DNA homopolymer tracts, poly(dA).poly(dT) and poly(dG).poly(dC), are the simplest of simple sequence repeats. Homopolymer tracts have been systematically examined in the coding, intron and flanking regions of a limited number of eukaryotes. As the number of DNA sequences publicly available increases, the representation (over and under) of homopolymer tracts of different lengths in these regions of different genomes can be compared.7 bases from 15,560 cleaned (redundancy removed) sequence documents were analyzed. Calculated frequencies of non-overlapping long homopolymer tracts were found over-represented in non-coding sequences of eukaryotes. Long poly(dA).poly(dT) tracts demonstrated an exponential increase with tract length compared to predicted frequencies. A novel negative slope was observed for all eukaryotes between their (G+C)% composition and the threshold length N where poly(dA).poly(dT) tracts exhibited over-representation and a corresponding positive slope was observed for poly(dG).poly(dC) tracts. Tract size thresholds where over-representation of tracts in different eukaryotes began to occur was between 4 – 11 bp depending upon the organism (G+C)% composition. The higher the GC%, the lower the threshold N value was for poly(dA).poly(dT) tracts, meaning that the over-representation happens at relatively lower tract length in more GC-rich surrounding sequence. We also observed a novel relationship between the highest over-representations, as well as lengths of homopolymer tracts in excess of their random occurrence expected maximum lengths.We carried out a survey of the extent of homopolymer tract over-representation (enrichment) and over-proportional length distribution (above expected length) primarily in the single gene documents, but including some whole chromosomes of 27 eukaryotics across the (G+C)% composition range from 20 – 60%. A total of 5.2 × 10We discuss how our novel tract over-representation observations can be accounted for by a few models. A likely model for poly(dA).poly(dT) tract over-representation involves the known insertion into genomes of DNA synthesized from retroviral mRNAs containing 3' polyA tails. A proposed model that can account for a number of our observed results, concerns the origin of the isochore nature of eukaryotic genomes via a non-equilibrium GC% dependent mutation rate mechanism. Our data also suggest that tract lengthening via slip strand replication is not governed by a simple thermodynamic loop energy model. D. discoideum  random model. The fslope is obtained from the inverse of P', where the slope [-log(P')] is obtained from the log  vs. N type plots in Figure st Order Markov chain behavior can describe some of the behavior of eukaryotic sequences, we have chosen here to compare the occurrence of tracts in real sequences to that of tracts in random DNA of equivalent base composition because the comparison is intuitively easy to grasp. The results from all 27 organisms are presented here in Figures fslope/fseq: where fseq is the actual base frequency tabulated from all bases comprising the sequences in the real sequence compartment) plotted versus the overall real (G+C)% of each individual DNA sequence compartment studied. The higher the frequency ratio in Figures We next wished to quantitatively compare for all 27 eukaryotes, the differences between the length N dependent frequencies of short tracts (N ≤ 6 bp) and long tracts (N ≥ 9 bp) of the type that we presented in Figure the eqn. b random the eqn. b random fslope values from the Figure For N ≥ 9 bp tracts in coding sequences Figure  &5B, theFor lengths N ≥ 9 bp in Figures , representing the predicted frequency of base i at length N based on random tract occurrence in DNA of equivalent base composition. Eqn.  of an eqn. [1b] plot. The fslope quantity, which represents an effective base frequency for that DNA, can be determined for a given set of data, and then compared to the real frequency for that base occurring in the sequences being examined, as we have previously described [the  an eqn. b plot. T, of a homopolymer tract of length N randomly occurring is calculated by the formula:The expected frequency,  is the fractional base composition of the tract base i within the DNA for that file, and N is the tract length.where base frequency i is then calculated as the ratio of observed to predicted frequencies, defined as "Representation"(R):The level of tract representation for base i tracts are "over-represented", while values less than 1 indicate tracts are "under-represented". The log(R) verses N plots presented in Figure R) were found using linear interpolation of the data. We term these N values, thresholds, corresponding to particular enrichments of tract occurrence.Values larger than 1 indicates base i, , given the base composition of the entire sequence, , is calculated by the formula:The maximum expected length of a homopolymer tract of base , to the longest expected length, . This parameter is defined as "Proportion (P)". Thus, we have the formula:The length of a given homopolymer tract can then be compared to its expected length by taking the ratio of the longest observed length, P values larger than 1 are called "over-proportional" and P values less than 1 are "under-proportional". P represents the tract length comparison on the x-axis of Figure R for tract frequency comparisons on y-axis. They are both important parameters for the evaluation of the frequency and length distributions of any tracts.YZ carried out the sequence data collection, clean-up, and analysis, and drafted the major part of the manuscript. JWB contributed both major programs Meltsim and Poly in this study, and also participated in analyzing data and drafted part of the manuscript. KAM conceived the study, participated in its design and coordination and revised and finalized the manuscript in all its revisions. All authors read and approved the final manuscript. The authors acknowledge the contribution from a reviewer of the concepts of retroelement insertion and differential polymerase selectivity as potential origins for the higher A & T tract frequencies than G&C tract frequencies that we describe here.


Hypertensive disorders in pregnancy are leading causes of maternal, fetal and neonatal morbidity and mortality worldwide. However, studies attempting to quantify the effect of hypertension on adverse perinatal outcomes have been mostly conducted in tertiary centres. This population-based study explored the frequency of hypertensive disorders in pregnancy and the associated increase in small for gestational age (SGA) and stillbirth.We used information on all pregnant women and births, in the Canadian province of Nova Scotia, between 1988 and 2000. Pregnancies were excluded if delivery occurred < 20 weeks, if birthweight was < 500 grams, if there was a high-order multiple pregnancy (greater than twin gestation), or a major fetal anomaly.The study population included 135,466 pregnancies. Of these, 7.7% had mild pregnancy-induced hypertension (PIH), 1.3% had severe PIH, 0.2% had HELLP , 0.02% had eclampsia, 0.6% had chronic hypertension, and 0.4% had chronic hypertension with superimposed PIH. Women with any hypertension in pregnancy were 1.6 (95% CI 1.5–1.6) times more likely to have a live birth with SGA and 1.4 (95% CI 1.1–1.8) times more likely to have a stillbirth as compared with normotensive women. Adjusted analyses showed that women with gestational hypertension without proteinuria (mild PIH) and with proteinuria  were more likely to have infants with SGA . Women with pre-existing hypertension were also more likely to give birth to an infant with SGA  or to have a stillbirth .This large, population-based study confirms and quantifies the magnitude of the excess risk of small for gestational age and stillbirth among births to women with hypertensive disease in pregnancy. Hypertensive disorders in pregnancy complicate approximately 10–16% of pregnancies and are leading causes of maternal, fetal and neonatal morbidity and mortality worldwide . DefinitWe, therefore, carried out a population-based study to quantify the frequency of hypertensive disorders in pregnancy and also the excess risk of SGA and stillbirth that is associated with this pregnancy complication. We investigated the way in which SGA and stillbirth were modified by other factors that also have a serious influence on SGA and stillbirth, for example, whether a twin gestation modifies the effect of hypertension on SGA, or whether smoking modifies the effect of hypertension on stillbirth.The study population included all births to residents of the province of Nova Scotia, Canada between 1988 and 2000. Information on these births was obtained from the Nova Scotia Atlee Perinatal Database. The Nova Scotia Atlee Perinatal Database includes several hundred variables containing maternal and newborn information, such as demographic variables, procedures, interventions, maternal and newborn diagnoses and morbidity and mortality information for every pregnancy and birth occurring in Nova Scotia hospitals and to Nova Scotia residents since 1988.Pregnancies were excluded if delivery occurred < 20 weeks, if birthweight was < 500 g, if there was a higher order pregnancy (greater than twin gestation), or a known major fetal anomaly. Information in the database on the type of hypertensive disorder is directly coded from medical charts. The diagnosis of hypertensive disorders in pregnancy was made if it occurred in the antepartum or postpartum period. Mild pregnancy induced hypertension (PIH) included physician-diagnosed mild pregnancy induced hypertension if in the chart, transient hypertension or a diastolic blood pressure exceeding 90 mmHg on two or more occasions in a 24-hour period. Severe pregnancy induced hypertension included physician-diagnosed severe pregnancy induced hypertension, diastolic blood pressure ≥ 110 mm Hg on at least two occasions within a 6-hour period, if magnesium sulfate was administered for seizure prophylaxis, if there was ≥ 2+ proteinuria, low platelets , and/or elevated liver enzymes . HELLP syndrome  included physician-diagnosed HELLP. Eclampsia included physician-diagnosed eclampsia or one or more convulsions not attributable to other cerebral conditions such as epilepsy or cerebral hemorrhage in a patient with hypertension. Chronic hypertensive disease included a history of hypertensive disease when not pregnant, prior to current pregnancy or prior to 20 weeks of the current pregnancy. Pregnancy induced hypertension superimposed on chronic hypertension included physician-diagnosed pregnancy induced hypertension superimposed on chronic hypertension or if there was hemolysis, elevated liver enzymes or low platelets.We first examined the maternal characteristics of study subjects with hypertension in pregnancy. For this analysis, hypertensive disorders in pregnancy were defined according to Nova Scotia Atlee Perinatal Database definitions: mild PIH, severe PIH, HELLP, eclampsia, chronic hypertension and chronic hypertension with superimposed PIH. Maternal characteristics which were considered included age, marital status, parity, prepregnancy weight, pregnancy weight gain, administration of antenatal steroids, smoking, drug abuse, the presence of anemia (Hgb < 10 gm%), gestational diabetes , pre-existing diabetes, and twin gestation.For the multivariate analyses, these database definitions were grouped to more closely approximate commonly used definitions such as those proposed by the Canadian Hypertensive Society and other international organizations . These gth percentile cut-off of a recently published Canadian fetal growth reference [Only live births were considered in the analysis of small for gestational age, while all births were considered for the stillbirth analyses. Small for gestational age was defined as birthweight for gestational age that was less than the sex-specific 10eference . StillbiEthical approval for the study was obtained from the Research Ethics Boards at Dalhousie University in Halifax, Nova Scotia, the Reproductive Care Program of Nova Scotia and the IWK Health Centre in Halifax, Nova Scotia.Exact binomial 95% confidence intervals were calculated for rates of hypertensive disorders in pregnancy. Descriptive analyses were carried out on maternal data to ascertain the association between maternal characteristics and hypertensive disorders in pregnancy. Categorical data between hypertensive and normotensive pregnancies were compared using chi-square and Fisher's exact tests, where appropriate.P < .05 and all tests were two-tailed. Statistical analyses were performed using the SAS programming package for Windows (Version 8.0).Logistic regression analyses were carried out to determine the adjusted odds ratios (OR) and 95% confidence intervals (CI) expressing the relationship between any hypertensive disorder in pregnancy and groups of hypertension  and the two dichotomous primary outcomes (SGA and stillbirth). Backward stepwise elimination of variables was carried out to identify all significant determinants of the outcome. Modification of the effect of hypertensive disorders on SGA or stillbirth was investigated, based on clinical understanding. Variables considered to be in the causal pathway between the determinant and the outcome  were not adjusted for in the model -24. WhenThe study population included 135,466 pregnancies. Of these, 7.7%  had mild PIH, 1.3%  had severe PIH, 0.2%  had HELLP, 0.02%  had eclampsia, 0.6%  had chronic hypertension and 0.4%  had chronic hypertension with superimposed PIH. The overall rate of hypertensive disease in pregnancy was 10.1% .Table Table P < .001) times more likely to have a live birth with SGA as compared with normotensive women. Women with gestational hypertension without proteinuria were 1.5  times more likely to have a live birth with SGA as compared with normotensive women. Similarly, women with gestational hypertension with proteinuria were 3.3  times more likely and women with pre-existing hypertension were 2.5  times more likely to have a live birth with SGA as compared with normotensive women.After controlling for potential confounders, women with any hypertensive disorder were 1.8  times more likely to have a stillbirth as compared with normotensive women (Table P < .001) times more likely to have a stillbirth as compared with normotensive women.After controlling for potential confounders, women with any hypertensive disorder were 1.4  was similar to that reported in the literature (10–16%). The rate of mild PIH in this population was 7.7%, compared with 6–7% in the literature. The rate of severe PIH (1.3%), HELLP (0.2%) and eclampsia (0.02%) were lower than expected (5–6%). The rate of pre-existing hypertension (0.6%) and pre-existing hypertension with superimposed PIH (0.4%) were lower than expected  ,26. ThesSmall for gestational age is a more appropriate measure of fetal growth than birthweight alone. Our study evaluated birthweight for gestational age and gender among live births to normotensive and hypertensive pregnancies using a recent population-based Canadian reference . Women wP values associated with the results be interpreted with caution.Chance and confounding are unlikely explanations for the results of our study, because of the large study size, and adjustment for potential confounders using logistic regression. This study was not able to correct for the degree of blood pressure control, which may have an effect on fetal growth. While retrospective studies in general are limited by the reliability of data, information in the Nova Scotia Atlee Perinatal Database is of high quality. Routine data checks and edits are made at the time of data collection, and validation  and reabth percentile) and stillbirth in comparison with women with normotensive pregnancies. Twin gestation and smoking were important modifiers of the effect of hypertension on SGA and stillbirth. This study allowed the quantification of risks of adverse outcomes in women with pregnancies complicated by hypertension, confirming associations in the published literature and allowing appropriate counseling and monitoring in the management of these women, as well as providing baseline risks which may be used in future intervention studies.This population-based cohort study demonstrates that women with hypertensive disease in pregnancy are at significantly higher risk of having pregnancies complicated by small for gestational age (<10None declared.VMA proposed the study, carried out the preliminary analyses and wrote the paper. All authors discussed the analyses, contributed to the intellectual content of the paper and approved the final version. VMA and KEM provided the maternal-fetal medicine perspective, KSJ provided the general medical and epidemiologic input, LAM provided the general and obstetrical medicine perspective, and AO provided the neonatal-perinatal perspective and epidemiologic input.The pre-publication history for this paper can be accessed here:


Aluminum has recently been recognized as a causative agent in dialysis encephalopathy, osteodystrophy, and microcytic anemia occurring in patients with chronic renal failure who undergo long-term hemodialysis. Only a small amount of Al(III) in dialysis solutions may give rise to these disorders.3O4). Then, metal complexing ligand alizarin yellow was covalently attached onto mPHEMA beads. Alizarin yellow loading was 208 μmol/g. These beads were used for the removal of Al(III) ions from tap and dialysis water in a magnetically stabilized fluidized bed.Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads in the size range of 80–120 μm were produced by free radical co-polymerization of HEMA and ethylene dimethacrylate (EDMA) in the presence of magnetite particles (FeAl(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 5.0. Comparison of batch and magnetically stabilized fluidized bed (MSFB) maximum capacities determined using Langmuir isotherms showed that dynamic capacity (17.5 mg/g) was somewhat higher than the batch capacity (11.8 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Al(III) ions could be repeatedly adsorbed and desorbed with these beads without noticeable loss in their Al(III) adsorption capacity.Adsorption of Al(III) demonstrate the affinity of magnetic dye-affinity beads. The MSFB experiments allowed us to conclude that this inexpensive sorbent system may be an important alternative to the existing adsorbents in the removal of aluminium. About 8% of the Earth's crust is comprised of aluminium. This element is the most abundant metal naturally present in air, soil and water. Consequently, environmental exposure to aluminium is potentially possible. Its ingestion is unavoidable since aluminium compounds are added not only to most water supplies but also to many processed foods and medicines. Aluminium is a known neurotoxicant. It enters the brain, where it contributes to some neuro-degenerative diseases including dialysis encephalopathy, osteomalacia, osteodystrophy, in particular those related to dialysis treatment of uremic subjects . Only a Positively charged aqua and hydroxy-monomeric forms have been found to be the most toxic species of aluminium to living organisms in the terrestrial and aquatic environments . GeneralThe selective removal of aluminium ions have been extensively investigated by applying several techniques -9. AmongThere have been several separation approaches performed under magnetic field . The mosIn the present study, we attempted to use alizarin yellow-attached magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads as specific adsorbent for aluminium removal from aqueous solutions in a magnetically stabilized fluidized bed. Al(III) adsorption on the alizarin yellow-affinity beads from aqueous solutions containing different amounts of Al(III) ions and at different pHs is reported here. Finally, reuse of the dye-affinity beads is also discussed.3O4, diameter < 1 μm) were obtained from Aldrich (USA). Alizarin yellow  was purchased from BDH . All other chemicals were obtained from Merck as analytical grade. All water used in the adsorption experiments was purified using a Barnstead  ROpure LP® reverse osmosis unit with a high flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANOpure® organic/colloid removal and ion exchange packed bed system.2-hydroxyethyl methacrylate (HEMA), was purchased from Sigma , and was purified by vacuum distillation under a nitrogen atmosphere. The comonomer, ethylene dimethacrylate  was used as the crosslinking agent. Magnetite particles  (10 ml). This reaction medium was boiled in rotary evaporator for 6 h. Then, 2.5 g alizarin yellow was dissolved in absolute ethanol (30 ml). Alizarin yellow-attachment process was performed in ethanol solution for 24 h. At the end of this reaction period, the alizarin yellow-attached beads were removed by filtration and washed with ethanol, water and tetrahydrofuran several times until all the unbound dye molecules were removed. The dye attached beads were stored at 4°C with 0.02% sodium azide to prevent microbial growth.Preparation and characterization of the alizarin yellow-attached mPHEMA beads were reported in our previous paper in detail . In ordeThe leakage of the alizarin yellow from the dye-attached beads was investigated within the media at the selected pH in the range of 2.0–7.0. These media were the same which were used in the Al(III) adsorption experiments. The medium with the dye attached magnetic beads was stirred for 24 h at room temperature. Then, magnetic beads were separated from the medium, and the alizarin yellow concentration was measured in the liquid phase by spectrophotometry at 500 nm.Al(III) adsorption studies were carried out in a magnetically stabilized fluidized bed. Beads suspended in pure water were degassed under reduced pressure (by using water suction pump) and magnetically stabilized into a column (10 cm × 0.9 cm inside diameter) equipped with a water jacket for temperature control. The vertically oriented magnetic field was produced by passing DC current through two modified Helmholtz coils (1.5 cm diameter × 2.5 cm thick) spaced 4 cm apart. At a current of 1.6 A (50 W), each coil produced a magnetic field of 40 Gauss. Equilibration of the column was performed by passing four column volumes of phosphate buffer (pH: 7.4) before injection of the Al(III) solution. In a typical adsorption system, 50 ml of the aqueous Al(III) solution was passed through the column containing magnetic beads, by a peristaltic pump for 2 h. After loading, the column was washed with deionized water to wash out Al(III) impurities. The concentrations of the Al(III) ions in the aqueous phases after the desired treatment periods were measured by using a graphite furnace atomic absorption spectrophotometer . Deuterium background correction was used. Pyrolitic graphite coated tubes were used for AAS measurements. The instrument response was periodically checked with known Al(III) solution standards. The experiments were performed in replicates of three and the samples were analyzed in replicates of three as well. For each set of data present, standard statistical methods were used to determine the mean values and standard deviations. Confidence intervals of 95% were calculated for each set of samples in order to determine the margin of error.In the first group of experiments, the flow rate of the aqueous solution  content of 50 mg/L) was changed between 0.5–3.0 mL/min. In the second group of experiments, Al(III) adsorption from aqueous solution was studied at different pH's (2.0–7.0). Adsorption isotherm was also obtained in the magnetically stabilized fluidized bed. Aqueous solutions containing different amount of Al(III) were used in these experiments. The changes in the Al(III) concentration with time was followed to obtain the adsorption curves. The amount of adsorbed Al(III) per dry magnetic beads was calculated by using the concentrations of the Al(III) in the initial solution and in the equilibrium.3 solution. In a typical desorption experiment, 50 ml of the desorption agent was recirculated through the magnetically stabilized fluidized bed containing dye-affinity magnetic beads for 1 h. The concentrations of the Al(III) ions in the desorption medium were measured by using a graphite furnace atomic absorption spectrophotometer. The desorption ratio was calculated from the amount of Al(III) adsorbed on the magnetic beads and the final Al(III) concentration in the desorption medium. In order to test the reusability of the dye-affinity magnetic beads, Al(III) adsorption-desorption procedure was repeated ten times by using the same magnetically stabilized fluidized bed.In all cases adsorbed Al(III) ions were desorbed using 0.1 M HNOAdsorption of Al(III) from aqueous solution was also investigated in batch experiments. Aqueous Al(III) solution (50 ml) was treated with the magnetic dye-affinity beads at room temperature, in the flasks agitated magnetically at an agitation speed of 600 rpm for 2 h. The suspension was brought to pH 5.0 by adding sodium hydroxide and nitric acid. The pH was maintained in a range of ± 0.1 units until equilibrium was attained. Polymer amount was kept constant at 100 mg per 50 ml. Al(III) determination was performed in water sample in an atomic absorption spectrophotometer coupled to a graphite furnace atomiser. Adsorption values (mg/g) were calculated as the difference in Al(III) ion concentration of the pre- and post adsorption solutions divided by the weight of dry magnetic affinity beads.3. The magnetic beads are crosslinked hydrogels. They do not dissolve in aqueous media, but do swell, depending on the degree of cross-linking and on the hydrophilicity of the matrix. The equilibrium swelling ratio (the ratio of the volumes of the microbeads before and after swelling) of the beads used in this study is 34%. The simple incorporation of water weaken the secondary bonds within the hydrogels. This enlarges the distance between the polymer chains and causes the uptake of water. It should be mentioned that the water uptake properties of the mPHEMA beads did not change after Alizarin Yellow attachment.mPHEMA beads (in the size range of 80–120 μm) carrying alizarin yellow were prepared as a specific affinity adsorbent for removal of Al(III) from the water which was used for preparation of dialysis solution. mPHEMA beads used in this study were prepared and characterized in our earlier study . The maiAfter the attachment of the dye  the size of the swollen beads did not change, but the colour became dark yellow, which is a clear indication of the incorporation of the dye molecules in the structure of the mPHEMA microbeads. As shown in our previous paper, the dye molecules were attached to the mPHEMA beads by covalent bonding via hydroxyl groups . The mPHThe adsorption capacity at different flow-rates are given in Figure Figure It should be noted that the nonspecific adsorption (adsorption on plain mPHEMA beads) of Al(III) ions was relatively low (0.63 mg/g). mPHEMA beads do not contain ion exchange or chelating groups. Preferred coordination structure and preferred coordinating ligand atom may be utilized for this adsorption. Al(III) ions may interact with Oxygen atoms as the ligand. Diffusion of Al(III) ions into the swollen polymeric structure and retention in the pores may also contribute to this nonspecific Al(III) adsorption.An adsorption isotherm is used to characterize the interactions of each molecule with the adsorbent. In this case it provides a relationship between the concentration of the Al(III) ions in the solution and the amount of Al(III) ions adsorbed on the solid phase when the two phases are at equilibrium. The Langmuir adsorption model assumes that the species are adsorbed at a fixed number of well-defined sites, each of which is capable of holding only one molecule. These sites are also assumed to be energetically equivalent, and distant from each other so that there are no interactions between molecules adsorbed on adjacent sites.Adsorption isotherms were used to evaluate adsorption properties. The Langmuir adsorption isotherm is expressed by Equation 1. The corresponding transformations of the equilibrium data for Al(III) gave rise to a linear plot, indicating that the Langmuir model could be applied in these systems and described by the equation:max. b . Ceq / (1 + bCeq)     (1)Q = Qeq is the equilibrium Al(III) concentration (mg/mL), b is the Langmuir constant (mL/mg) and, Qmax is the maximum adsorption capacity (mg/g). This equation can be linearized so thatwhere Q is the adsorbed amount of Al(III) (mg/g), Ceq/Q = 1/(Qmax. b) + Ceq/Qmax.     (2)Ceq versus Ceq/Q was employed to generate the intercept of 1/Qmax.b and the slope of 1/Qmax.The plot of Cmax) data for the adsorption of Al(III) was obtained from the experimental data. The correlation coefficient (R2) was 0.989. The Langmuir adsorption model can be applied in this affinity adsorbent system. Maximum adsorption capacities determined using Langmuir isotherms show that dynamic capacity (25.3 mg/g) was somewhat higher than the batch capacity (12.6 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands.The maximum adsorption capacity  adsorption of this alizarin yellow-attached mPHEMA beads is also shown in Figure Competitive adsorption of the metal ions from tap water in Ankara and dialysis water (reverse osmosis) was also investigated. The water containing different amounts of each metal ion was treated with dye beads in MSFB. Table The World Health Organization (WHO) and the European Community (EC) guide values for Al(III) ions for tap water is 200 ng/ml ,27. Al ions are disrupted and subsequently Al(III) ions are released from the solid surface into the desorption medium. Therefore, we conclude that HNO3 is a suitable desorption agent for the dye adsorbents, and allows their repeated use. In order to show the reusability of the dye-attached mPHEMA beads, adsorption-desorption cycle was repeated ten times by using the same sample of affinity adsorbent. As can be seen from Figure Desorption ratios were very high (up to 97.6%) with the eluant system and under conditions used. When HNOAs can be seen in Figure The medical relevance of aluminium has stimulated the development of cost and time effective separation techniques including polymeric carriers. Magnetic adsorbents have several potential advantages over conventional adsorbents -32. The


The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2–ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2–ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses. Upon HIV-1 infection of T cells, Nef activates Rac by binding the DOCK2-ELMO1 complex. In this way, Nef influences multiple aspects of T cell function, including inhibition of lymphocyte chemotaxis Primate lentiviruses persist in the host by active replication and can reemerge from latent reservoirs that are established in cells of the immune system . The infThe lentiviral accessory protein Nef is a multifunctional regulator that is important for rapid progression to AIDS . One key+ T cells from transgenic mice are hypersensitive to stimulation via the T cell antigen receptor (TCR)  , as are  T cells  and cell T cells . Nef was T cells  and acti T cells . Recent  T cells . The not+ Jurkat T cells that stably express the extensively studied patient-derived HIV-1 Nef protein NA7  via DOCK2 using Ni–NTA resin and eluted with imidazole A, lane 4(G2∇HA)), and therefore functionally defective, did not associate with DOCK2, ELMO1, or Rac1  bind DOCK2, ELMO1, and Rac2. We also tested a Nef protein from a strain of pathogenic SIV, mac239, that is important for rapid progression to AIDS in experimentally infected rhesus macaques . Nef andGTP) binds the p21-binding domains (PBD) of PAKs directly  or a control nef-deleted vector (H-Δ). Since HIV-1 Env protein may activate DOCK2-controlled signaling pathways through binding to chemokine receptors such as CXCR4, env-defective, VSV-G-pseudotyped viruses were used in these experiments. We cultured 98% pure populations of CD4+ T cells isolated from the peripheral blood leukocytes of healthy donors for 5 d in the presence of IL-7 and then transduced them with Nef-expressing H-NA7 or control H-Δ virus. The purity of the infected populations and the efficiency of transduction were assessed 4 d later by flow cytometric analysis of CD4 expression on the cell surface and intracellular p24 Gag expression, respectively. As shown in + T cells were productively infected. Notably, the unusually low level of CD4 on the surface of cells infected with H-NA7 virus was due to robust downregulation of cell surface CD4 by NA7 Nef . NS1 and NS1ELMO1 cells were infected with a lentiviral vector expressing HIV-1 NA7 Nef (FUGWCNA7) or with a control empty vector (FUGW). Cell extracts were prepared from the infected populations, and PBD–GST pulldown assays were performed to determine the fraction of activated Rac. In agreement with a previous report , NS1 cells contain a small but readily detectable pool of activated Rac in spite of the lack of detectable ELMO1 expression, which is most likely generated by ELMO1-independent mechanism(s)  A, lane 1anism(s) A. In conO1 cells A, lane 4Next, we studied Rac activation by Nef in HEK 293 cells, which do not express endogeneous DOCK2. Combinations of Nef, DOCK2, ELMO1, and either Rac1 or Rac2 were expressed in HEK 293 cells by transient transfection. The expression of Nef in the absence of DOCK2 had little effect on the activation of either Rac isoform . Signifi∇HA) did not significantly affect Nef functions in other assays, were tested for their effects on Rac activation in Jurkat T cells  nor the productively infected (Gag-positive) cells, produced detectable amounts of IL-2 ) abolished the inhibition of migration  and, as controls, wild-type Rac1 and Rac2 in Jurkat T cells and measured their migration to SDF-1  is not constitutively expressed in Jurkat T cells, we transiently expressed CCR5 and GFP marker from a bicistronic vector either alone or together with HIV-1 NA7 Nef. Using this vector, CCR5 expression levels were positively correlated with GFP marker protein expression A. We theNef is a multifunctional adaptor protein that modulates signal transduction and protein-sorting machineries. We purified to near homogeneity an abundant Nef-associated protein complex from T cells and identified by mass spectroscopy its major subunits as DOCK2 and ELMO1, a bipartite Rac activator . NotablyWhy were proteins previously reported to associate with Nef not detected in our studies? One possible explanation is that previously reported associations are unstable under the biochemical purification conditions used in our studies. This likely explains the apparent absence of clathrin adaptors in Nef preparations purified in our studies, since we know that Nef binds AP-2/AP-1 clathrin adaptors only weakly in the salt and pH conditions used here (data not shown). It is also possible that in some cases, epitope tags may be buried and therefore inaccessible to the monoclonal antibodies (mAbs) used for immunoaffinity purification. Moreover, some of the previously reported associations, especially those with protein kinases, such as PAKs, were best detected by an ultrasensitive in vitro kinase assay  and, as + T lymphocytes even in the absence of stimulation with antigen or chemokines. These data indicate that Nef targets a critical switch, DOCK2–ELMO1, that regulates Rac GTPases downstream of chemokine receptors and the TCR and uses it to modulate the downstream processes they control. Thus, the interaction of Nef with DOCK2 and ELMO1 provides an important mechanism by which Nef may impact pathogenesis by primate lentiviruses.Our observations strongly argue that DOCK2–ELMO1 is the major upstream regulator used by Nef to activate Rac in T cells and that through this interaction Nef can activate Rac in CD4Our data strongly suggest that DOCK2–ELMO1 is the major activator of Rac targeted by Nef in T lymphocytes. This model is supported by the observations that Nef physically associates with a complex that contains DOCK2–ELMO1 and Rac, that specific mutations in Nef simultaneously disrupt its ability to bind this complex and to activate Rac, and that Nef fails to activate Rac in the absence of ELMO1. Although previous reports implicated Vav, a Rac1 guanine nucleotide exchange factor (GEF), as the critical downstream effector that Nef binds directly to activate PAK , our dat+ T cells did not alter IL-2 production in standard activation protocols was unexpected, because previous genetic evidence linked Nef binding to DOCK2–ELMO1 and the ensuing activation of Rac GTPases to the the ability of Nef to facilitate T cell activation. Specifically, the same mutations that we observed to disrupt Rac activation through the DOCK2–ELMO1 complex were previously reported to disrupt the stimulatory effect of Nef on aspects of T cell activation (DOCK2(−/−) and Rac2(−/−) mice is associated with defective immunological synapse formation and depressed antigen-specific responses  Nef, the myristoylation signal was disrupted by insertion of the HA epitope (ANATYPYDVPDYAG) at glycine G2. The full-length human ELMO1 cDNA , DOCK2 cDNA , and cDNAs encoding wild-type and mutant forms of Rac1 and Rac2  were subcloned into pCG plasmids containing N-terminal c-Myc (EQKLISEEDL) or polyhistidine (HHHHHHH) epitope tags, using standard techniques. pBABE ELMO1 contains an N-terminal c-Myc epitope-tagged ELMO1 cDNA subcloned into the pBABE-neo vector. H-NA7 was constructed by substituting the HIV-1 NL4–3 nef coding sequence with that of HIV-1 NA7 nef . H-Δ is based on H-NA7 with a deletion that removes residues 1–34 of Nef and prevents expression of Nef protein. For chemotaxis assays, cDNAs encoding HIV-1 NA7 Nef, human CCR5 , and Rac proteins were subcloned into the pCGCG bicistronic vector that directs the expression of GFP from an internal ribosomal entry site element (Sequences encoding variant and mutant Nef proteins tagged at their C-termini with a peptide (hf) containing the HA and FLAG epitopes (DTYRYIYANATYPYDVPDYAGDYKDDDDK) were subcloned into pBABE-plasmids . In NA7, by calcium phosphate coprecipitation, and viral supernatants were used to infect a Jurkat T cell subline , provide10 Jurkat T cells stably expressing NA7-hf (or control cells) were lysed for 1 h in 200 ml of LB buffer  . Extracts were precleared with protein G–agarose (Roche) for 1 h and incubated with 12CA5 mAb crosslinked to protein G–agarose beads for 4 h. The immunoprecipitate was washed extensively with LB, and bound proteins were eluted by competition with HA peptide (0.2 mg/ml)  in LB for 45 min at 30°C. The eluate was incubated with anti-FLAG M2 affinity gel beads (Sigma) overnight, and the immunoprecipitate was washed extensively with LB and then LB modified to contain 0.1% Triton X-100 (FEB buffer). Proteins were eluted with FEB containing FLAG peptide (0.2 mg/ml) (Sigma) for 45 min at 30°C. The eluate was concentrated on Microcon centrifugal filter devices  with a cutoff of 8 kDa.Unless stated otherwise, all reactions were performed at 4°C. Approximately 1.8 × 10Nef and associated proteins were separated by 8%–17% SDS-PAGE and visualized using SYPRO stain . Visible bands were excised and processed for identification by mass spectrometry. The samples were analyzed by LC-MS/MS as described previously . Spectranef coding sequence under control of the CMV promoter, subcloned downstream of the Woodchuck-responsive element in the FUGW lentiviral vector  by density gradient separation on Lymphocyte Separation Medium , and CD4+ T lymphocytes were isolated using CD4+ T Cell Enrichment Columns . Replication incompetent HIV-1 particles pseudotyped with VSV-G were produced by calcium phosphate transfection of HEK 293 cells and used to infect >98% pure populations of CD4+ T lymphocytes that were cultured in the presence of IL-7 . Cells were recovered from wells by vigorous pipetting, fixed, and permeabilized using Cytofix/Cytoperm Kit (BD Bioscience PharMingen). Intracellular IL-2 and p24 Gag were revealed simultaneously with PE-conjugated rat anti-human IL-2 mAb,  and FITC-conjugated KC57-RD1 mAb , respectively.Anti-CD3 mAb and anti-CD28 mAb , alone or in combinations, were immobilized on 12-well microtiter plates  in PBS overnight at 4°C. Wells were washed three times with PBS, and 5 × 10HEK 293 cells were transfected by calcium phosphate coprecipitation with 20 μg of pCG plasmids expressing epitope-tagged DOCK2, ELMO1, Rac2 or Rac1, and/or tagged Nef proteins and a control empty vector. Cells were lysed 48 h posttransfection in LB buffer. To isolate Nef and associated proteins, extracts were incubated overnight with anti-FLAG M2 Affinity Gel (Sigma), the immunoprecipitates were washed four times with LB buffer, once with LB containing 0.5 M LiCl, and proteins were eluted with FLAG peptide as described above. To isolate DOCK2 and associated proteins, extracts were incubated with Ni–NTA agarose  and proteins were eluted from the precipitate with 250 mM imidazole. DOCK2–Nef complexes were isolated from imidazole eluates with anti-FLAG M2 affinity gel as described above. Eluted proteins proteins were separated by 16% SDS-PAGE, electroblotted onto PVDF membrane (Millipore), and immunoblotted with the following antibodies: anti-c-Myc mAb , anti-FLAG M2 mAb , 12CA5 mAb (1:5000), or rabbit serum raised to a DOCK2-specific peptide (GDKKTLTRKKVNQFFKTM). Immune complexes were revealed with HRP-conjugated antibodies specific for the Fc fragment of mouse or rabbit immunoglobulin G.  and ECL .2 (RB buffer). Extracts were incubated for 2 h with 40 μg of recombinant PAK1 PBD–GST (kindly provided by Linda Van Aelst) bound to glutathione–agarose beads (8 mg/ml) (Roche), and beads were washed extensively with RB buffer. Bound proteins and aliquots of extracts were separated by 16% SDS-PAGE. Rac was immunoblotted with anti-c-Myc epitope mAb , or with anti-Rac mAb , CDC42 was detected with sc-87 rabbit antibody , and Nef was detected with rabbit serum raised against HIV-1 HxB3 Nef  accession numbers of the genetic loci discussed in this paper are DOCK2 (LocusLink ID 1794), ELMO1 (LocusLink ID 9844), Rac1 (LocusLink ID 5879), and Rac2 (LocusLink ID 5880).The LocusLink (


There is a controversy about the additional benefit of various supplemental interventions used in clinical practice to further enhance the effectiveness of respiratory rehabilitation in patients with Chronic obstructive pulmonary disease (COPD). The aim of this research was to assess randomised controlled trials (RCTs) testing the additional benefit of supplemental interventions during respiratory rehabilitation in COPD patients.Systematic review with literature searches in six electronic databases, extensive hand-searching and contacting of authors. Two reviewers selected independently eligible RCTs, rated the methodological quality and extracted the data, which were analyzed considering the minimal important difference of patient-important outcomes where possible.We identified 20 RCTs whereof 18 provided sufficient data for analysis. The methodological quality was low and sample sizes were too small for most trials to produce meaningful results . Data from five trials showed that supplemental oxygen during exercise did not have clinically meaningful effects on health-related quality of life while improvements of exercise capacity may be even larger for patients exercising on room air. RCTs of adding assisted ventilation, nutritional supplements or a number of anabolically acting drugs do not provide sufficient evidence for or against the use any of these supplemental interventions.There is insufficient evidence for most supplemental interventions during respiratory rehabilitation to estimate their additional value, partly due to methodological shortcomings of included RCTs. Current data do not suggest benefit from supplemental oxygen during exercise, although the methodological quality of included trials limits conclusions. To appropriately assess any of the various supplemental interventions used in clinical practice, pragmatic trials on respiratory rehabilitation of COPD patients need to consider methodological aspects as well as appropriate sample sizes. Chronic obstructive pulmonary disease (COPD) has a large impact on health-related quality of life (HRQL) and represents a major health burden in industrialized and developing countries -4. A sysPhysical exercise is the central component of respiratory rehabilitation programs because it reverses peripheral muscle dysfunction, a highlClinicians, who consider these supplemental interventions during respiratory rehabilitation programs, should know their benefits and downsides. They need evidence from RCTs directly comparing respiratory rehabilitation with or without supplements in order to carefully discuss these benefits and downsides with their patients. Therefore, we conducted a systematic review of pragmatic RCTs comparing the effects respiratory rehabilitation with and without any supplemental intervention to assess their added value in HRQL and exercise capacity improvement.We performed electronic database searches in MEDLINE , EMBASE , PEDRO  and the Cochrane Central Register of Controlled Trials . We also searched the Science Citation Index database  and the "related articles" function of PubMed  by entering all included studies. In addition, we hand searched the bibliographies of all included studies, of reviews on respiratory rehabilitation or physical exercise in patients with COPD that were identified in the literature search, as well as the Proceedings of the International Conferences of the American Thoracic Society and the congress of the European Respiratory Society to identify further relevant studies. We also contacted authors in the field to ask for published or unpublished data.We included RCTs investigating any supplemental intervention added to respiratory rehabilitation that included a standardized physical exercise program. We focused on standardized exercise programs because only these allow reproduction in clinical practice. A standardized exercise protocol was defined as the use of an identical exercise activity for all patients  at measurable exercise intensity . We included studies if more than 90% of study participants patients had COPD according to the following criteria: (1) a clinical diagnosis of COPD, (2) irreversible airways obstruction and (3) one of the following: (a) best recorded FEV1/FVC ratio of individual patients < 0.7; (b) best recorded FEV1 of individual patients < 70% of predicted value. We considered the following outcome measures: HRQL as measured by generic (e.g. SF-36) or disease-specific (e.g. St. George Respiratory Questionnaire) questionnaires, symptom scales, functional exercise capacity as measured walk tests and results from cardiopulmonary exercise testing. We did not apply any language restrictions.We excluded studies that compared any exercise program versus usual care (i.e. no exercise) or studies that used unstandardised exercise protocols (e.g. home exercise programs).The bibliographic details of all retrieved articles were stored in a Reference Manager file. We removed duplicate records resulting from the various database searches. Two members of the review team independently scrutinized the titles and abstracts of all identified citations  using multivariable regression models  where clinical and methodological items would act as explanatory variables. No pooling was undertaken in the presence of significant source heterogeneity.Whenever possible, for each outcome, estimates and confidence limits was related to its minimal important difference. We asseData were analyzed using STATA .Figure Table 3 see  shows a 2 retention during walking tests despite high oxygen flow of 5 l/minute.The characteristics of the five trials on supplemental oxygen -29 are sTwo trials,31 evaluGarrod assessedJohnson evaluate2 (22 in group with supplement and 30 in group with placebo) the difference between groups was 27 meters (95% CI 1–53) in the incremental and 121 seconds (95% CI -44–286) in the endurance shuttle walk test. Patients with the carbohydrate-rich diet increased their body weight compared to the placebo group by 1.23 kg (95% CI 0.42–2.05), which occurred mainly because of an increase of the fat mass . There was a dropout rate of 40% in the group with and of 16% in the group without carbohydrate-rich diet.We identified two RCTs that assess the additional benefit of nutritional supplements during respiratory rehabilitation  for peripheral muscle function. One trial[Assisted ventilation also aims at increasing oxygen availability during exercise, but the trials indicated no additional benefit. An exception may represent overnight non-invasive positive pressure ventilation. This treatment may improve quality of sleep as well as daytime gas levels and respiratory muscle function thereby providing a better milieu Table 3: Internal validity of included studiesClick here for fileAppendix: The appendix lists all studies that were excluded after full text assessment. The full reference and the reason for exclusion are given.Click here for fileTable 1: Characteristics of randomised controlled trials investigating supplemental oxygen and assisted ventilationClick here for fileTable 4: Effect of assisted ventilation on HRQL and exercise capacityClick here for fileTable 2: Characteristics of randomised controlled trials investigating drug and nutritional supplementsClick here for fileTable 5: Effect of drug and nutritional interventions on HRQL and exercise capacityClick here for file


Mixed evergreen forests form the smallest, most widely distributed and fragmented biome in southern Africa. Within South Africa, 44% of this vegetation type has been transformed. Afromontane forest only covers 0.56 % of South Africa, yet it contains 5.35% of South Africa's plant species. Prior to this investigation of the indigenous forests on the Blyde River Canyon Nature Reserve (BRCNR), very little was known about the size, floristic composition and conservation status of the forest biome conserved within the reserve. We report here an inventory of the forest size, fragmentation, species composition and the basic floristic communities along environmental gradients.A total of 2111 ha of forest occurs on Blyde River Canyon Nature Reserve. The forest is fragmented, with a total of 60 forest patches recorded, varying from 0.21 ha to 567 ha in size. On average, patch size was 23 ha. Two forest communities – high altitude moist afromontane forest and low altitude dry afromontane forest – are identified. Sub-communities are recognized based on canopy development and slope, respectively. An altitudinal gradient accounts for most of the variation within the forest communities.BRCNR has a fragmented network of small forest patches that together make up 7.3% of the reserve's surface area. These forest patches host a variety of forest-dependent trees, including some species considered rare, insufficiently known, or listed under the Red Data List of South African Plants. The fragmented nature of the relatively small forest patches accentuates the need for careful fire management and stringent alien plant control. Originacies km-2. Cooper cies km-2 conducteTo begin to understand the nature and distribution of forests in the BRCNR landscape, we need to have an accurate surface area inventory, a species composition list, and an identification and classification of the communities. Standard ecological fieldwork coupled with remotely sensed data and GIS allow for an efficient analysis. Prior to this investigation of the indigenous forests on BRCNR very little was known about the size and floristic composition of the forest biome conserved within the reserve. In comparison, the grassland biome has received much attention on BRCNR with a monitoring program set up to assess the status of the grasslands. We report here an inventory of forest size, fragmentation, species composition and the basic floristic communities along environmental gradients.BRCNR is situated on the northern Drakensberg Escarpment, Mpumalanga Province, South Africa . The flammable nature of this species, compared to natural forest margin species, allows grassland fires to penetrate forests, resulting in a reduction in forest patch size. With a large edge effect resulting from many small forest patches, there is a need for careful fire management and stringent alien plant control.Some severely over-utilized forest patches are currently in a state of recovery; however, current and future damage from invasive trees is a threat to this recovery. Some of the forest patches on BRCNR have a forest margin largely comprised of the invasive black wattle . Forest boundaries were marked on 1:10 000 orthophotos, and the data were digitized onto the Mpumalanga Parks Board's GIS program, SPANS . Total fTwenty-two plots 0.04 ha each) were subjectively distributed throughout two of the five largest forest patches occurring on BRCNR (Figure  ha each Eigenvalues produced from the TWINSPAN and DECORANA programs are a measure of the degree of separation in the data. According to Jongman, ter Braak & van Tongeren , low eigThe 22 relevés were analyzed for a circumscription of possible plant communities. Firstly, a complementary analysis was run using the hierarchical classification program TWINSPAN  and the Specific species responses to environmental variables are a useful way of displaying the impact a certain environmental variable may have on a species. Direct ordinations relate species presence to environmental variables on the basis of species and environmental data from the same set of sample plots . A scattML conducted the field work and the map digitization. Both authors performed the statistical analyses. Both authors read and approved the final manuscript.Appendix 1. Forest flora in 22 relevés on Blyde River Canyon Nature ReserveClick here for file


Although shoulder pain is a commonly encountered problem in primary care, there are few studies examining its presenting characteristics and clinical management in this setting.We performed secondary data analysis of 692 office visits for shoulder pain collected through the National Ambulatory Medical Care Survey (Survey years 1993–2000). Information on demographic characteristics, history and place of injury, and clinical management  were examined.Shoulder pain was associated with an injury in one third (33.2% (230/692)) of office visits in this population of US primary care physicians. Males, and younger adults (age ≤ 52) more often associated their shoulder pain with previous injury, but there were no racial differences in injury status. Injury-related shoulder pain was related to work in over one-fifth (21.3% (43/202)) of visits.An x-ray was performed in 29.0% (164/566) of office visits, a finding that did not differ by gender, race, or by age status. Other imaging  was infrequently performed .Physiotherapy was ordered in 23.9% (135/566) of visits for shoulder pain. Younger adults and patients with a history of injury more often had physiotherapy ordered, but there was no significant difference in the ordering of physiotherapy by gender or race. Examination of the use of intraarticular injection was not possible with this data set.These data from the largest sample of patients with shoulder pain presenting to primary care settings offer insights into the presenting characteristics and clinical management of shoulder pain at the primary care level. The National Ambulatory Medical Care Survey is a useful resource for examining the clinical management of specific symptoms in U.S. primary care offices. Shoulder pain is a common clinical problem in the ambulatory setting. The one year prevalence of shoulder pain is as high as 50% in the general population, and 50% of those afflicted consult a physician ,2. As maExamining the clinical management of shoulder pain in primary care settings, where the vast majority of patients present, is essential to improving the quality of care and to understanding the associated costs . HoweverThe National Ambulatory Medical Care Survey offers a means of studying how common clinical conditions are managed by United States primary care physicians in a large, nationally representative sample. We sought to examine the presenting characteristics and clinical management of patients presenting to primary care physicians for evaluation of shoulder pain. In addition to offering insights on the clinical management of shoulder pain, this investigative strategy serves as a model for using this national data set to examine the quality of musculoskeletal care.Data for this study comes from the National Ambulatory Medical Care Survey (NAMCS), 1993 to 2000. Conducted by the National Center for Health Statistics , the NAMCS survey uses a multistage probability sample design . Using tFor each selected patient visit, the physician completes a visit form that details patient, physician, and clinical information. Patient information includes demographics, insurance status, and up to three reasons for the visit. Physician information includes self-selected specialty, geographic location, and if the practice is in a metropolitan area. Information on clinical management includes which diagnostic and therapeutic maneuvers took place at the time of the visit or were ordered as a result of the visit.Up to three reasons for the office visit were solicited by the survey. Using the NAMCS categorization scheme for reason for visit, we extracted all visits for which shoulder pain (number 14900–14950) was a reason for the visit . We limiEach visit is assigned a weight derived from the probability of being sampled, to account for regional and specialty sampling bias as well as nonresponse. Sampling weights are often used to produce national estimates based on the available sample. Because the weighting scheme of NAMCS was not based on symptom, and the decision to analyze data based on a single specific condition such as shoulder pain precludes the use of weights to produce national estimates, we present our other results as unweighted analyses (Korn). All analyses were conducted using JMP-SAS  and Stata .This sample of patients with shoulder pain was characterized by age, race, and gender. We reported whether the shoulder pain was the result of an injury, whether the injury was work related, and how the injury states differed by demographic characteristics. Younger versus older adults were designated by using the median age of 52 years of age to separate age groups. ICD-9 diagnosis codes were reported for each patient ).Clinical management of the shoulder pain was examined by analyzing the proportion of patients for which plain x-rays and more advanced imaging  and the proportion of patients for whom physiotherapy was ordered. Demographic differences for imaging and physiotherapy orders were examined.We attempted to characterize the treatment of shoulder pain by examining (1) whether physiotherapy was ordered by the physician and (2) whether the physician administered an intraarticular steroid injection. For the physiotherapy issue, specific question was part of the survey during survey years 1995–2000. However, determining whether a corticosteroid was administered was not possible. The survey solicits whether an office surgical procedure was performed during the office visit, but the performance of a minor procedure such as arthrocentesis is not usually considered a surgical procedure. The coding of up to six medications administered or prescribed during the office visit in the NAMCAS survey should allow identification of corticosteroid medications used in a intraarticular injection. However, the route of administration  is not specified by the survey, and the availability of a given corticosteroid in various preparations makes for uncertainty in assigning as truly including determining whether an intraarticular injection took place during an office visit.Figure The mean age of the patients seen at these visits was 53.3 ± 17.8 years, and 54.3% (376/692) of the visits were for female patients. White patients outnumbered blacks (84.6% (586/692) versus 12.1% (84/692) in this data set. The proportion of patients whose shoulder pain was a result of an injury was 33.2% (230/692). Males, and younger adults were more likely to have had an injury associated with the shoulder pain , but there were no race differences in injury association . For the survey years 1995–2000 when the specific question was asked, the proportion of office visits for shoulder pain from injury that were related to work was 7.6% (43/566) of all visits to these primary care physicians and 21.3% (43/202) of those associated with injury.During the survey years 1995–2000 when the specific question was posed regarding the performance or ordering of an x-ray, 164/566 (29.0%) of the visits results in an x-ray order, a finding that did not differ by gender , by race , or by age status . Whether an x-ray was performed was not associated with a history of injury 27.2% versus 30.0%). Advanced imaging  was performed in 6.5% (37/566) of visits.Physiotherapy was ordered in the case of 23.9% (135/566) of visits. There was no significant difference in the ordering of physiotherapy by patient gender  or by patient race , but younger adults were more likely to have physiotherapy ordered . Patients with a history of associated injury were more likely to receive an order for physiotherapy . As discussed above, it was not possible to determine whether intraarticular steroids were administered.The labeling of the years 2000–2010 as the "Bone and Joint Decade" is, in part, a worldwide plea for better understanding and management of common musculoskeletal conditions . AlthougTo our knowledge, the only published studies of shoulder pain in the primary care setting come from the Netherlands and the United Kingdom. Van Der Windt et al examined the characteristics and management of intrinsic shoulder disorders for 349 patients from eleven Dutch general practices during a one year period (1995) . More reThe demographic characteristics of patients of these primary care settings were similar to those of other studies of shoulder pain in primary care settings ,11 with The proportion of patients whose shoulder pain was a result of an injury was 33.2% 230/692) in this study, higher than in the van der Windt study where 12% of patients gave a history of injury and 13% of strain/overuse with unusual activities [2 in thisImaging was performed in 29.0% of patients in this study of U.S office visits, a marked contrast to the British study of primary care management where only 2% of the patients presenting with shoulder pain underwent x-ray studies . BecauseA specific question about the clinician's actions allowed us to determine that physiotherapy was ordered a rate of 24%, comparable to the rate of 30% reported for British general practitioners . InteresSeveral limitations of using this data set for investigation of shoulder pain deserve mention. A precise diagnosis for the shoulder pain would be desirable. The large number of ICD-9 diagnosis codes assigned by the clinician illustrates well the problems in defining and managing this syndrome. However, this problem of imprecise diagnosis of shoulder pain is well known and is not unique to this data set .In addition to seeking insights into the clinical management of shoulder pain, we were interested in exploring whether this data set based on a symptom complex was possible and meaningful. The NAMCS, a series of annual surveys conducted since 1990 in the United States, has been utilized to study health service utilization, patients with known diagnoses, and prescribing behaviour of office-based physicians. To our knowledge, there are no published studies focusing on specific symptoms. The advantages of using this data set for analyzing clinical management of symptoms includes a well organized and standardized classification system of symptoms, a large number of office visits, and the systematic sampling strategy. While our attempt to explore meaningful clinical issues was hampered by the nature of the data set, we nevertheless succeeded in offering insights into the presenting characteristics and clinical management of shoulder pain for this population of patients.Shoulder pain was associated with an injury in one third of office visits in this population of US primary care physicians. Males, and younger adults were more likely to relate their shoulder pain to injury, but there were no racial differences in injury status. Shoulder pain from injury was related to work in over one-fifth of office visits. An x-ray was performed in nearly one third of office visits, a finding that did not differ by gender, race, or by age status. Other imaging  was infrequently performed. Physical therapy was ordered in one quarter of visits for shoulder pain. Younger adults and patients with a history of injury were more likely to have physiotherapy ordered but there was no significant difference in the ordering of physiotherapy by gender or race. Examination of the use of intrarticular injection was not possible with this data set. The National Ambulatory Medical Care Survey is a useful resource for examining the clinical management of specific symptoms in U.S. primary care offices.The author(s) declare that they have no competing interests.JW conceived of the study, and participated in its design andcoordination, and drafted the manuscript.RM and RW participated in formulating the analysis strategy.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


A series of studies aimed at developing methods and systems for analyzing epigenetic information in cells are presented. The role of the epigenetic information of cells, which is complementary to their genetic information, was inferred by comparing the predictions of genetic information with the cell behaviour observed under conditions chosen to reveal adaptation processes and community effects. Analysis of epigenetic information was developed starting from the twin complementary viewpoints of cells regulation as an 'algebraic' system  and as a 'geometric' system . The knowlege acquired from this study will lead to the use of cells for fully controlled practical applications like cell-based drug screening and the regeneration of organs. Knowledge about living organisms increased dramatically during the 20th century and has produced the modern disciplines of genomics and proteomics. Despite these advances, however, there remains the great challenge of learning how the different living components of the cell are integrated and regulated. As we move into the post-genomic period, the complementarity of genomics and proteomics will become apparent and the connections between them will be exploited. However, neither genomics nor proteomics alone can provide the knowledge needed to interconnect the molecular events in living cells. The cells in a group are individual entities, and differences arise even among cells with identical genetic information that have grown under the same conditions. These cells respond to perturbations differently. Why and how do these differences arise? Cells are the minimum units containing both genetic and epigenetic information which are used in response to environmental conditions such as interactions between neighbouring cells and of changes in extracellular conditions. To understand the rules underlying the possible differences occurring in cells, we need to develop methods for simultaneously evaluating both the genetic information and the epigenetic information Fig. . In othee.g., cell population, network pattern, or nutrient conditions) by using the on-chip single-cell cultivation chip [In 1999 the author moved to the Univ. of Tokyo and began his research on the "determination of genetic and epigenetic cellular control processes". To understand the meaning of the genetic variability and the epigenetic correlation of cells, we have developed the on-chip single-cell-based microcultivation method. As shown in Fig. ion chip -10 or byion chip -14. Finaion chip . In thisThe aim of our project is to develop methods and systems for analyzing the epigenetic information in cells. The project is based on the idea that, although genetic information makes a network of biochemical reactions, the history of the network as a parallel-processing recurrent network was ultimately determined by the environmental conditions of cells, which we call epigenetic information. As described above, if we are to understand the events in living systems at the cellular level, we need to keep in mind that epigenetic information is complementary to genetic information.The advantage of this approach is that it bypasses the complexity of underlying physicochemical reactions which are not always completely understood and for which most of the necessary variables cannot be measured. Moreover, this approach shifts the view of cell regulatory processes from the basic chemical ground to the paradigm of a cell as an information-processing unit working as an intelligent machine capable of adaptation to changing environmental and internal conditions. It is an alternative representation of the cell and can bring new insight into cellular processes. Moreover, models derived from such a viewpoint can directly help in the more traditional biochemical and molecular biological analyses of cell control.The basic part of the project is the development of on-chip single-cell-based cultivation and analysis systems for monitoring the dynamic processes in the cell. In addition we have employed these systems to examine a number of other processes eg; the variability of cells having the same genetic information, the inheritance of non-genetic information between adjacent generations of cells, the cellular adaptation processes caused by environmental change, the community effect of cells and network pattern formation in cell groups Figs.  and 4. ATo understand the variability of cells having the same genetic information and to observe the adaptation processes of cells, we need to compare the sister cells or the direct descendant cells directly Fig. . For thaE. coli cells derived from a common ancestor. The four series of interdivision times varied around the overall mean value, 52 min (dashed line); the mean values of the four cell lines a, b, c, and d were 54, 51, 56 and 56 min, showing differences rather small compared with the large variations in the interdivision times of consecutive generations. This result supports the idea that interdivision time variations from generation to generation are dominated by fluctuations around the mean value, and it was evidence of a stabilized phenotype that was subsequently inherited. To explore this idea, we examined the dependence of interdivision time on the interdivision time of the previous generation. We grouped the interdivision time data into four categories and determined their distributions  and the community effect of cells is to create a fully controlled network by using cells on the chip Fig. . We haveAs explained above, the agarose-microchamber cell-cultivation system includes an apparatus for photothermal etching. Photothermal etching is an area-specific melting of the agarose microchambers by spot heating using a focused laser beam and a thin layer made of a light-absorbing material such as chromium (since agarose itself has little absorbance at 1064-nm). We made the three-dimensional structure of the agar microchambers by using a photo-thermal etching module. Figure By using this photothermal etching method, we can change the neural network pattern on a multi-electrode array chip during cultivation. Figure The agarose microchamber system can also be used to observe the dynamics of the synchronizing process of two isolated rat cardiac myocytes. Figure We have newly developed and have just started to use a series of methods for understanding the meaning of genetic information and epigenetic information in a simple cell model system. The most important expected contribution of this project is to reconstruct the concept of a cell regulatory network from the 'local' (molecules expressed at certain times and places) to the 'global' . Knowledge of epigenetic information, which we can control and change during their life, is complementary to genetic information, and those two kinds of information are indispensable for living organisms. This new kind of knowlege has the potential to be the basis of a new field of science.KY conceived of the study, its design and coordination.


The myasthenia gravis is twice as common in women as in men and frequently affects young women in the second and third decades of life, overlapping with the childbearing years. Generally, during pregnancy in one third of patients the disease exacerbates, whereas in two thirds it remains clinically unchanged. Complete remission can occur in some patients.To describe the clinical course, delivery and neonatal outcome of 18 pregnant women with the diagnosis of myasthenia gravis. Retrospective chart review of pregnant patients with myasthenia gravis, followed at the National Institute of Perinatology in Mexico City over an 8-year period. Data was abstracted from the medical records on the clinical course during pregnancy, delivery and neonatal outcome.From January 1, 1996 to December 31, 2003 18 patients with myasthenia gravis were identified and included in the study. The mean ± SD maternal age was 27.4 ± 4.0 years. During pregnancy 2 women (11%) had an improvement in the clinical symptoms of myasthenia gravis, 7 women (39%) had clinical worsening of the condition of 9 other patients (50%) remained clinically unchanged. Nine patients delivered vaginally, 8 delivered by cesarean section and 1 pregnancy ended in fetal loss. Seventeen infants were born at mean ± SD gestational age of 37.5 ± 3.0 weeks and a mean birth weight of 2710 ± 73 g. Only one infant presented with transient neonatal myasthenia gravis. No congenital anomalies were identified in any of the newborns.The clinical course of myasthenia gravis during pregnancy is variable, with a significant proportion of patients experiencing worsening of the clinical symptoms. However, neonatal transient myasthenia was uncommon in our patient population. Myasthenia gravis (MG) is an acquired, neuromuscular, autoimmune disease that presents clinically with weakness and fatigue of the skeletal muscles. The disorder is characterized by a decrease of the number of acetylcholine receptors in the neuromuscular plates, due to an autoimmune process mediated by antibodies directed against the alpha-subunit of the nicotine receptor of the acetylcholine . The disPapazian  reportedIn anti acetylcholine-receptor (anti-AchR) antibody-associated AMC, fetal or neonatal death is common. The possible mechanisms could be crossing of maternal antibodies through the placenta with consequent blockage of the function of the fetal isoform of the AchR leading to fetal paralysis causing AMC. In the typical form of TNMG the usual clinical findings include poor sucking and generalized hypotonia .Other reported clinical manifestations are week cry (60% to 70%), facial diplegia or paresis (37 to 60%), swallowing and sucking difficulties (50 to 71%), and mild respiratory distress -11. PtosThe purpose of our present study was to report on the clinical course, delivery and neonatal outcome of pregnant women with the diagnosis of myasthenia gravis, followed in our perinatal center.From January 1,1996 to December 31, 2003, 18 pregnant women with MG were treated during pregnancy and delivered at the National Institute of Perinatology, a tertiary referral center in Mexico City, Mexico. The clinical course of the disease during pregnancy, labor and post-partum period was reviewed, as well as the neonatal period in the 17 infants born to MG mothers. All clinical data was ascertained after reviewing and collecting data from the patient's medical records. The diagnosis of myasthenia gravis was made on clinical grounds and confirmed by positive edrophonium chloride and electromyography tests ,14. TranAfter reviewing the medical records of patients the following criteria was used to define clinical change of MG during pregnancy: a) the first was the type and dosage of medications that the patient received before, during and after pregnancy. Data was collected on the type and doses of medications administered to the patient during the 3 periods, b) the second parameter was the stage of the disease according the Osserman's classification before, during and after pregnancy.The criteria for improvement, unchanged or worsening of MG during pregnancy were the following: 1) Remission: those patients that presented a total disappearance of the symptoms (Osserman's stage 0) and who did not require any specific medication, 2) Improvement: patients who had clinical improvement of the symptoms and decrease of the dosage of the medications that they received before pregnancy by 30% or more, 3) No change: patients with no clinical change in their symptoms (According to Osserman's classification) and same doses of medications compared with before pregnancy. 4) Deterioration: patients who had a deterioration of the disease (worsening of the Osserman's stage) and an increase in the dosages of medications compared with before the pregnancy, or the need for immunosuppressant drugs such as azathioprine and/or prednisone.The Osserman's classification used in this study was the one used by the Myasthenia Gravis Foundation of America: grade I: any ocular muscle weakness; grade II: mild weakness affecting other than ocular muscles; III: moderate weakness affecting other than ocular muscles; IV Severe weakness affecting other than ocular muscles; and grade V: Defined by tracheal intubation, with or without mechanical ventilation, except when employed in routine postoperative management .In the first two trimesters all patients were seen in the clinic once a month, every 15 days between 32 and 36 weeks, and weekly after 36 weeks of gestation. During every visit the dosage of the medications, and the Osserman's stage were reviewed. All patients were seen by a team of obstetricians and clinical neurologists.Descriptive statistics was used to compute the results.During the study period 18 pregnant women with MG were seen at the hospital and had the medical records available for review. The mean ± SD maternal age was 27.4 ± 4.0 years. Before pregnancy 3 patients (17%) were in remission (Osserman's stage 0) and 15 patients (83%) were classified as Osserman's stage II. All the patients were clinically stable before pregnancy. Of the 15 patients with in stage II, 13 (86%) used pyridostigmine, one used pyridostigmine plus steroids (7%), and one used pyridostigmine, azathioprine and steroids. Thymectomy was performed in 17 patients (94%) before the pregnancy. The mean length of time from the start of symptoms to thymectomy was 24.0 months (range: 1–168 months). Other clinical conditions were also diagnosed in 5 patients (28%) before pregnancy: 3 (17%) had impaired glucose tolerance and 2 autoimmune thyroiditis (11%). Serum antibodies against the human acetylcholine receptor assayed by standard RIA were positive in 14 patients (77%). The patients became pregnant at a mean of 2 years post-thymectomy. In our center we prefer to do the thymectomy first and then when the patient is stable we recommend the pregnancy. This is not a generalized protocol in many centers but the majority of our patients were in good conditions before the pregnancy.During pregnancy 9 patients (50%) did not change the clinical status compared with before pregnancy, 2 11%) had improvement and 7 (39%) had worsening of the MG. Of the seven patients who deteriorated, one did so in the first trimester and six in the second trimester. Only one patient experienced a myasthenic crisis during pregnancy. During pregnancy 11 patients (61%) received pyridostigmine, one patient (6%) received pyridostigmine plus steroids and another (6%) received pyridostigmine, steroids, and azathioprine. The pregnancy in two patients (11%) was complicated by eclampsy; one woman was diagnosed with chorioamnionitis and another with thrombocytopenia. Pregnancy duration was 37.5 ± 3.0 weeks (range 29–41 weeks). The clinical characteristics of patients is shown in Table 1% had imTable Seventeen infants were born at mean ± SD gestational age of 37.5 ± 3.0 weeks and a mean birth weight of 2710 ± 73 g. Only one infant presented with transient neonatal myasthenia gravis. This baby presented with sucking difficulties, which resolved spontaneously by day 7 and did not require any specific treatment. The patient with myasthenic crisis delivered by spontaneous vaginal delivery at 37 weeks. The weight of the newborn was 2800 g without complications. No congenital anomalies were identified in any of the newborns.Myasthenia gravis is not rare among women of reproductive age, the reported incidence ranges from 1:10,000 to 1:50,000 . LiteratSchlezinger  describeOur study showed a frequency of worsening of 33%, being an intermediate frequency compared with the reported frequencies of 15 to 55%. The majority of our patients showed that the worsening occurred in the second trimester as in the Batochi et al  series. Regarding the newborns, our study showed that their weight is lower compared with other studies ,7. This In conclusion the present literature in pregnant patients with myasthenia gravis is somewhat limited. It consists mostly of case reports and case series. Our study adds to the body of literature showing that about third of our patients deteriorated during pregnancy, which was observed in the second trimester. Our cesarean section rate was high and the rate of TNMG was relatively low.The author(s) declare that they have no competing interests.JFTZ. Has made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data.LHR. Has made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data.VS. Has made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data.BE. Has been involved in drafting the article or revising it critically for important intellectual contentODS. Has been involved in drafting the article or revising it critically for important intellectual contentAll authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: