection is a tractable and informative approach to determining immunological functions of single genes from heterologous organisms. In the case of the filarial ALT proteins, our data suggest that they may participate in the Th2 bias observed in the response to parasite infection by modulating cytokine-induced signalling within immune system cells. Brugia malayi is a causative agent of the disease lymphatic filariasis, which afflicts over 100 million people in tropical countries. Mosquito-borne infective stage larvae gain entry to the human body during a blood meal, and establish long-lived infections characterised by down-regulation of host T cell and macrophage reactivity -labelled cDNA was measured by phosphorimaging of spots of interest. Little difference was seen in hybridization for iNOS, IL-10, IL-12 , which was cloned into the pSSU vector and electroporated into L. mexicana. These transfectants expressed immuno-reactive protein . We are now using this system to analyse the gene expression profiles of macrophages infected with MIF-transfected parasites in both in vitro and in vivo settings.We have also extended the use of this transfection system to the analysis of other parasite genes that are hypothesised to be immunomodulatory. For example, filarial parasites produce homologues of mammalian macrophage migration inhibitory factor, MIF ,40, a cyLeishmania transfection system can be used in parallel for in vitro and in vivo experiments, and the importance of an in vivo read-out to assay gene effects within the immune system as a whole. In addition, our work has demonstrated that Leishmania transfection for helminth gene analysis is equally applicable for two completely unrelated, but immunologically important, gene families.These data illustrate the facility with which the L. mexicana is ideally suited to the study of macrophage modulation by genes predicted to function in this environment. Parallel investigations can also be envisaged by transfection of L. major, which will permit a more thorough analysis of T cell modulation to be undertaken. For example, resistance to L. major is clearly associated with development of a parasite-specific Th1 response, and it is possible that parasite genes that inhibit Th1 differentiation will alter the course of L. major infection in vivo. These studies are now under way.The application of this new strategy to transfection of alt-transfected parasites has resonance for initial host susceptibility, and longer-term propensity for chronic infection, as well as the ability to eliminate the blood-borne Mf stage.Macrophages are known to be a critical cell population in the immune response to filarial parasites at successive points in time. First, they participate in innate defences against invading larvae ,36; secoA key outcome of the present study is that ALT expression is associated with up-regulation of GATA-3 and SOCS-1. GATA-3 is a pivotal transcription factor for the development and function of the Th2 pathway ,44 and hLeishmania parasites, because macrophages from SOCS-1 null mice require 100-fold less IFN-γ to clear infection dCTP. The radiolabelled cDNA probes generated from non-infected and infected cells were hybridized to identical membranes containing the mouse cDNA arrays. Following hybridization, high stringency washes were performed and the membranes were subjected to autoradiography. Quantification was carried out using a PhosphorImager and data analyzed with ImageQuant v1.2.Gene expression was analysed using the Mouse Cytokine Expression Array (R&D systems). The mouse cytokine-specific primers were first annealed to the total RNA, which was then reverse transcribed in the presence of SuperScript II  and [α-alt-2 infected macrophages cDNA were used to produce a standard curve in each reaction. The abundances of GATA-3 and SOCS-1 were expressed as ratios of amplified product to the control, mouse S29 ribosomal protein. Primers for RT-PCR analysis were as follows: GATA-3: 5'-CTA CGG TGC AGA GGT ATC C-3' and 5'-GAT GGA CGT CTT GGA GAA GG-3'; SOCS-1: 5'-ACC TTC TTG GTG CGC GAC AGT CGC CAA-3' and 5'-GGA ACT CAG GTA GTC ACG GAG TAC-3'; and S29 ribosomal protein: 5'-ATG GGT CAC CAG CAG CTC TAC-3' and 5'-GTC CAA CTT AAT GAA GCC TAT-3'.Total RNA was extracted in Trizol, as described above, and single-stranded cDNA was synthesized using MMLV reverse transcriptase (Stratagene). Relative quantification of the expression of the genes of interest was measured by real-time PCR using the LightCycler . PCR amplifications were performed in 10 μl volumes containing 1 μl cDNA, 2.5 mM MgCl2, 3 μmM primers and the LightCycler-DNA SYBR Green I mix (Qiagen). The reaction was performed in the following conditions: 15 min activation step at 95°C for one cycle, 15 s denaturation at 95°C, 20 s annealing of primers at 50°C and 15 s elongation at 72°C, for 50 cycles. The fluorescent DNA binding dye SYBR Green was monitored after each cycle at 80°C. Five serial 1:2 dilutions of alt, abundant larval transcript gene or mRNA; BMM, bone marrow-derived macrophages; FACS, fluorescence-activated cell sorter; L3, third-stage infective larva; NO, nitric oxide.ALT, abundant larval transcript protein; Leishmania transfection methodology. TA, CCB and RMM jointly conceived the transfection strategy for Brugia genes, critically assessed progress, and made revisions to the draft manuscript. The overall study was co-ordinated by RMM who also completed the draft of the manuscript. All authors read and approved the final manuscript.NG-E designed and led the experimental work, including molecular constructs, cell transfection, immunological assays, array and RT-PCR analyses. She also drafted the manuscript. CB and LP-L made constructs in pSSU, maintained Leishmania cultures, participated in electroporation procedures, macrophage infection, in vivo infection and dilution assays. TA designed the transfection system and devised the


We establish that the occurrence of protein folds among genomes can be accurately described with a Weibull function. Systems which exhibit Weibull character can be interpreted with reliability theory commonly used in engineering analysis. For instance, Weibull distributions are widely used in reliability, maintainability and safety work to model time-to-failure of mechanical devices, mechanisms, building constructions and equipment.We have found that the Weibull function describes protein fold distribution within and among genomes more accurately than conventional power functions which have been used in a number of structural genomic studies reported to date.β for protein fold distributions varies between genomes and may reflect differences in rates of gene duplication in evolutionary history of organisms.It has also been found that the Weibull reliability parameter The results of this work demonstrate that reliability analysis can provide useful insights and testable predictions in the fields of comparative and structural genomics. Px ≈ xγ-, where x is the number of links connected to each network node and γ is the value of the exponent typically varying in the range of 2–3 [Recent advances in networks theory have demonstrated a key role of uneven distributions occurring in many natural processes. It has been found that seemingly unrelated systems such as economic, professional, sexual and social networks, airline routing, power lines connections, language networks and internet hyperlinks all exhibit a power law decay of the cumulative distribution e of 2–3 . The hetThe applicability of the scale free networks has been examined in numerous structural genomics studies. It has been proposed that the genomic occurrence of protein families, superfamilies and folds can follows an asymptotic power law:SDF(GO) = aGOb -    (1)SDF(GO) is survival distribution function of genomic occurrence GO of a certain protein family, superfamily and fold. These findings have laid the foundation for characterizing the evolution of the protein universe in terms of a growing scale-free system in which individual genes are represented as nodes of a propagating network [, where  network -7.b estimated in the study generally fall below the 2–3 range typical for scale-free systems  .In our previous work , we haveigations ). Table The established lowered values of the power exponent and modest accuracy of the power law dependences (1) encouraged us to seek alternative approaches to more accurately describe protein folds distributions.The Weibull distribution is a general-purpose statistical function defined within Extreme Value Theory  and wideα is a scaling factor and β is a shape parameter also known as the slope [, where he slope .i.e it utilizes time-to-failure (or time under the testing stress) to assess the reliability of a system and to forecast its stability through parameters of the characteristic life span α and shape β. A typical Weibull experiment is based on application of disruptive stress to multiple samples representative of the population until the tested objects achieve a state of failure and produce time-to-failure numbers. The corresponding time-to-failure values form heterogeneous Weibull distributions described by (2).The Weibull analysis operates on life data, i.e. to fail. Considering these analogies, we anticipated that the Weibull logistic can provide some natural explanations for highly heterogeneous abundance of protein folds in genomes. To test this hypothesis we used two independent approaches to examine whether the genomic occurrence of protein topologies and domains can indeed be adequately described by the Weibull function.The distribution of protein folds in a genome can be viewed much like the behavior of a mechanical system under disruptive testing. It is feasible to stipulate that the increase of genomic abundance of any protein fold occurs under evolutionary pressure. Some folds are able to expand their genomic occurrence over a course of evolution others have higher probability to be lost through genetic drift and other random events, SDF(x) of the genomic occurrences of protein topologies and homologous superfamilies into the Weibull dependence (2). The corresponding Weibull shape parameters have been established by solving the equation  while the scaling factors have been calculated as .First of all, we employed the maximum likelihood (ML) method  to fit tSDF(GO) parameters reproduced within equation (2) through α and β values estimated by the ML approach. It is obvious that these computed blue curves labeled as 'Weibull analytical' resemble the experimental distributions (marked in red) very precisely.The ML method allowed very accurate description of the distribution of protein folds among the genomes. Figures α and β values estimated by the ML approach have been collected into Table 2 (see The corresponding le 2 see .SDF(x) leads to the equation of a straight line:The second way of examining applicability of the Weibull function (2) was based on notion that the double logarithmic transformation of the SDF(x)) = β log(x) - β log(α)     (3)log(- log(SDF(GO) data to estimate the Weibull coefficients α and β and squared correlation coefficients r2 which all have also been collected into Table 2 (marked as 'Weibull by plotting').We performed the transformation (3) on the experimental r2 from Table 2 demonstrate very high accuracy of the linear dependences (3) established from the survival distributions of CATH folds in the studied genomes. These parameters also allow comparing the accuracy of double logarithmic dependences (3) with accuracy of simple logarithmic dependences derived from the power law model (1):The values of the estimated squared correlation coefficients log(SDF) = a - b*log(GO)     (4)SDF(GO) functions for individual genomes, superkingdoms and for the combined set of proteins. The comparison of r2 values from Table 2 and Table 1 established from the linear functions (3) and (4) respectively, reveals that for all studies cases  the statistical quality of Weibull dependences (3) is much better than of power law function (4). Figures SDF(x) curves with remarkable accuracy. Apparently, the distributions calculated from (3) (labeled as 'Weibull by plotting') are much closer to the experimental distributions than the power law curves (labeled as 'power law') computed within the conventional power function (1). Apparently, that the Weibull distributions established by the double logarithmic representations (4)  and the preference should, perhaps, be given to more stringent ML-derived data.As it has been mentioned earlier, the dependences (4) have been estimated for the Although the estimated statistical criteria clearly demonstrate the suitability and superiority of a Weibull function over a power function in describing protein fold distributions, we decided to examine several additional criteria characteristic of the Weibull distribution. As it has been suggested by Romeu  there arr2 values from the columns marked as 'Weibull by plotting' are all contained within the range ~0.95–0.98 what demonstrates that the 'Weibull papers' do indeed describe protein folds distribution in the studied genomes with high accuracy. Figures As it can be seen from Table 1 the estimated β derived by analytical maximum likelihood approach.The data from Table 2 demonstrate that the estimated slopes of the 'Weibull papers' are very close to the values of GOβ distributions through the power factors β. The exponential character of the resulting distribution has been examined by several statistical tests and in all cases has been confirmed. The observed medians of the exponential distributions GOβ accumulated in Table 3  from shape and scale parameters [Thus, all four specific criteria studied indicate that the genomic occurrence of protein topologies and domains can be characterized as true Weibull distributions. To support this notion further we have also considered another important property of the he Weibull distribution – the dependence of its median  and analytical (ML) approaches. The corresponding 'MDN Calctd' values have been collected into Table 3 along with the observed medians of the Weibull distributions (marked 'MDN Obsrvd'). The estimated high quality linear dependences between the theoretical and observed medians are present on figures To assess the applicability of this condition, we calculated Weibull medians using sets of Thus, multiple independent tests have demonstrated that occurrence of protein folds in genomes obey the Weibull distribution and therefore can be interpreted in terms of the reliability theory what can provide additional insight into folds evolution.The very fact that we were able to assign the Weibull character to the distributions of the CATH protein topologies and homologous superfamilies within genomes ultimately implies that parameters of genomic occurrence can be classified as extreme values. According to the Extreme Values Theory the Weibull distribution will successfully model life systems for which many competing similar failure processes are "racing" to failure and the first to reach it produces the observed failure time . In regaβ > 1 characterizes a life system that increasingly deteriorates. If the shape parameter is smaller then unity (β <1), there is a reliability growth as the failure rate of the system decreases with time [β does indeed describe the "skewdness" of the fold distribution, for example Caenorhabditis elegans has the lowest calculated value β among the studied organisms, whilst this genome has also been characterized for its recent expansion and duplication of several gene families [β (according to the reliability theory characterizing the genome of C. elegans as the most stable amongst the studied) may reflect the fact that chances of loosing some lower abundant fold families are lower for C. elegans (considering that >70% of the translated ORFs C. elegans genome have been covered by the sequence-structure threading we have assumed that the recently duplicate genes are accordingly represented in the results). In this context, the reliability of a proteome can be viewed as its ability to maintain and expand its composition without loss of protein folds.On another hand, according to reliability theory a Weibull distribution with ith time . It is nfamilies . Presumaβ <1 i.e. to have more balanced (less heterogeneous) folds representation in their genomes. The fact that most β values presented in Table 2 fall below the unity threshold demonstrates that, in general, the reliability of genome fold composition increases with time, i.e. less protein folds reach the failure state  as an organism evolves.We can speculate that life systems that enjoy evolutionary success will tend to minimize β shape parameters for topologies distributions across the three superkingdoms. All three linear dependences ln(- ln(SDF(GO))) ~ ln(GO) for Bacteria, Eukaryote and Archaea presented on Figures Interestingly, little difference is observed has been found between the i.e. more abundant folds tend to expand their genomic presence even further causing lesser abundant folds to extinct.As it has been already mentioned above, it is difficult to decide at this point whether the observed Weibull character of protein folds distribution can be placed in a larger context. We can only speculate that protein folding preferences may lead to a greater abundance of favorable protein configurations and to extinction of those folds which are less favorable. Such selection may represent a mechanism of evolutionary quest for searching for better protein folds. In any case, the observed phenomenon illustrates the act of natural selection in determination of the protein fold repertoires and that the propagation of protein folds in a genome occurs in a competitive manner, It also remains to be seen whether some other properties of genomes and proteomes can also be described by the Weibull statistics. In our studies we plan to use the Weibull approach to examine other distributions such as genomic occurrence of transcriptional promoters and regulatory elements, levels of gene expression and occurrence of protein domains per gene, among others.etc in the CATH can be adequately described by the Weibull law. Thus, it has been previously demonstrated that another widely used folds library – SCOP does indeed obey the power low [Another possible development for the reliability analysis in structural genomics might be to investigate whether the standard libraries of proteins folds themselves can be adequately described by the Weibull function. As it has been stipulated, in the study we have used the CATH standard library of protein folds, which is one of the most accepted and used protein folds classifications. Ii is not unfeasible, that the representation of protein architectures, topologies, homologous superfamilies, ower low . Such obTo summarize the current work, it is possible to conclude that the use of the Weibull distribution allows more accurate description of protein topologies and domains distributions within and among genomes than power function used in conventional structural genomic studies. In addition, we were able to establish the Extreme Values relationships for protein folds distributions to demonstrate that the protein fold repertoire of an organism most likely occurs as a result of the competition amongst folds. This may reflect a mechanism of natural selection searching for an optimal protein structures when more evolutionary favorable folds tend to populate the entire genomic space and cause the extinction of lesser favorable protein configurations.Use of a Weibull function allows describing cumulative distribution of protein topologies and domains within individual genomes and superkingdoms with higher accuracy compared to the conventional power function used in the related studies. The developed approach may be applied to quantification of the distribution of different properties of genomes and can be particularly useful for assessing and comparing fold distributions between different organisms and possible impact of the "reliability" of organisms due to a higher redundancy in their fold composition.In general, the results of investigation demonstrate the feasibility and importance of using the reliability analysis to improve the bioinformatics analysis of proteomes.The prediction of the protein folds has been conducted using the THREADER2 program . The CATThe translated protein sequences for 33 complete genomes downloaded from the NCBI and ENSEMBL databases have been processed in this manner. The threading computations have been paralleled for processing on a Beowulf cluster consisting of 52 dual processor blades . The automated control was implemented by our own PVM-supporting Perl scripts enabling to distribute and query the individual threading processes over multiple computer servers.GO. At the next step these numbers have been converted into the fractional values. After that the survival distribution functions SDF(x) have been computed for genomes, superfamilies and for the combined set of proteins. The SDF(GO) numbers have been calculated for each integer GO value in the range from 0 to the maximal GO estimated within the set .After the occurrences of distinct classes, architectures, topologies and homologue family representatives have been established within the individual genomes, superkingdoms and in total, the corresponding survival distributions have been computed. First of all, we have established the counts of protein architectures, topologies and domains (homologues families) with a given genomic occurrence SDF(GO)~GO functions has been conducted by the SAS 9.0 statistical package (SAS Inc.). The power law dependences SDF(GO) = aGOb -have been analyzed as a logarithmic transformsThe fitting of the SDF(GO) = a - b*GOLog(where the fitting has been conducted for the linear function.SDF(GO) = exp(-aGOb) have been fitted using both non-linear approximation (by maximum likelihood method) and by the linear fitting of the double logarithmic transform:The Weibull – like dependences SDF(x)) = β log(x) - β log(α)log = a GOClick here for fileα, β and medians  of Weibull distribution of survival functions of genome occurrences  established by maximum likelihood and plotting methods.Parameters Click here for fileStatistical parameters for 'Weibull papers' for genomic occurrences of protein topologies and domains.Click here for file


Antiretroviral drugs change the lives of patients with HIV/AIDS—if they have access to them. Most patients in resource-poor countries cannot afford the drugs. Major initiatives are under way to expand access to antiretrovirals in developing countries, but the number of individuals in need of the drugs currently vastly exceeds the supply, and will continue to do so for the foreseeable future. These circumstances make for difficult decisions about treatment allocation. David Wilson and Sally Blower have shown how it is possible to design an equitable antiretroviral allocation strategy, that is, to come up with a plan that would give each individual with HIV an equal chance of receiving antiretrovirals. Their novel spatial model enables them to model the “spatial diffusion” of antiretrovirals in a resource-constrained country.Based on the premise that only a limited number of drugs will be available and only a limited number of health-care facilities can be used for drug distribution (each of them serving the population in a specific area), they determine an optimal equitable allocation strategy. They then apply this approach to a practical example—the equitable allocation of antiretrovirals to patients with HIV/AIDS in the South African province of KwaZulu–Natal. Using data from a detailed rollout plan for antiretrovirals designed by the South African government, they come up with an allocation strategy that differs substantially from the current governmental plan for the province.KwaZulu–Natal has a total of 54 health-care facilities, of which 17 are assigned to allocate antiretrovirals under the current plan. It is the largest province in South Africa, with a population of about 9.4 million, and it has more people with HIV than any other province . Wilson and Blower assume that the available amount of antiretrovirals can treat 10% of the individuals with HIV in KwaZulu–Natal. Modeling the 17 health-care facilities and the 51 communities of individuals with HIV, they determine the amount of drugs to allocate to each facility to achieve equitable access by patients throughout the province. They then extend the analysis assuming that additional health-care facilities could be made available to distribute drugs. They conclude that in order to achieve the greatest degree of treatment equality, all 54 health-care facilities should be used, and they should, on average, each serve the population within a radius of 50 km.Wilson and Blower discuss how their model can be adjusted and therefore used by policy makers in resource-constrained countries to determine a scientifically based allocation strategy for limited resources based on a number of specific objectives. They also recognize that there are other considerations that influence ethical treatment allocation besides equity, for example, the desire to maximize epidemic reduction, or the imperative to give priority to the least advantaged individuals They believe that their model can be adjusted and therefore “used by policy makers to determine an optimal scientifically based allocation strategy” for a number of specific objectives. Another possibility would be to apply the equity strategy to allocate drugs to particular health-care facilities , and then take additional ethical considerations into account at the community level.


Invasion is an important early step of cancer metastasis that is not well understood. Developing therapeutics to limit metastasis requires the identification and validation of candidate proteins necessary for invasion and migration.We developed a functional proteomic screen to identify mediators of tumor cell invasion. This screen couples Fluorophore Assisted Light Inactivation (FALI) to a scFv antibody library to systematically inactivate surface proteins expressed by human fibrosarcoma cells followed by a high-throughput assessment of transwell invasion.Using this screen, we have identified CD155 (the poliovirus receptor) as a mediator of tumor cell invasion through its role in migration. Knockdown of CD155 by FALI or by RNAi resulted in a significant decrease in transwell migration of HT1080 fibrosarcoma cells towards a serum chemoattractant. CD155 was found to be highly expressed in multiple cancer cell lines and primary tumors including glioblastoma (GBM). Knockdown of CD155 also decreased migration of U87MG GBM cells. CD155 is recruited to the leading edge of migrating cells where it colocalizes with actin and αv-integrin, known mediators of motility and adhesion. Knockdown of CD155 also altered cellular morphology, resulting in cells that were larger and more elongated than controls when plated on a Matrigel substrate.These results implicate a role for CD155 in mediating tumor cell invasion and migration and suggest that CD155 may contribute to tumorigenesis. Metastasis is responsible for greater than 90% of cancer-related deaths . It is tBoth invasion and dispersal are complex processes that require migration of individual cells from the tumor core into surrounding tissue and the extracellular matrix. Cell migration requires a coordinated orchestration of complex events including polarization, protrusion, adhesion, de-adhesion, and retraction . Many ceIn the post-genome era, global strategies are being developed to identify new players in complex biological processes such as tumor cell invasion. Microarray experiments have identified many differentially expressed genes that may contribute to enhanced invasion -10, but To complement these approaches, we have developed a high-throughput, acute protein inactivation strategy called fluorophore-assisted light inactivation (FALI) that allows for direct assessment of protein function through the systematic inactivation of proteins -16. We h2 atmosphere.HT1080 human fibrosarcoma, Hs27 human fibroblasts, and U87MG human glioblastoma cells were obtained from ATCC. Normal human astrocytes were obtained from Cambrex Bioproducts. Cells were maintained in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin . HT1080 cells were additionally supplemented with 0.1 mM non-essential amino acids (Invitrogen). All cells were grown at 37°C under a humidified 7% COMonoclonal antibodies used for FALI were as follows: β1-integrin ; CD155 . Two additional CD155-specific monoclonal antibodies  were generated by fusing specific binding scFvs selected from our library to a human IgG backbone. ScFv 1A2 was used as a negative control in the migration assay. 1A2 was selected for binding to the surface of HT1080 cells (see scFv Library Generation) and was confirmed to bind to the surface of HT1080 and U87MG cells by immunocytochemistry, though its protein target is not known. Antibodies used for immunocytochemistry and immunoblotting were as follows: CD155 ; αv-integrin ; ErbB2 . Actin was visualized with rhodamine-phalloidin (Molecular Probes). Fluorophore-labeled secondary antibodies were obtained from Molecular Probes; peroxidase-labeled secondary antibodies were from Cell Signaling.E. coli TG-1 resulting in a library of 107 independent clones. HTl080-specific scFvs were selected by immunopanning phage against fixed HT1080 lysate, resulting in 2760 binders. These scFvs were recloned into expression vector pXP7 (containing his- and E-tags) and expressed in TG-1 cells. Bacterial lysates containing scFv were prepared and tested for binding to the surface of fixed HT1080 cells by ELISA. This additional round of selection yielded 595 HT1080 surface binders. These scFvs were confirmed to bind to the surface of HT1080 by immunocytochemistry on live cells. For FALI/screening, his-tagged scFvs were purified from bacterial lysates using Ni-NTA Superflow resin (Qiagen).Spleen RNA was harvested from HT1080 immunized mice as described . Immunog5 lux) blue-filtered light  using a high-powered slide projector . A replicate control plate was kept in the dark for 1 h.Antibodies or scFvs were conjugated with FITC (Molecular Probes) at pH 9.5 at room temperature as previously described . Cells w5 cells were labeled with cell tracker orange, incubated with 20 μg/ml of FITC-labeled scFv, irradiated (or kept in the dark) with blue-filtered light, and 5 × l04 cells were loaded onto matrigel-coated  polycarbonate membranes . Each sample was assayed in triplicate in at least two independent experiments. scFvs that showed a significant change in invasion (p < 0.01 and change > 2 standard deviations) were assayed a third time with a new scFv preparation. U87MG and HT1080 migration assays were performed essentially as above, but excluded matrigel and used 8μM, 96-well Fluoroblok membranes (BD Biosciences). Cell invasion or migration was quantified using a fluorescent plate reader  and confirmed by visualization under an inverted fluorescent microscope.FALI invasion assays with HT1080 cells were done as described previously ,16 but uHomo sapiens in the NCBI and SwissProt databases. Alternatively, the extracted peptide fragments were analyzed by nano electrospray mass spectrometry (nanoES-MS) on a Q-STAR QqTOF mass spectrometer (PE SCIEX). Relevant ions were selected for CID (collision induced dissociation)-MS and the obtained fragment ion data used for Peptide Sequence Tag database search.To identify scFv protein targets, candidate scFv genes were re-cloned into expression vector pXP14, containing a strep-tag. Purified scFvs were coupled to StrepTactin Sepharose (50 μg/50 μl resin) and the washed scFv-beads were added to 1 mg HT1080 lysate. The scFv-target complexes were eluted  and the immunoprecipitated proteins were analyzed by SDS-PAGE and silver staining. In a parallel experiment, the immunoprecipitated proteins were subjected to deglycosylation using N-glycosidase F prior to SDS-PAGE analysis. Stained bands were excised and subjected to in-gel tryptic digestion. The peptide fragments were extracted from the gel, desalted on ZipTip μC18, the eluted peptides spotted on a Teflon-coated MALDI target, let dry and overlayed with 1 μl of a 3.5 mg/ml solution of α-Cyano-hydroxycinnamic acid. The samples were analyzed on a STR-DE Voyager MALDI mass spectrometer (Applied Biosystems) and the obtained peptide masses were used for protein identification via peptide mass fingerprint, searching all entries for the species A double stranded siRNA oligonucleotide targeting CD155 (5'-CAACUUUAAUCUGCAACGUdTdT-3') was chemically synthesized (Dharmacon Research) and transfected into HT1080 cells using Oligofectamine (Invitrogen) following manufacturers instructions using 200 nM siRNA per 10 cm dish. Cells were incubated with siRNA in OptiMEM (Invitrogen) for 6 hrs after which time normal growth media was added. Cells were then incubated for 48–72 h to achieve >80% knockdown of CD155. Control cells were transfected with a scrambled siRNA oligonucleotide at matching concentration. Cells were then inserted into the migration assay described above or used for morphology experiments.3 cells were loaded onto glass coverslips coated with Matrigel . After 2 hours of incubation at 37°C/7% CO2, cells were fixed and immunostained for CD155 using mAb D171. Cells were visualized with a Nikon Diaphot 200 microscope and images taken of >100 transfected cells (containing fluorescent oligo). Analysis of images was performed with OpenLab software (Improvision). Measurements of cell area, perimeter, and shape were made for each condition.HT1080 cells were co-transfected with siRNA (scramble control or CD155-specific) and fluorescently labeled oligonucleotide  for 48 hours. Cells were detached with Versene, and 5–15 × 108-chamber slides  were coated with matrigel (2 μg/ml) and blocked with FCS. 100,000 cells were plated in each well and grown to confluency. A 20-gauge needle was used to create a linear wound and cells allowed to recover for 3 h at 37°C. Cells were fixed and processed for immunocytochemistry as described below.Cells were fixed in PBS/4% paraformaldehyde/4% sucrose, blocked in PBS/0.01% triton-x-100/10%FBS, and incubated in primary antibody for 1 h at rt. Appropriate species-specific secondary antibodies conjugated to Alexa 488 or 594 were used to visualize antibody staining on a Leica SP2 confocal microscope using LCS software. Antibodies: CD155 (D171), αv-integrin (AB1930), actin , ErbB2 (6562). Secondary antibodies alone were used to control for non-specific staining. For primary tissue, paraffin sections from a human tissue library were stained with biotinylated anti-CD155 human mAb 1D8 at 200 μg/ml and visualized with streptavidin-hrp and NovaRed substrate. For glioblastoma tumors, we used tissue microarrays, made from 20 primary tumors arrayed in quadruplicates of 1 mm cores with a Beecher Instrument arrayer. Positive staining was visualized with DAB substrate.Pelleted and PBS-washed cells were lysed in NP40 lysis buffer  with protease inhibitors (Roche) at 4°C. Lysates were cleared by centrifugation and quantified using the DC Protein Assay (BioRad). 30 μg lysate was separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were blocked in 5% nonfat dry milk in PBS and probed with primary antibody overnight. Antibody binding was detected with peroxidase-conjugated secondary antibodies  and visualized using ECL substrate (PerkinElmer).We generated a recombinant single chain variable fragment (scFv) antibody library that recognized proteins on the surface of HT1080 fibrosarcoma cells, a highly invasive tumor cell line. ScFvs were selected from a phage display library generated from mice immunized with fixed HT1080 cells . Genes eThe target of group I was α3βl-integrin, a promiscuous extracellular matrix binder that plays multiple roles in cell adhesion, morphology, migration, and invasion . IdentifIn order to determine whether this result was specific to invasion through matrigel or due to effects on cell migration, we repeated the transwell experiment in the absence of matrigel using several different CD155-specific monoclonal antibodies, either previously characterized  or newly generated by fusing the variable regions from our CD155-binding scFvs to a human IgG backbone . FALI of CD155 significantly inhibited transwell migration by 20 to 23% .High levels of CD155 expression were observed in HT1080 and U87MG cells whereas the protein was only weakly expressed in their non-tumor counterparts Fig. . Since HGiven our finding that CD155 appeared to be overexpressed in our model tumor cell lines, we evaluated CD155 expression levels in normal and cancerous human tissue. We performed immunohistochemistry on paraffin sections taken from a tissue library using mAb 1D8. In normal tissue we observed moderate staining in kidney, plasma cells, liver, lung, theca interna of the ovary, and testis (data not shown). No staining was observed in normal adrenal, bladder, brain, breast, colon, heart, pancreas, placenta, prostate, skin, skeletal muscle, small intestine epithelium, spleen, stomach, thymus, thyroid, or uterus (at least two samples examined per tissue). In cancer tissue, we observed extensive staining in a subset of samples taken from several different tumor types Fig. . These iTo address the role of CD155 in migrating cells, we employed a modified wound-healing assay and examined the sub-cellular localization of CD155. HT1080 cells were plated onto chambered tissue culture slides coated with a thin layer of Matrigel ECM substrate and grown to near confluence. A linear wound was made using a 20-gauge needle and the cells were allowed to recover for 3 h. Cells were fixed and immunostained to visualize CD155. CD155 was found to preferentially localize to the leading edge of cells, though some staining in trailing edges and cell-cell contacts could also be seen Fig. . In cellGiven our findings that CD155 was important for cell migration and that it localized to the leading edge of migrating cells, we asked whether CD155 might co-localize with other proteins known to be involved in motility. We used immunofluorescence and confocal microscopy to visualize CD155 along with actin and αv-integrin. CD155 colocalized extensively with actin ruffles at the leading edge of migrating cells Fig. , suggestThe previous section showed that CD155 localizes to the leading edge of migrating cells and colocalizes with known mediators of motility. Since cell shape changes are often associated with changes in motility and/or adhesion, we next investigated whether knockdown of CD155 by RNAi affected cellular morphology. HT1080 cells were transfected with either a CD155-specific or scrambled control siRNA (200 nM) along with a fluorescent, non-specific oligo (10 nM) to identify transfected cells. 48 hours after transfection, cells were plated at low density onto coverslips that had previously been coated with Matrigel, fixed, and immunostained for CD155 to confirm protein knockdown. Only cells containing fluorescent oligo were selected for analysis. CD155 knockdown cells had significantly larger perimeters and appeared more irregular in shape than control cells  tumors for which migratory behavior is poorly understood [Our studies so far have been conducted using HT1080 fibrosarcoma cells. To assess the generality of CD155 function in cancer, we asked whether it could regulate the migration of other tumor cells derstood . U87MG GWe developed a functional proteomic screen to identify surface proteins involved in tumor cell invasion  and herein vitro migration and a pronounced change in cellular morphology with cells becoming more elongated and irregular in shape. CD155 localizes to the leading edge of migrating tumor cells and co-localizes with actin ruffles and αv-integrin, suggesting that CD155 may act in motility and/or cell-substrate adhesion. We also observed elevated expression of CD155 in a number of different cancer cell lines and primary tumors, suggesting a link between CD155 and tumorigenesis.Knockdown of CD155 by FALI or by RNAi resulted in impaired Tage4 gene [The endogenous function of CD155 is not well understood. CD155 is a type I transmembrane glycoprotein first identified based on its ability to mediate the binding of poliovirus to host cells . It is age4 gene . Nectinsge4 gene ,25. Whilge4 gene , suggestin vitro suggesting that CD155 may mediate cell-substrate adhesion [Several proteins have been found to interact with CD155. The ECM protein vitronectin binds CD155 adhesion . Our finadhesion  and suggadhesion . Our expadhesion . We specadhesion . CD47 apadhesion . CD155 hadhesion , the dynadhesion , and alsadhesion . Future Our results, in which CD155 inhibition reduces migration, are also consistent with a model in which transient interactions between CD155 and ECM result in pro-migratory signals. For example, it is known that binding of integrins to ECM proteins induces pro-migratory signaling through clustering of associated kinases . CD155 iCD155 expression has been reported widely to be restricted to primates ,36, but ) using the unique identifier AACCACCCAG supports the idea that expression of the CD155 gene may be elevated in several tumor types including colon, brain, kidney, pancreas, lung, and stomach  from Xerion Pharmaceuticals, AG.KS contributed to study design, data interpretation, carried out the migration experiments with FALI and RNAi and the characterization of CD155 expression, and drafted the manuscript. BE contributed to study design, data interpretation, carried out the invasion screen and morphology experiments, and helped revise the manuscript. JS contributed to the invasion screen. CZ and CU generated and characterized the scFv library. CT performed the IP and mass spec to identify CD155. MS helped with the morphology experiments. JR and DL performed the GBM tissue staining and analysis and provided helpful discussion. LI and DJ contributed to study design, data interpretation, and editing of the manuscript.The pre-publication history for this paper can be accessed here:


The pulse oximeter, a medical device capable of measuring blood oxygen saturation (SpO2), has been shown to be a valuable device for monitoring patients in critical conditions. In order to incorporate the technique into a wearable device which can be used in ambulatory settings, the influence of motion artifacts on the estimated SpO2 must be reduced. This study investigates the use of the smoothed psuedo Wigner-Ville distribution (SPWVD) for the reduction of motion artifacts affecting pulse oximetry.The SPWVD approach is compared with two techniques currently used in this field, i.e. the weighted moving average (WMA) and the fast Fourier transform (FFT) approaches. SpO2 and pulse rate were estimated from a photoplethysmographic (PPG) signal recorded when subject is in a resting position as well as in the act of performing four types of motions: horizontal and vertical movements of the hand, and bending and pressing motions of the finger. For each condition, 24 sets of PPG signals collected from 6 subjects, each of 30 seconds, were studied with reference to the PPG signal recorded simultaneously from the subject's other hand, which was stationary at all times.The SPWVD approach shows significant improvement (p < 0.05), as compared to traditional approaches, when subjects bend their finger or press their finger against the sensor. In addition, the SPWVD approach also reduces the mean absolute pulse rate error significantly (p < 0.05) from 16.4 bpm and 11.2 bpm for the WMA and FFT approaches, respectively, to 5.62 bpm.The results suggested that the SPWVD approach could potentially be used to reduce motion artifact on wearable pulse oximeters. Wearable medical devices are capable of continuously monitoring an individual's vital signs in real time. These devices are particularly important to the world's increasingly aging population, whose health conditions have to be assessed regularly or monitored continuously. The devices can warn individuals of symptoms of deterioration, e.g. alerting them when their blood pressure is increasing to a level above a predetermined threshold. The devices can also automatically notify emergency services in critical situations. In order to make wearable devices practical, a series of technical problems have to be solved. For example, these devices need to be miniature in size, must possess a user-friendly interface and be efficient in power consumption. Most importantly, these devices need to have a low failure rate and must report minimal false alarms. In other words, these devices are required to provide an accurate estimate of the monitored vital sign under normal daily life situations. This leads to the important topic on the reduction of motion artifacts -4. In thThe paper is organized as follows. Section 2 reviews the techniques commonly used for attenuating motion artifacts in pulse oximetry. Section 3 discusses the basic theory for SpO2 computation and the techniques used in this study for reducing motion artifacts. Section 4 compares the performance of two time-frequency techniques, i.e. the short-time Fourier transform (STFT) and the SPWVD. Section 5 presents the protocol and the results of an experiment to assess motion artifact reduction in real data. Section 6 discusses the performance of the SPWVD approach as compared to the traditional time domain and spectral methods. Lastly, the major findings of this paper are summarized in section 7.SpO2 is commonly monitored by a pulse oximeter, which has been widely adopted as a standard measure during anesthesia, neonatal care and post-operative recovery ,6. PulseA number of attempts have been made in the past decade to improve the accuracy of pulse oximeters when subjects move. Typical methods can be generally classified into three categories: (1) based on an independent measure of motion; (2) based on a model of the ideal signal or the noise; and (3) based on features recognized from the corrupted signal. For techniques based on an independent measure of motion, one or more transducers  are employed to record the user's motion. By assuming that the artifact is a linear addition to the pulsatile photoplethysmographic (PPG) signal, the original signal can be reconstructed from the corrupted signal -11. ThisA recently proposed PPG artifact reduction methodology was based on the inversion of a nonlinear physical artifact model and could significantly reduce the effect of changes of probe coupling ,12. Howeet al. calculated the derivative of a signal and identified a portion of it as a motion artifact whenever the ratio of adjacent positive and negative peaks of the derivative is below a threshold ,     (1)εHbO2 and εHb are the extinction coefficients of oxygenated and de-oxygenated hemoglobin, and s, c, and d represent SpO2, total concentration of hemoglobin and the optical path length respectively.where, R, i.e. the normalized ratio of the red to the infrared transmitted light intensity. That is,By using two light sources – red and infrared lights – and calculating a normalized ratio of the AC component to the DC component for each light source, SpO2 can be computed from the ratio of ratios R. In this study, SpO2 is estimated directly from equation (3).In practice, SpO2 can be obtained from equation (3) directly or by an empirical equation that relates SpO2 and By calculating the ratio of the AC components and the ratio of the DC components of the two light sources, SpO2 can be obtained from every single pulse of a PPG signal. To stabilize the reading, the weighted moving average (WMA) is often used . TypicalBased on the hypothesis that cardiac rate can be estimated more easily by spectral analysis than time domain analysis, techniques in the frequency domain have been widely investigated as alternatives in pulse oximetry. For example, the FFT and discrete cosine transform (DCT) were proposed for estimating SpO2 -16. Thesx(t) is given as:The Wigner-Ville distribution (WVD) of a signal x(t) and x*(t) are the time series of the signal and its complex conjugate respectively.where The problem of the WVD is the so-called cross-term interference, which appears as frequencies that lie between the frequencies of any two strong components. In order to suppress cross-term interference, the smoothed pseudo WVD is often used:h and g are equivalent to smoothing the WVD in the frequency and time domain respectively. Selection of the window is a compromise between the joint time-frequency resolution and the level of cross-term interference. Common choices of window include the rectangular and Kaiser windows [g(t) and h(τ).The two windowing operations  windows -21. In oR and SpO2. Moreover, as the rate of change of SpO2 is relatively slow, SpO2 that changed by more than 2% per second was considered to be physiologically impossible, and was rejected from the calculation [The maximum magnitude within the cardiac frequency band of the SPWVD in each second was used for SpO2 computation. Since SPWVD represents energy distribution, the square root of the magnitude was used for calculating the ratio of ratios culation .The performance of SpO2 computation by the SPWVD approach was evaluated in a simulation using PPG signals collected from a volunteer under modest random motions. For comparison, SpO2 was also estimated from the spectrogram obtained by STFT.The noise-mixing-composition (NMC) method was applied to mimic the clinical situation and to induce a range of signal-to-noise ratios (SNR) . To syntS and artifact episode N and a mix parameter ε, which was adjusted to achieve the desired SNR.with the basis episode 512 samples of the PPG signal were recorded at 20 Hz from a volunteer. The signal was contaminated with in-band noise but with the cardiac frequency recognizable. The artifact noise was extracted by a filtering technique in the frequency domain. The spectrogram of the PPG signal was calculated using an FFT-based algorithm. Coefficients of the spectrum within 0.5 Hz of the primary cardiac frequency or its harmonics were set to zeros. The inverse FFT of the modified spectrum allowed us to obtain a pure artifact noise. Typical spectra of the contaminated signal (solid) and the resulting pure artifact noise (starred) are shown in Figure ε in equation (6). The undisturbed signal was also estimated from equation (6) by setting ε = 0. SpO2 were estimated from the set of synthesized signals in an 8-second period at 1-second interval by using the STFT and SPWVD approaches. The mean SpO2 error during the complete 25.6 seconds is shown in Figure A set of synthesized signals with different SNR values was obtained by changing the value of the mix parameter Figure It should be noted that the pulse rate was predetermined in the simulation, which helped both approaches to determine the cardiac frequency band more accurately. In practical situations, the electrocardiogram can be recorded simultaneously and used as a reliable pulse rate estimator. As for the computational cost, the SPWVD approach can be implemented efficiently by making use of its symmetry properties, and thus, it can reduce the computational cost to a quarter of that of the STFT technique .The purpose of this experiment is to compare the performance of three different methods  in estimating SpO2 on subjects when they are (a) in a resting position and (b) in motion.Six healthy subjects participated in the study. Four kinds of motions have been investigated: horizontal movement (M1) and vertical movement (M2) of the hand, as well as the bending motion (M3) and pressing motion (M4) of the finger. These motions were selected because they are some of the common movements attributable to the motion artifact in pulse oximetry ,24. SubjThe collected signals were separated into an AC and a DC component. The AC component was filtered out by a 4th order Butterworth band-pass filter with cut-off frequencies at 0.5 Hz and 20 Hz. The ratio of the DC components was computed directly in the time domain and the same value was used for the three different approaches, i.e. the WMA, FFT and SPWVD approach. On the other hand, a different ratio of the AC components was computed using each of the three approaches.To evaluate the performance of the different approaches, the SpO2 bias and precision, the pulse rate error, the dropout rate and the SpO2 performance index (PI) were calculated. The bias and precision are defined as the mean and standard deviation of the difference between reference and estimated SpO2 respectively. The pulse rate error is the difference between reference and estimated pulse rate. The dropout rate and SpO2 PI are evaluation parameters adopted from previous work by S.J. Barker . The droTable However, the SPWVD approach shows significant improvement in both SpO2 and pulse rate estimation as compared to the WMA and FFT approaches when subjects were in motion. SpO2 estimated from the SPWVD, WMA and FFT approaches differed from the reference by -1.07 ± 2.42%, -1.31 ± 3.58% and -1.42 ± 3.18%, respectively. The mean absolute pulse rate error is reduced significantly (p < 0.05) from 16.4 bpm and 11.2 bpm for the WMA and FFT approaches, respectively, to 5.62 bpm for the SPWVD approach. The SpO2 PI also has the highest SpO2 PI (91%). Both the SPWVD and FFT approaches achieve 0.0% dropout rate. The WMA approach sometimes failed to give a reading during bending or pressing motions (dropout rate = 4.6%), which would lead to instrument "dropout" or "freeze" in clinical situations.Figure For the estimation of pulse rate, 90% of the pulse rate error falls in the error band  when the SPWVD approach is used  in pulse rate estimation when subjects were in motion. Although such a significant improvement is not found in the estimation of SpO2, this is attributed to the fact that erroneous SpO2 estimates above the 100% upper bound were always rejected. It is hypothesized that when patients with SpO2 much lower than 100% are recruited as subjects for evaluating the different approaches, the performance of each approach will be more notably different from each other. However, this hypothesis remains to be proven in a clinical study involving a significantly large patient population.Estimation of SpO2 by a time-frequency representation, the SPWVD, has been investigated in this study. The approach has been tested on four kinds of motions that are found in common movements associated with motion artifacts in pulse oximetry ,24, i.e.The author(s) declare that they have no competing interests.YSY designed and carried out the experiment, analyzed and interpreted the data, and drafted the manuscript. CCYP helped to analyze and interpret the data, and assisted in drafting the manuscript. YTZ conceived of the study, and participated in its design and coordination and helped to finalize the manuscript. All authors read and approved the final manuscript.


Autonomic nervous system (ANS) dysfunction is present in up to one third of patients with tetanus. The prognostic value of ANS dysfunction is known in severe tetanus but its value is not well established in mild to moderate tetanus.Medical records of all patients admitted with tetanus at two academic tertiary care centers in Karachi, Pakistan were reviewed. The demographic, clinical and laboratory data was recorded and analyzed. ANS dysfunction was defined as presence of labile or persistent hypertension or hypotension and sinus tachycardia, tachyarrythmia or bradycardia on EKG. Patients were divided into two groups based on presence of ANS dysfunction (ANS group and non ANS group). Tetanus severity was classified on the basis of Ablett criteria.Ninety six  patients were admitted with the diagnosis over a period of 10 years. ANS group had 31 (32%) patients while non ANS group comprised of 65 (68%) patients. Both groups matched for age, gender, symptom severity, use of tetanus immunoglobulin and antibiotics. Twelve patients in ANS group had mild to moderate tetanus (Ablett I and II) and 19 patients had severe/very severe tetanus (Ablett III and IV). Fifteen (50%) patients in ANS group required ventilation as compared to 28 (45%) in non-ANS group (p = 0.09). Fourteen (47%) patients died in ANS group as compared to 10 (15%) in non ANS group (p= 0.002). Out of those 14 patients died in ANS group, six patients had mild to moderate tetanus and eight patients had severe/ very severe tetanus. Major cause of death was cardiac arrhythmias  in ANS group and respiratory arrest  in non ANS group. Ten (33%) patients had complete recovery in ANS group while in non ANS group 35(48%) patients had complete recovery (p= 0.05).ANS dysfunction was present in one third of our tetanus population. 40% patients with ANS dysfunction had only mild to moderate tetanus. ANS dysfunction, irrespective of the need of mechanical ventilation or severity of tetanus, predicted poor outcome. Tetanus is still a major health problem in developing world. Care of this potentially fatal, though preventable, disease has been revolutionized with advent of mechanical ventilation. However, still a significant minority, at least 10%, would die despite every effort , howeverAll patients admitted to two tertiary care centers in Karachi, Pakistan over a period of 10 years, were identified through ICD-9 coding system of the hospital medical records. The charts were reviewed retrospectively. The demographic, clinical, laboratory data was recorded and analyzed.The autonomic dysfunction was defined as presence of labile or persistent hypertension (>140/90 mmHg) or hypotension (<90/60 mmHg) and persistent sinus tachycardia (heart rate >100 bpm), tachyarrhythmia or bradycardia (heart rate <50 bpm) alternating with tachycardia on EKG. These values were recorded at resting state, not during tetanic spasm. Brief episodes of tachycardia or hypertension are common in ICU and could be related to pain or anxiety. The values in ICU were mostly recorded in sedated and relaxed position. Continuous automated blood pressure and heart rate monitoring was done in all ICU patients. Blood pressure and heart rate were checked every four hour for the ward patients. The data regarding arterial lines is not available. The values recorded during tetanic spasms were not included in the study. The decision to admit these patients was based mostly on availability of ICU beds. Patients with only locked jaw without any autonomic abnormalities on admission were electively admitted to ward.Severity of tetanus was classified based on Ablett criteria. The poor outcome was defined as either death or partial recovery at the time of discharge from the hospital. Partial recovery was defined as neck pain, back pain, walking difficulty and abnormal gait at discharge, in various combinations. Complete recovery was defined as absence of all of the symptoms and normal gait on examination at discharge.t test) methods.The data was analyzed on SPSS version 10.0. Statistical analysis employed descriptive and univariate (chi-square and Ninety six patients were identified. Sixty four (67%) were men and 32 (33%) were women. Their mean age was 44 (11–75) years. Identifiable risk factors were present in only 49 (51%) patients, recent trauma being most common which was noted in 27(28%) of the patients. Other risk factors were skin wounds, either infected or uninfected; 10 (11%), needle injury; 8 (9%) and recent surgery; 8 (9%).Most common clinical features were trismus; 89 (93%), neck stiffness; 73 (76%), muscle spasms; (67) 70%, followed by dysphagia; 57 (59%), back stiffness; 42 (44%), dysarthria; 19 (20%), abdominal pain; 10 (11%) and subjective breathing difficulty; 7 (8%).All patients received TIG  and almost all (99%) received benzodiazepine infusion. About one third (34%) of the patients received paralytics and 4 (5%) were also given magnesium sulphate. Majority of patients  were given antibiotics.The EKG abnormalities include sinus tachycardia in 44 (46%) patients, cardiac ischemia in 5 (5%), sinus bradycardia in 2 (2%) and heart block in 1 (1%) patient. Blood pressure abnormalities include persistently elevated blood pressure in 8 (8%) patients, persistent hypotension in 2 (2%) and labile hypertension in 14 (15%).Forty five (47%) patients recovered fully, 27 (28%) had partial recovery and 24 (25%) patients died. Major cause of death was cardiac arrhythmias  followed by respiratory arrest . All of the patients who had respiratory arrest were in general wards and never intubated. Fifty eight (60%) patients admitted to ICU. 43 (44%) required mechanical ventilation. Mean hospital stay was 17 (1–63) days. The hospital stay was prolonged in patients who required ventilation (25 vs 9 days)ANS group had 31 (32%) patients while non ANS group comprised of 65 (68%) patients.Severity of tetanus in ANS group was mild to moderate (Ablett I and II); 12 patients and severe/very severe (Ablett III and IV); 19 patients. 15 patients out of these 19 with severe tetanus required mechanical ventilation. Fourteen of 31 (47%) patients died in ANS group as compared to 10/62 (15%) in non ANS group (p= 0.002)  declare that they have no competing interests.MW: conceptualization, data analysis, manuscript preparation. BAK: data entry and analysis, statistical analysis, manuscript preparation. NT: data acquisition, manuscript preparation. RS: data acquisition, data analysis, manuscript preparation. NAS: conceptualization, manuscript preparation. NS: conceptualization, manuscript preparation.The pre-publication history for this paper can be accessed here:


Little is known about teaching medical ethics across cultural and linguistic boundaries. This study examined two successive cohorts of first year medical students in a six year undergraduate MBBS program.The objective was to investigate whether Arabic speaking students studying medicine in an Arabic country would be able to correctly identify some of the principles of Western medical ethical reasoning. This cohort study was conducted on first year students in a six-year undergraduate program studying medicine in English, their second language at a medical school in the Arabian Gulf. The ethics teaching was based on the four-principle approach  and delivered by a non-Muslim native English speaker with no knowledge of the Arabic language. Although the course was respectful of Arabic culture and tradition, the content excluded an analysis of Islamic medical ethics and focused on Western ethical reasoning. Following two 45-minute interactive seminars, students in groups of 3 or 4 visited a primary health care centre for one morning, sitting in with an attending physician seeing his or her patients in Arabic. Each student submitted a personal report for summative assessment detailing the ethical issues they had observed.All 62 students enrolled in these courses participated. Each student acting independently was able to correctly identify a median number of 4 different medical ethical issues (range 2–9) and correctly identify and label accurately a median of 2 different medical ethical issues (range 2–7) There were no significant correlations between their English language skills or general academic ability and the number or accuracy of ethical issues identified.This study has demonstrated that these students could identify medical ethical issues based on Western constructs, despite learning in English, their second language, being in the third week of their medical school experience and with minimal instruction. This result was independent of their academic and English language skills suggesting that ethical principles as espoused in the four principal approach may be common to the students' Islamic religious beliefs, allowing them to access complex medical ethical reasoning skills at an early stage in the medical curriculum. Medical ethics has increasingly become a common component of the undergraduate curriculum at many medical schools, often within a defined humanities program -3. In 19Medical ethics teaching can occur within the traditional model of medical education, where a separate program of clinical instruction follows several years of medical sciences . HoweverAlthough Islam has a long tradition of ethical reasoning, there had been little impetus to establish courses in ethics within medical school curricula in the Arabian Gulf. The Faculty of Medicine and Health Sciences (FMHS) was almost alone in having done so, an initiative introduced by expatriate family physicians from New Zealand who had undertaken training in Western medical ethics. Ethics had been taught at the FMHS since 1997 in a vertically integrated program that spanned all levels of the curriculum, based on the four-principle approach developed by Beauchamp and Childress ,13 as moEnglish has become the lingua franca of medicine, with most international medical societies, publications and meetings conducted in English ,17. ThisThere is a significant body of literature describing how people from different cultures utilise different approaches to the clinical application of medical ethics ,19. HoweThe aim of this study was to investigate whether Arabic speaking students studying medicine in an Arabic country, albeit with the language of instruction being English, would be able to correctly identify some of the principles of Western medical ethical reasoning, a discipline that challenged significant cultural and linguistic boundaries.The FMHS at the United Arab Emirates (UAE) University offers a six-year undergraduate program in medicine. Students are admitted after a one-year post high school tertiary orientation year, which includes additional English language instruction. Although all students are citizens of the UAE, speaking Arabic as their first language, the course is conducted in English therefore students are required to meet an English language proficiency level in order to be accepted into the medical school. Faculty members come from across the globe, with approximately 1/3 with English as their first language, 1/3 with Arabic as their first language and 1/3 with a different primary language.All medical students participated in an introductory course in medical ethics, conducted in the first three weeks of first year and taught by a non-Muslim native English speaker from New Zealand with no knowledge of the Arabic language. Apart from his qualifications in Family Medicine, he had also undergone training in Western medical ethics. Although the course was respectful of Arabic culture and tradition, the content of the introductory course focused on Western medical ethical reasoning and excluded an analysis of Islamic medical ethics.Based on the principles of adult learning, the educational strategy focused on the use of real clinical encounters after minimal orientation, followed by an interactive debriefing session. Rather than provide a sound basis for ethical reasoning, the aim of the introductory course was to introduce the students to the central importance of ethical principles in medical practice, knowing that a more complete and integrated understanding would develop over the ensuing six years of the course. Following two 45-minute interactive seminars, students in groups of 3 or 4 visited a primary health care centre for one morning, sitting in with an attending physician seeing his or her patients. In the absence of any clinical understanding at this early point in their training, the students were encouraged to focus all their energies on medical ethics.Following the visit to the primary health care centre each student submitted a personal report for summative assessment in which they described the ethical situations they had observed and then provided an appropriate label according to the four-principal approach. Although the seminars were in held in English, the clinical sessions were conducted in Arabic. When questioned by the investigator (SM), the clinical staff stated they had never received formal instruction in medical ethics. The clinical staff were discouraged from indicating ethical issues to the students; the educational process was for the students to identify these on their own. Following submission of their personal report, a third seminar of the introductory course was held with the same teacher where students were invited to share and discuss their experiences.This study examined two cohorts of first year students, 33 students from the 2002 – 2003 academic year and 29 students from the 2003 – 2004 academic year . Each cohort experienced the same admission criteria, underwent the same curriculum, was taught ethics by the same native English-speaking teacher and utilised the same group of primary health care physicians.Although both experienced educators and educational researchers, the investigators (VY and SM) did not teach medical ethics at this level of the curriculum. Each investigator coded all personal reports independently and any discrepancies were resolved by mutual agreement. A positive score for labelling any of the four principles and the subsets of autonomy was only allocated when the accompanying description corresponded with the label given. Merely listing a label was not coded as positive.As an indicator of general academic ability, the students' score on a multidisciplinary unit test held at the end of the fifth week of year one was also considered. The results of this test have been considered as a variable in the study to determine if there was a correlation between general academic ability and the students' competency in identifying ethical principals and to avoid associated bias. This multidisciplinary unit test covered all topics taught in the first 5 weeks including anatomy, chemistry, medical physics and medical ethics. The pass mark was set at 75%. The medical ethics component contributed 5% of the total mark.As not all students had the same clinical experience, each individual student may not have had the opportunity to observe the same range of ethical issues during their clinical experience. For example, some students may not have seen an example of competence to detail in their report. Hence, as this manuscript concerns each individual student's ability to report what he or she as an individual had observed in an experience not shared across the whole group, only numbers of ethical issues identified by individual students are reported rather than the total numbers of students per ethical issue.A third variable included was their score in a standardised test of English, the Test of English as a Foreign Language (TOEFL) , used inThe Statistical Package for the Social Sciences was used for analysing the results . Simple The Research Ethics Committee of the United Arab Emirates University Faculty of Medicine and Health Sciences, which complies with the ethical rules for human experimentation that are stated in the Declaration of Helsinki, approved the project.All members of each class participated in this study. Participant demographics are detailed in Table The similarities of issues identified and described across the group of students who attended the same clinic were highly suggestive that the events described actually occurred. However, there were sufficient differences in identified principles, evidence provided and labels given, to indicate that reports were written independently.Table The four cohorts of students displayed a wide range of TOEFL scores ranging from the low 400 s through the mid 600 s. There was a moderate correlation between the TOEFL score and the multidisciplinary unit test score . There was no statistically significant correlation between the TOEFL score and the number of ethical issues identified. There were weak statistically significant correlations between the multidisciplinary unit test scores and ethical issues identified for 'total number of issues correctly identified'  and 'number of autonomy issues correctly identified' . There were no other statistically significant correlations between test scores and ethical issues identified.This study has demonstrated that these first year students were able to identify medical ethical issues in a clinical setting after minimal instruction. Although introductory courses by their very nature aim to build on the students' previously accumulated knowledge, understanding and experience, especially the unstructured kind based on life experience, the students in this study were able to develop this skill with very minimal instruction, when compared to the normal length of introductory medical ethics courses .Feldman et al found important differences in the ethical practices and beliefs of Internists in the USA and China, even amongst those practicing Western medicine and suggested that some basic bioethical principles may be culturally based rather than universal . One impAlthough the students in this study demonstrated that they were able to use an imported model of ethical principles, this study did not directly address whether the students embraced the underlying conceptual framework, as the educational objectives of the introductory course were primarily to arouse interest and discussion amongst the students. However, informal review of the performance in examination settings of earlier cohorts of students who underwent similar educational programs suggests that by the end of their pre-registration medical training students do internalise the concepts and appear to understand their clinical applications. Further studies could investigate the impact of ethical training on their eventual behaviour as independent clinicians.This study had an underlying a priori assumption that the students would not be successful in identifying ethical issues due to a combination of the brief nature of the introductory seminars, their relative youth and inexperience with the adult learning model and being at the beginning of their medical training. Hence, the relatively high number of issues identified and the acceptable accuracy in labelling suggests that these students did demonstrate a reasonable level of skill even in the absence of a control group to help validate this conclusion. With little research available on the impact of early education in medical ethics, further studies utilising a control group would prove beneficial to determine what issues students would have been able to identify with no training.With ethics and professionalism being globally recognised as an integral component of primary medical degree curriculum, the results of the project suggest that early exposure may be beneficial in that it would provide a solid base in ethics, an essential requirement for their later clinical training. If ethical principles are clearly established early in the students' training, all future clinical training can be considered from an ethical standpoint.Jackson found that corporate managers' ethical decision-making was more influenced by their home country than by the country in which they resided . This suA number of researchers have suggested that the four principles of Western medical ethics have always existed in Islam -30. IslaThis study found at best a weak correlation between general academic ability and the number and accuracy of ethics issues identified. This suggests that even academically weak students were able to grasp the concepts being taught. Perhaps this outcome was enhanced by two factors; utilizing an adult learning model where students worked together in groups  undergoing real rather than simulated experiences and the incorporation of their Islamic religious beliefs and self developed ethical reasoning.The students in this study had a broad range of English language skills as suggested by the large range in TOEFL results. Although there is no clear evidence that TOEFL correlates with the ability to conceptualise theoretical constructs presented in English, there is strong evidence that TOEFL correlates with grade point average at a North American Universities ,33, and This study only addressed the ability of medical students at the onset of their medical training to assimilate and conceptualise the basic principles of ethical reasoning. In particular, no assessment was made of the long-term impact of this course on 'moral enculturation' as this study has only described one component of a longitudinal six-year course in medical ethics.This study has demonstrated that these students could identify medical ethical issues based on Western constructs, despite learning in English, their second language, being in the third week of their medical school experience and with minimal instruction. This result was independent of their academic and English language skills suggesting that ethical principles as espoused in the four principal approach may be common to the students' Islamic religious beliefs, allowing them to access complex medical ethical reasoning skills at an early stage in the medical curriculum.None declared.SM conceived the study, prepared the ethics application, coded the student's material, analysed the results, prepared and reviewed the manuscript. VY prepared the ethics application, prepared the coding sheet, coded the student's material, analysed the results, prepared and reviewed the manuscript.The pre-publication history for this paper can be accessed here:


Chronic non-cancer pain is a common problem that is often accompanied by psychiatric comorbidity and disability. The effectiveness of a multi-disciplinary pain management program was tested in a 3 month before and after trial.Providers in an academic general medicine clinic referred patients with chronic non-cancer pain for participation in a program that combined the skills of internists, clinical pharmacists, and a psychiatrist. Patients were either receiving opioids or being considered for opioid therapy. The intervention consisted of structured clinical assessments, monthly follow-up, pain contracts, medication titration, and psychiatric consultation. Pain, mood, and function were assessed at baseline and 3 months using the Brief Pain Inventory (BPI), the Center for Epidemiological Studies-Depression Scale scale (CESD) and the Pain Disability Index (PDI). Patients were monitored for substance misuse.Eighty-five patients were enrolled. Mean age was 51 years, 60% were male, 78% were Caucasian, and 93% were receiving opioids. Baseline average pain was 6.5 on an 11 point scale. The average CESD score was 24.0, and the mean PDI score was 47.0. Sixty-three patients (73%) completed 3 month follow-up. Fifteen withdrew from the program after identification of substance misuse. Among those completing 3 month follow-up, the average pain score improved to 5.5 (p = 0.003). The mean PDI score improved to 39.3 (p < 0.001). Mean CESD score was reduced to 18.0 (p < 0.001), and the proportion of depressed patients fell from 79% to 54% (p = 0.003). Substance misuse was identified in 27 patients (32%).A primary care disease management program improved pain, depression, and disability scores over three months in a cohort of opioid-treated patients with chronic non-cancer pain. Substance misuse and depression were common, and many patients who had substance misuse identified left the program when they were no longer prescribed opioids. Effective care of patients with chronic pain should include rigorous assessment and treatment of these comorbid disorders and intensive efforts to insure follow up. Chronic, non-cancer pain, defined as pain of greater than 3 months duration, is a common, important health issue. The prevalence of chronic pain ranges from 20% to 60% . The preThe optimal approach to treating chronic pain is controversial -13. The In large part, uncertainty about how to best manage patients with chronic pain stems from a lack of research in this area . The limChronic pain management is complex. Like other chronic diseases, chronic pain is multi-faceted, and it is attended by its own set of comorbidities. It is complicated by substantial psychological and functional impairment in the forms of depression, disability, and loss of livelihood -30. It iGeneralists, even if well-versed in the biopsychosocial model of disease, often feel unprepared to manage chronic pain. They may lack the training in using certain pharmacological regimens, such as combining chronic opioids with psychiatric and pain modulating agents ,32. TheyWe developed a disease management program to improve the management of chronic pain in opioid-treated patients attending an academic general medicine practice. Traditional models of office-based care focus on diagnonsis and acute management of medical problems in the context of a single provider-patient relationship. In contrast, disease management emphasizes: (1.) The use of multi-disciplinary teams providing integrated care; (2.) Evidence-based algorithms; (3.) Interval visits to monitor response to therapy; and (4.) Information systems that permit tracking of outcomes and adjustment of therapy -39. We aIn designing the program, we reviewed existing research, contacted experts, and conducted informal assessments of the main barriers to effective pain management in our practice . BarrierPatients were eligible if they had pain of greater than 3 months duration and were either taking or considering opioid therapy. Attending and resident physicians were encouraged to refer patients if they were having difficulty managing their pain or if they suspected misuse of opioid medications. We publicized the program through educational conferences with residents and attendings and through informal communication within our practice.After obtaining informed consent, a research assistant administered a comprehensive baseline assessment to gather socio-demographic data and review the medical history with an emphasis on the medical management of pain. Validated measures of pain, disability and psychological status were included. Using an 11 point scale, the Brief Pain Inventory (BPI) asked patients to rate their pain at the time of the interview and at its worst, least, and average over the past month ,43. The Patients were managed by a multi-disciplinary team consisting of the patient's primary care physician, a clinical pharmacist, a program assistant with training in health behavior, and a psychiatrist with sub-specialization in pain management who saw patients with the team one half-day per week. One nurse was dedicated to checking-in study patients and obtaining urine specimens. At entry, all patients signed a Medication Contract (Appendix A)  and provThe clinical pharmacist or psychiatrist modified or titrated a patient's pain medications in consultation with the primary care physician. During titration of medications, patients returned at one month intervals. Medical management adhered to published guidelines and expert opinion on the management of chronic, non-cancer pain ,52-54. T• Longer-acting opioids  were initiated in patients who had been receiving short-acting agents that were only partially effective.• Short-acting, potent opioids  were prescribed for breakthrough pain.• Longer-acting opioids were titrated at interval visits.• Less costly, generic medications (e.g. methadone and long-acting morphine) were preferred over proprietary products of equal or lesser efficacy .• Tricyclic anti-depressants, gabapentin, and other agents were used adjunctively, especially for neuropathic pain.To address psychiatric comorbidity, patients with depression and other complex psychiatric conditions (e.g. psychotic depression and bipolar disorder with substance misuse) received psychiatric evaluation. Depression was diagnosed based on clinical interview and CESD score. In addition, primary care physicians could request psychiatric consultation on patients who had unaddressed psychiatric problems. The clinical pharmacist, psychiatrist and primary care physicians used CESD scores to guide treatment of patients scoring in the depressed range. The program, however, did not employ a strict protocol for depression treatment.As defined in our medication agreement with participants, we prospectively monitored substance misuse through clinical history, review of medications, communication with pharmacies and providers, and urine toxicological screening (UTS). Medications were documented in the electronic medical record and our program database. Discrepencies and inconsistencies were discussed with the patient's primary provider. We contacted a patient's pharmacy to verify procurement of medications, and, if substance misuse was suspected, we contacted additional pharmacies to ascertain whether or not a patient was receiving opioids from multiple sources. A UTS was obtained at each visit and was correlated with the patient's reported history of medication use. In collaboration with our institution's toxicologist, results of the UTS were verified using the appropriate confirmatory assays. For example, the presence or absence of "opiates" on the UTS was confirmed using gas chromatography. In addition, all positive results for amphetamines were confirmed with gas chromatography due to the possibility of assay interference from other medications ,51.We defined serious substance misuse prospectively as any of the following: 1. Cocaine or amphetamines detected on UTS; 2. Procurement of opioids from more than one provider on a regular basis ("doctor collecting"); 3. Diversion of opioids; 4. UTS negative on at least two occasions for prescribed opioids in the context of a reported history that the patient was taking the medication as prescribed (We considered repeatedly "negative" urines as an indicator of possible diversion.); 5. UTS positive on at least two occasions for opioids not prescribed by our practice (an inappropriate or inconsistent urine). A positive cannabinoid finding on UTS was not defined as serious substance misuse for the purposes of our study, but we counseled patients to refrain from marijuana use.Patients were advised at entry into our program (and in the Medication Contract) that serious violations of the contract would result in discontinuation of opioids. Past instances of serious misuse were not subject to sanction. We constituted a formal practice-wide committee to evaluate and respond to suspected misuse. It consisted of the practice director, two attending physicians, a clinical pharmacist, two resident physicians, and a nurse. The committee deliberated through secure email and considered the violations defined above. Patients committing serious substance misuse were offered referral to substance abuse experts at our institution. In most cases, opioid therapy would be reconsidered in 6 months if the patient participated in substance abuse counseling (The practice policy addressing substance misuse is included as Appendix B.).At 3 month follow-up, a research assistant reassessed each patient's clinical status. Pain, disability, and mood scores were re-measured using the instruments previously described. The research assistant was not blinded to study participation status.Descriptive statistics are reported as means and percents. Paired t-tests were used to compare changes in pain, disability and depression scores from baseline to 3 month follow-up. McNemar's test was used to measure differences in proportions of patients receiving treatment for depression at 0 and 3 months. We also compared changes in pain scores based on changes in opioid dose. All analyses were performed using Stata 7.0 .The research protocol was approved by the University of North Carolina School of Medicine Committee on the Protection of the Rights of Human Subjects. The funding sources had no role in the collection or interpretation of the results.Between December 2002 and May 2003, 85 patients agreed to participate in the study. Table The average age of patients was 51 years, 60% were male, and 78% were white, most (83%) had an income less than $20,000 per year and 65% were disabled. Forty-four percent had a history of illicit substance use . All patients had pain of at least 3 months duration and 90% had pain for greater than 1 year. At baseline 93% were receiving opioids. At 3 month follow up, 97% were receiving opioids.Table Table At 3 month follow up, BPI pain scores improved by 12% to 15%, and all reductions were statistically significant. The mean depression score improved from 24.0 to 18.0 (p < 0.001), and the proportion of patients scoring in the depression range decreased from 79% to 54% (p = 0.003). We did not correct for multiple comparisons because of the exploratory nature of our analyses.Some authors have demonstrated that the conventional cutoffs for the CESD  may lack specificity in patients with chronic pain and have proposed a CESD threshold of 27 for diagnosing depression in this population . Using tThe mean daily opioid dose in milligram equivalents of morphine increased from 72 mg per day to 91 mg per day . Forty-eight percent of patients had their opioid dose increased over 3 months. In these patients, the mean opioid equivalent increased from 53 mg to 105 mg per day. No clear relationship emerged between opioid dosing and improvements in pain, disability, and depression scores, after adjusting for baseline pain, disability and depression (Table Twenty-seven patients 32%) committed some form of serious substance misuse Table .. Althou% committWe found that a multi-disciplinary, primary care-based, disease management program can improve pain, depression and disability scores in opioid-treated patients with chronic pain in a 3 month uncontrolled trial. The improvements across all outcomes support an improved quality of life resulting from the intervention. These improvements were obtained using an approach that balanced the potential benefits and adverse effects of opioids.We hyothesize that these improvements resulted from the combined effects of systematizing pain management and treating depression. Improvements appear independent of opioid dosing. The clinical significance of the 12% to 15% improvement in pain scores is unclear. Uncontrolled trials in specialty pain clinics have reported a 20% to 25% reduction in pain scores . Some reThe improvement in depression scores was clinically important and may reflect combined effects of intensification of pharmacological management for depression and pain and the systematization of care. Although the reciprocal relationship between pain and depression has been established in previous studies, the effect that treating one condition has on the other has not been well-assessed . One recTo our knowledge, this is the first study to prospectively examine the effects of multi-discplinary pain management on the outcomes of pain, disability, depression, and substance misuse in an academic primary care practice caring for a wide range of patients. Previous studies conducted in a military clinic and a heWe documented a high prevalence of substance misuse (32%). We did not assess addiction per se. The prevalence of substance misuse and addiction in patients receiving chronic opioids is unclear and depends on the populaton under study. Some authors have asserted that addiction and substance misuse are uncommon consequences of opioid use for pain. One widely cited reference estimated the prevalence of addiction at approximately 4 in 10,000 treated patients . Others ® and other prescription drugs among teens and young adults  CIS NOTE."The pre-publication history for this paper can be accessed here: