Metabolic pathway analysis has been recognized as a central approach to the structural analysis of metabolic networks. The concept of elementary (flux) modes provides a rigorous formalism to describe and assess pathways and has proven to be valuable for many applications. However, computing elementary modes is a hard computational task. In recent years we assisted in a multiplication of algorithms dedicated to it. We require a summarizing point of view and a continued improvement of the current methods.We show that computing the set of elementary modes is equivalent to computing the set of extreme rays of a convex cone. This standard mathematical representation provides a unified framework that encompasses the most prominent algorithmic methods that compute elementary modes and allows a clear comparison between them. Taking lessons from this benchmark, we here introduce a new method, the binary approach, which computes the elementary modes as binary patterns of participating reactions from which the respective stoichiometric coefficients can be computed in a post-processing step. We implemented the binary approach in FluxAnalyzer 5.1, a software that is free for academics. The binary approach decreases the memory demand up to 96% without loss of speed giving the most efficient method available for computing elementary modes to date.The equivalence between elementary modes and extreme ray computations offers opportunities for employing tools from polyhedral computation for metabolic pathway analysis. The new binary approach introduced herein was derived from this general theoretical framework and facilitates the computation of elementary modes in considerably larger networks. The background section presents the importance of computing elementary modes for metabolic system analysis, its computational difficulties and the existence of various known algorithms. A theoretical section brings these algorithms into a unified framework. In a following section we introduce a new approach, called the binary approach. Although relying on concepts introduced in the theoretical section, this section gives enough practical details to be stand-alone for the implementer. Results obtained from example networks and a conclusion section close the article.m metabolites and q reactions. Reactions may involve further metabolites that are not considered as proper members of the system of study. The latter metabolites, considered to be buffered, are called external metabolites in opposition to the m metabolites within the boundary of the system, called internal metabolites. The stoichiometry matrix N is an m × q matrix whose element nij is the signed stoichiometric coefficient of metabolite i in reaction j with the following sign convention: negative for educts, positive for products. Some reactions, called irreversible reactions, are thermodynamically feasible in only one direction under the normal conditions of the system. Therefore, reaction indices are split into two sets: Irrev (the set of irreversible reaction indices) and Rev (the set of reversible reaction indices). A flux vector (flux distribution), denoted v, is a q-vector of the reaction space q, in which each element vi describes the net rate of the ith reaction. Sometimes we are interested only in the relative proportions of fluxes in a flux vector. In this sense, two flux vectors v and v' can be seen to be equivalent, denoted by v ≃ v', if and only if there is some α > 0 such that v = α · v'.We consider a metabolic network with Nv = 0. Thermodynamics impose the rate of each irreversible reaction to be nonnegative. Consequently the set of feasible flux vectors is restricted toMetabolism involves fast reactions and high turnover of substances compared to events of gene regulation. Therefore, it is often assumed that metabolite concentrations and reaction rates are equilibrated, thus constant, in the timescale of study. The metabolic system is then considered to be in quasi steady state. This assumption implies P = {v ∈ q : Nv = 0 and vi ≥ 0, i ∈ Irrev}     (1)P is a set of q-vectors that obey a finite set of homogeneous linear equalities and inequalities, namely the |Irrev| inequalities defined by vi ≥ 0, i ∈ Irrev and the m equalities defined by Nv = 0. P is therefore – by definition – a convex polyhedral cone  where NRev consists of all columns of N corresponding to reversible reactions. Note that if v is a flux vector of S and v' is its reconfiguration then Nv = N'v'.Let v' ∈ Irrev ∪ Rev × {-1;+1} is such that for any reversible reaction index i ∈ Rev at least one of the two coefficients v'i,+1)   the set of reconfigured EMs of Sthe set of two-cycles made of a forward and a backward reaction of S' derived from the same reversible reaction of Sb) Proof: see Methods.Thus, the set of EMs of the original network is equivalent (up to the two-cycles) to the set of EMs in the reconfigured network and therefore can be seen as a reduced set of extreme rays of the pointed convex polyhedron as defined by:P = {v' ∈ q + |Rev| : N'v' = 0 and v' ≥ 0}     (5)Hence, EMs computation can be derived from any extreme ray enumeration algorithm applied to the reconfigured network and followed by vector back-configuration and the elimination of meaningless vectors, namely the two-cycles.extreme currents. Thus, extreme currents are identical to the EMs in the reconfigured network and, hence, also (up to the 2-cycles) equivalent to the EMs from the original networkNote that exactly the same procedure – splitting reversible reactions into two irreversible ones – was carried out also in the original work of Clarke  on stabiIn the following we present a simple yet efficient algorithm for extreme ray enumeration, the so-called Double Description Method . We showgenerating matrix R of a pointed polyhedral cone P(A) is a matrix such that P(A) = {x ∈ d : x = Rλ for some λ ≥ 0}. The pair  is called a Double Description pair, or DD pair. As mentioned above, the extreme rays form the unique set of minimal generating vectors of P(A) and thus, considered as set of d-vectors, the extreme rays of P(A) form the columns of a generating matrix R that is minimal in terms of number of columns. The pair  is then called a minimal DD pair.A Ak, Rk) from a minimal DD pair , where Ak is a submatrix of A made of k rows of A. At each step the columns of Rk are the extreme rays of P(Ak), the convex polyhedron defined by the linear inequalities Ak. The incremental step introduces a constraint of A that is not yet satisfied by all computed extreme rays. Some extreme rays are kept, some are discarded and new ones are generated. The generation of new extreme rays relies on the notion of adjacent extreme rays. Here again, for the sake of simplicity, we adopt a characteristic . Then the following statements are equivalent:r and r' are adjacent extreme rays(a) r" is a ray of P(A) with Z(r) ∩ Z(r') ⊆ Z(r") then either r" ≃ r or r" ≃ r'(b) if P is pointed, A has full rank and contains a nonsingular submatrix of order d denoted by Ad. Hence,  is a minimal DD pair which works as initialization and leads directly to step k = d. Note that there is some freedom in choosing a submatrix Ad or some alternative starting minimal DD pair.The initialization of the double description method must be done with a minimal DD pair. One possibility is the following. Since Ak - 1, Rk - 1) is a minimal DD pair and consider a kth constraint defined by a not yet extracted row of A, denoted Ai•. Let J be the set of column indices of Rk - 1 and rj, j ∈ J, its column vectors, i.e. the extreme rays of P(Ak - 1), the polyhedral cone of the previous iteration. Ai• splits J in three parts , with equality (zero ray) or does not satisfy it (negative ray):Assume J- = {j ∈ J : Ai•rj < 0}Rk is ensured in considering all positive rays, all zero rays and new rays obtained as combination of a positive and a negative ray that are adjacent to each other T and can thus be used for Aq. The matrix Aq-1 = I-1 = I gives the q extreme rays that obey to these q independent constraints and works as initialization of R.The matrix m linear inequalities defined by Nr ≥ 0 and -Nr ≥ 0, i.e. m equalities: Nr = 0. The processing of an equality constraint is done in a single pass by only keeping rays of J0 instead of J+ ∪ J0. This is achieved by replacing the line R' ← {rj : j ∈ J+ ∪ J0} with R' ← {rj : j ∈ J0} in the part "Processing of constraints in a given order" in Table Arj (required for the Gaussian combination step) are explicitly stored throughout the algorithm (in the left-hand side of the tableau ; b = ; c = a(b). Vector c reads then .• c(1:3) = ; here, "1:3" expresses "from 1 to 3", thus, 5, 8 and 9 are assigned to the first three elements of vector mat.• mat = 3; value 3 is assigned to the element in the second row and fifth column of matrix mat1 is copied into the third row of matrix mat2. Here, the colon operator ":" expresses "all elements of the respective dimension" (here: columns). Of course, it must be ensured that mat1 and mat2 have the same number of columns.• mat1 = mat2; the values of the fifth row of matrix mat are assigned to a which is now a 3-element vector.• a = mat; the first three elements of the seventh row of matrix a and shifts all elements behind one position back, i.e. vector a reads now .• a= ; a(2)= ; deletes the second element of The pseudo-code given in Figure length(a); if a is a vector  then length returns the number of elements in a.• c = find(a); if a is a vector  then find returns all positions in a which are not zero. Example: find returns .• c = or returns the result of the logical OR operation applied element-wise to a and b. a and b can be scalars, vectors or matrices and must have the same size. Example: if a = , b =  then or returns . In the pseudo-code, we use this routine exclusively for OR-operations of bit masks (arrays with only "ones" and "zeros").• c = zeros returns a matrix of size m × n filled with zeros.• c = null(a) returns a null-space matrix of matrix a.• c = intersect returns the intersection of elements in vectors a and b.• c = all(b) returns "1" if all entries in vector b are not zero and "0" otherwise.• c = EM(s): Elementary Mode(s) also known as Elementary Flux Mode(s).Both authors contributed equally to this work, the starting idea of the binary approach coming from a discussion between them. JG mainly established the relationships between extreme ray and elementary modes computation. SK mainly devised and implemented the binary null-space algorithm. Both authors prepared the manuscript jointly.


MnSOD promoter contains an activator protein-2 (AP-2) binding site that modifies transcription of MnSOD. Mutations have been identified in the proximal region of the promoter in human tumor cell lines. One of these mutations (-102C>T) has been shown to change the binding pattern of AP-2 leading to a reduction in transcriptional activity. The aim of our study was to develop a method to identify and determine the frequency of this (-102C>T) polymorphism in human tissues.Manganese superoxide dismutase (MnSOD) plays a critical role in the detoxification of mitochondrial reactive oxygen species constituting a major cellular defense mechanism against agents that induce oxidative stress. The A new TaqMan allelic discrimination genotype method was successfully applied to genomic DNA samples derived from blood, buccal swabs, snap frozen tissue and paraffin blocks. The polymorphism was shown to be in Hardy-Weinberg Equilibrium in an evaluation of 130 Caucasians from Warsaw, Poland: 44 (33.8%) were heterozygous and 6 (4.6%) were homozygous for -102T.MnSOD -102C>T polymorphism in human subjects by a novel Taqman allelic discrimination assay. This method should enable molecular epidemiological studies to evaluate possible associations of this polymorphism with malignancies and other diseases related to reactive oxygen species.This report represents the first description of the  Three distinct types of SODs have been identified in human cells: 1) a homodimeric cytosolic CuZnSOD [Antioxidant enzymes such as superoxide dismutase (SOD) protect cells from oxidative stress. Generation of reactive oxygen species (ROS) has been implicated in the etiology of a diversity of human diseases, including cancer, aging22, athero CuZnSOD , 2) an e CuZnSOD , and 3)  CuZnSOD .Numerous reports indicate a relative deficiency of superoxide dismutase catalytic activity, including mitochondrial MnSOD, in many types of solid tumors ,10. InteFurther evaluation of MnSOD suggests that it is critically important in maintenance of mitochondrial function. Mice with deficiency of this enzyme exhibit progressive cardiomyopathy, neurodegeneration and perinatal death. These sMnSOD mitochondrial targeting sequence has been associated with risks to various diseases including breast cancer[Genetic polymorphism in the st cancer,18, lungst cancer, cardiomst cancer and Parkst cancer.A reduction of MnSOD activity has been shown to exist in many types of human cancer cells when compared to normal cells . A recenMnSOD gene is localized to chromosome 6 (6q25). The MnSOD promoter region is characterized by a lack of TATA or CAAT boxes but the presence of a GC rich region containing multiple SP-1 and AP-2 binding sites [MnSOD promoter sequence (MnSOD -102C>T) has been shown to change the binding pattern of AP-2 leading to a reduction in transcriptional activity. However the presence of this polymorphism has not been reported in human tissue.The human ng sites . Furthersome 6 6q. The MnSMnSOD promoter.In this study we developed a TaqMan allelic discrimination assay to reliably genotype DNA from many tissues  for the -102C>T polymorphism in the We confirmed the presence of the -102C>T single nucleotide polymorphism in human subjects and submitted the sequence variant to Genbank. The genReactive oxygen species in the form of superoxide radicals, hydrogen peroxide, and hydroxy-radicals are formed during incomplete reduction of molecular oxygen during normal respirations. The production of reactive oxygen species remains relatively stable during normal physiologic respirations. A significant increase in the production of reactive oxygen species such as superoxide radicals can be greatly increased as a result of metabolic disorders or more commonly from exposure to toxins such as cigarette smoke, well-cooked meat, urban residency, and excessive alcohol consumption.MnSOD gene in E. coli significantly increased mutation frequency and cell death when bacteria were grown under aerobic conditions [MnSOD gene has led to detrimental effects. Polymorphisms of the human MnSOD gene have been found in the promoter region, the sequence coding for mature protein, and the mitochondrial targeting sequence. Initial evaluation of the five prime flanking regions from human tumor cell lines indicated that there were no major additions or deletions in the five prime flanking regions of the human MnSOD gene [MnSOD gene [Under normal physiologic conditions, superoxide radicals are detoxified by superoxide dismutase. Among the three SODs, MnSOD has been demonstrated to be the only form that has been essential for survival of aerobic life . Inactivnditions . This haSOD gene . HoweverSOD gene . This chSOD gene , no evalEvaluation of the -102C>T polymorphism is complicated by difficulty in PCR because of the excessive GC rich region in which this polymorphism exists. This location, upstream from the transcription start site was extremely difficult to identify through multiple PCR-restriction fragment length polymorphism (RFLP) assays, which failed to adequately digest at this polymorphism site, and led to multiple false negative results. We found only the highest quality DNA (i.e. blood) was able to be evaluated using a PCR RFLP assay with only 50% genotyping success. This failure to accurately reproduce the PCR-RFLP assay , led us The TaqMan allelic discrimination assay provided results that were confirmed by automated DNA sequencing and blind repeat genotyping. Although we did not test it use on DNA from multiple tissues from the same individual, it was successful for DNA samples derived from buccal swabs and paraffin blocks. It has significant advantages over RFLP analysis, allele-specific amplification, allele-specific hybridization, and oligo-nucleotide ligation assay techniques. The reasons for this advantage come from the reduction in labor intensive work up, the lack of need for special handling of radioactive probes, and the ability to modify this technique to evaluate multiple polymorphisms in this gene. In addition as more significant polymorphisms within the MnSOD gene are discovered, this technique will facilitate detection within the MnSOD gene.The limitations of this technique ultimately come from the quality of DNA that is available and the significant initial expense that is required for a TaqMan assay instrumentation.MnSOD -102C>T polymorphism in human subjects by a novel Taqman allelic discrimination assay. This method should enable molecular epidemiological studies to evaluate possible associations of this polymorphism with malignancies and other diseases related to reactive oxygen species.This report represents the first description of the Most DNA samples (130) were isolated from buffy coats of Caucasian controls derived from a population-based case-control study of stomach cancer carried out in Warsaw, Poland as previously described . To testDNA extraction from paraffin sections was performed after tissue sections  were cut from paraffin blocks. Samples were removed from paraffin through a sequential extraction with histaclear, 100% ethanol and acetone, and dried under vacuum. The pellet was incubated overnight with proteinase K  at 55°C. After heating at 100°C for 10 min, digestion was sequentially extracted with phenol, phenol/chloroform and chloroform. DNA was precipitated with the addition of 3X volume 95% ethanol.MnSOD promoter region. A MGB quencher probe was utilized on the 3' end by a linker arm. TaqMan Universal PCR Master Mix (Applied Biosystems) was used to prepare the PCR. The 2X mix was optimized for TaqMan reactions and contained AmpliTaq-Gold DNA polymerase, AmpErase, UNG, dNTPs with UTP and a Passive Reference. Primers, probes and genomic DNA were added to final concentrations of 300 nM, 100 nM, and 0.5–2.5 ng/μl respectively. Controls (no DNA template) were run to ensure there was no amplification of contaminating DNA. Reference control DNA was also utilized to verify the polymorphisms identified. The amplification reactions were carried out in an ABI Prism 7700 Sequence Detection System (Applied Biosystems) with two initial hold steps  and 50 cycles of a two step PCR . The fluorescence intensity of each sample was measured at each temperature change to monitor amplification of the 278 base pair MnSOD promoter region. The -102 nucleotide was determined by the fluorescence ratio of the two SNP-specific fluorogenic probes. The fluorescence signal increases when the probe with the exact sequence match binds to the single stranded template DNA and is digested by the 5'-3' exonuclease activity of AmpliTaq-Gold DNA polymerase (Applied Biosystems). Digestion of the probe releases the fluorescent reporter dye (either FAM or VIC) from the quencher dye. As shown in figure MnSOD promoter region.SNP-specific polymerase chain reaction (PCR) primers and fluorogenic probes Table  were desTwenty samples with genotypes C/T (4 samples), T/T (3 samples), and C/C (13 samples), some of which were derived from paraffin-embedded tissues, were all confirmed by automated DNA sequencing. These sequence-confirmed samples served as reference standards for the remaining samples. In addition, 10% of the samples were genotyped blind a second time with identical results obtained.RM: Participated in design of study and manuscript preparationKH: Participated in genotyping samplesMD: Participated in design of methods of assayQL: Participated in statistical analysisBM: Participated in design of methods of assayJL: Participated in sample collectionNR: Contributed to the study design and the analysis and interpretation of the dataDH: Participated in design of study and manuscript preparation


Reports of the use of psyllium, largely in hypercholesterolemic men, have suggested that it lowers serum cholesterol as a result of the binding of bile acids in the intestinal lumen. Widespread advertisements have claimed an association between the use of soluble fibre from psyllium seed husk and a reduced risk of coronary heart disease. Given the purported mechanism of cholesterol-lowering by psyllium, we hypothesized that there would be a greater effect when psyllium is taken with breakfast than when taken at bedtime. Secondarily, we expected to confirm a cholesterol-lowering effect of psyllium in subjects with "average" cholesterol levels.Sixteen men and 47 women ranging in age from 18 to 77 years [mean 53 +/- 13] with LDL cholesterol levels that were normal or slightly elevated but acceptable for subjects at low risk of coronary artery disease were recruited from general gastroenterology and low risk lipid clinics. Following a one month dietary stabilization period, they received an average daily dose of 12.7 g of psyllium hydrophilic mucilloid, in randomized order, for 8 weeks in the morning and 8 weeks in the evening. Change from baseline was determined for serum total cholesterol, LDL, HDL and triglycerides.Total cholesterol for the "AM first" group at baseline, 8 and 16 weeks was 5.76, 5.77 and 5.80 mmol/L and for the "PM first" group the corresponding values were 5.47, 5.61 and 5.57 mmol/L. No effect on any lipid parameter was demonstrated for the group as a whole or in any sub-group analysis.The timing of psyllium administration had no effect on cholesterol-lowering and, in fact, no cholesterol-lowering was observed. Conclusions regarding the effectiveness of psyllium for the prevention of heart disease in the population at large may be premature. A cholesterol lowering effect has been reported for a variety of soluble dietary fibres -5. In FeThe accumulation and concentration of bile in the gallbladder is a continuous process. In rats which lack a gallbladder, the biliary excretion rate of bile salts is maximal at night  and bilePatients identified in gastroenterology practices as requiring long-term treatment with psyllium, typically for chronic constipation or the irritable bowel syndrome, were invited to participate in the study. In addition, individuals who had received dietary counselling in a lipid clinic regarding cholesterol-lowering and who subsequently had cholesterol levels deemed not to require further intervention because they met targets set out in clinical practice guidelines were invited by clinic staff to participate. Subjects were deemed ineligible if they were under age 18 years, were under active treatment for hyperlipidemia, had total cholesterol greater than 7.00 mmol/L, required alterations in dosage of medications which might have an effect on lipid levels, had had a gastrectomy, had any disease which is associated with hyperlipidemia, were receiving a bile acid binding resin, or if they did not eat breakfast regularly.The study was approved by the Conjoint Health Research Ethics Board of the University of Calgary, Faculty of Medicine. Subjects were given a description of the study indicating our interest in comparing the relative efficacy of hs versus am dosing with psyllium without indicating the specific hypothesis, and were asked to sign a consent form.Gastroenterology patients were given a high fibre diet sheet as part of their therapeutic regimen and were asked to take this on a continuing basis, beginning one month prior to the initiation of the study. The diet sheet emphasized dietary sources containing predominantly insoluble fibre. Lipid clinic patients had all received in-depth counselling regarding dietary measures for hypercholesterolemia, including a high fibre regimen, and had implemented their dietary changes at least one month before beginning the study. An unsweetened psyllium preparation, "Novo-Mucilax" [NovoPharm], providing three grams of hydrophilic mucilloid per 6.2 gram powder, and a scoop known to provide at least ten grams of psyllium were provided. Containers were numbered and weighed at the conclusion of each test interval. Subjects were randomized to initially take a scoop full of psyllium either with breakfast or at bedtime. Using a crossover design, psyllium was taken in the morning or evening for eight weeks and at the alternate time for the subsequent eight weeks.Determinations of serum total cholesterol, LDL, HDL and triglycerides were made before beginning psyllium, at eight weeks and at sixteen weeks after commencing its use. A trained dietician obtained a dietary history and patients were weighed at the beginning and at the conclusion of the study. All lipid determinations were undertaken following a 14 hour fast and analyses were done in a central laboratory.After initial data inspection based on boxplots and summary measures, cholesterol values at 8 and 16 weeks were examined using analysis of variance, taking into account treatment, period, and between and within subject effects in accordance with the cross-over design. The pattern of change from baseline to 16 weeks was evaluated using paired t-tests.Of 86 subjects beginning the study, 33 of those referred from the gastroenterology clinics and 30 of those referred from the lipid clinic completed it. Of those withdrawing, eight did so because they could not tolerate the psyllium or it was felt to interfere with prescribed medication, 2 had elevated lipids, 4 did not complete all the required blood work, 5 were unable to comply with the protocol because of work or lifestyle changes and 4 developed intercurrent diseases which precluded completing the protocol.The age range of subjects was 18 to 77 years, mean 53 +/- 13 years, including 16 men and 47 women. The mean dose of psyllium taken was 12.7 +/ - 2.3 g for morning dosing and 12.7 +/- 2.2 g when taken in the evenings.Values for total, LDL, HDL cholesterol and triglycerides at baseline, eight and sixteen weeks for various subgroupings are tabulated in tables The mean caloric intake or the intakes of fat or fibre did not change significantly during the study table . There wWe failed to prove our hypothesis that administration of psyllium in the morning would have a greater cholesterol-lowering effect than it would in the evening. Not only was there no observable difference in lipid levels between the crossover periods but the daily ingestion of a greater daily dose than the 10.2 g of psyllium for which the FDA allows health claims to be made  had no eWe used a crossover design since this was the most appropriate one for the primary question being addressed; accordingly, our study did not include a control group. However, the nature of the study should not have provided any motivation for study subjects to adopt any new lifestyle or dietary changes beyond those implemented well before the introduction of psyllium. Observational data has been shown to provide valid information, which is consistent with that observed in randomized, controlled trials ,18. The Failure of lipid-lowering by psyllium has also been demonstrated in twenty hypercholesterolemic children , in twenPublished studies include few normocholesterolemic subjects. Cholesterol reduction was observed in 7 normal men  and in 5A meta-analysis of 17 studies of patients with hypercholesterolemia has suggested a small but significant cholesterol-lowering effect of psyllium . All of Several factors may contribute to the difference between our observations and those of others. A meta-analysis has demonstrated that the initial level of cholesterol was highly predictive of the subsequent reduction of cholesterol by oat bran . A greatThe small increase in the weight of subjects is believed to be have resulted from reduced physical activity. In a meta-analysis of the effect of weight reduction on lipids, predominantly through dietary change, a reduction in total cholesterol of 0.05 mmol/L and of 0.02 mmol/L in LDL cholesterol per kilogram of weight lost was identified . DietaryA small cholesterol-lowering effect of psyllium appears to occur in hypercholesterolemic individuals, at least in men and possibly postmenopausal women. The notion of a benefit accruing to the general population requires additional study. The promotion of foods containing psyllium as reducing the risk of heart disease for the population at large  may be pThe timing of psyllium administration had no effect on cholesterol-lowering and, in fact, no cholesterol-lowering was observed. Conclusions regarding the effectiveness of psyllium for the prevention of heart disease in the population at large may be premature.The authors declare that they have no competing interests.GVR carried out the study design, data review and writing. EAS carried out the study design and data review. RB carried out the study design, data review and statistical analysis. ALE carried out the study design and data review.


One of the cornerstones of immune system function is movement. When word spreads that a virus has entered the body, chemical signals tell lymphocytes to proliferate and travel to the site of infection. Efforts to combat HIV have focused on understanding how the virus disrupts this immune response in the hopes of developing drugs to block its replication as well as vaccines to control the virus itself. Toward this end, scientists are investigating how each of the virus's nine genes—which all appear to have multiple functions—contribute to HIV infection.When HIV infects a cell, viral enzymes copy its RNA genes into DNA, which can then invade the infected cell's chromosomes. The viral DNA might lay dormant or it might use the cell to reproduce more viruses, which go on to infect other cells. The course of infection is determined by interactions between circulating T cells and antigen-presenting cells (cells that present evidence of infection), like macrophages, which may unwittingly aid the virus by transferring it to the T cells. Macrophages, for example, produce proteins that tell T cells to come check out an infection.A viral protein called Nef sparked intensive research after observations that patients with a rare strain of HIV lacking Nef took a very long time to develop AIDS symptoms. Nef has been linked to molecules involved in macrophage- and other antigen-signaling pathways and may use the molecules to appropriate these pathways for its own ends—enhancing virulence by facilitating viral replication. How Nef does this is not entirely clear. Now Jacek Skowronski and his colleagues at Cold Spring Harbor Laboratory in New York have identified the key molecules that Nef enlists to coopt the signaling machinery of immune cells.To understand how this might happen, biochemically speaking, Skowronski's lab first needed to determine which molecules Nef associates with. An adaptor protein, Nef does not directly catalyze reactions, but binds to enzymes that do. The researchers identified two proteins, DOCK2 and ELMO1, that form a complex with Nef. DOCK2 regulates enzymes, called Rac1 and Rac2, that are required for normal lymphocyte migration and antigen-specific responses. ELMO1 has also been shown to help DOCK2 activate Rac. Because DOCK2 activates Rac as part of two different signaling pathways—one activated by the T cell receptor, which mediates T cell activation, and one by a chemokine receptor, which controls T cell migration—the researchers investigated whether Nef could affect these important pathways by modulating Rac activity. They found that Nef in fact activates Rac by binding to the DOCK2–ELMO1 complex. And they went on to show that HIV uses these components of the chemokine receptor pathway to disrupt T cell migration. To generate an effective immune response, it is crucial that T cells travel to sites within lymphatic tissues where they interact with other lymphocytes. By inhibiting T cell migration, the researchers propose, Nef prevents these critical interactions, thereby providing a mechanism for stifling the immune response.These results, the authors argue, provide the biochemical evidence that Nef targets a protein “switch” that can interfere with important aspects of T cell function. In this way, Nef subverts the immune response pathways controlled by receptors on the surface of T cells to effectively disarm the immune system and turn T cells into viral replication factories. Understanding how Nef interacts with these proteins to spread infection could lay the foundation for valuable new therapies aimed at inhibiting and arresting HIV infection by blocking Nef-mediated effects.


Multicellular organisms contain a complete set of genes in nearly all of their cells, each cell harboring the potential to make nearly any protein in their genome. The same holds true for a single-celled bacterium or yeast. Yet a cell activates only a fraction of its genes at any given time, calling on a number of different mechanisms to activate the right genes at the right time. To metabolize sugar, for example, a cell needs to synthesize proteins involved in sugar metabolism, not protein repair, and vice versa. In a new study, Jason Brickner and Peter Walter report a mechanism for gene activation that depends on shuttling DNA to a particular location within the nucleus.In organisms whose cells have nuclei (eukaryotes), genomes lie within the nucleus  but also interact with the inner nuclear membrane. Transcription factors activate gene expression by binding to a promoter sequence in the gene's DNA. The physical structure of DNA—which is packaged with proteins into chromatin—affects gene expression by controlling access to DNA. Where chromatin exists in the nucleus also influences gene expression. Heterochromatin—stretches of highly condensed chromatin—typically lines the nuclear periphery, and genes bundled into heterochromatin are typically silent. Active transcription generally occurs in the less condensed euchromatic regions. But since euchromatic regions are also silenced when they associate with heterochromatin along the membrane, it is thought that delivering chromatin to the nuclear periphery regulates transcriptional repression. Brickner and Walter, however, found evidence of the opposite effect—recruiting genes to the nuclear periphery can promote their activation—suggesting that nuclear membrane recruitment plays a much broader role than previously suspected in gene regulation.INO1, which encodes inositol 1-phosphate synthase, an enzyme involved in phospholipid (fat) biosynthesis. INO1 is also a target gene of the “unfolded protein response,” which is triggered when unfolded proteins accumulate in the endoplasmic reticulum, a subcellular organelle where secreted proteins are folded. The INO1 gene contains a regulatory element  within its promoter region that responds to inositol availability. Genes under the control of this element are transcriptionally repressed by a repressor, Opi1, and activated by two transcription factors, Ino2 and Ino4. The presence of unfolded proteins sets off a chain of events to relieve Opi1 repression and allow activation of INO1.To explore the consequences of chromatin location, the authors focused on a yeast gene called INO1 promoter. Opi1 associates with the chromatin, restricting the INO1 locus to the nucleoplasm and repressing transcription. Induction of the unfolded protein response bumps Opi1 off the chromatin and, with Opi1 out of the way, INO1 travels to the membrane and transcription proceeds. Crucially, the authors show that artificial recruitment of INO1 to the nuclear membrane can be enough to activate the gene. There are several mechanistic aspects of this model to figure out still, but Brickner and Walter argue that for INO1, gene recruitment to the nuclear membrane promotes its activation. In light of other recent work, this phenomenon may be emerging as a more general mechanism for regulating eukaryotic gene expression.Through a series of genetic and biochemical studies, Bricker and Walter show that Ino2 and Ino4 are always bound to the


The development of enzyme replacement therapy for Gaucher disease was a triumph of translational medicine. What were the key steps in its development? What are the controversies surrounding its use? Gaucher disease is the most common lysosomal storage disorder . A deficGaucher disease is an inherited metabolic disorder in which harmful quantities of a fatty substance called glucocerebroside accumulate in the spleen, liver, lungs, bone marrow, and, in rare cases, the brain. There are three common forms.Type 1 is the most common. Clinical features include easy bruising, anemia, low blood platelets, enlargement of the liver and spleen, bone disease, and, in some instances, lung impairment. There are no signs of brain involvement. Problems may begin early in life, be delayed until adulthood, or not occur at all.In type 2, liver and spleen enlargement are apparent by three months of age, and there is extensive and progressive brain damage. These patients usually die by two years of age.In type 3, liver and spleen enlargement is variable, and signs of brain involvement, such as seizures, become apparent gradually.Until 1990, treatment consisted only of palliative measures such as splenectomy and hip replacement. The development of enzyme replacement therapy for Gaucher disease, that is, exogenous administration of the missing enzyme, is a triumph of translational medicine. At the same time, powerful commercial interests may have been influential in physicians adopting a high-dose rather than a low-dose treatment schedule. Moreover, the high cost of enzyme replacement therapy forces us to consider what society can afford in the way of palliative treatments for very rare diseases.The possibility that the therapeutic replacement of enzymes missing from lysosomes could be achieved was first raised by de Duve forty years ago when he wrote: “Any substance that is taken up intracellularly by an endocytic process is likely to end up within lysosomes. This obviously opens up many possibilities for interaction, including replacement therapy” .Type 1 Gaucher disease, the most common type, seems a particularly suitable target for enzyme replacement therapy because of the lack of central nervous system involvement . By the 1970s, the underlying enzyme deficiency had been identified, and methods had been developed to purify the enzyme from human placenta in a high state of purity. Three groups of investigators then attempted to treat the disease by infusing exogenous enzyme.In the United States, at the National Institutes of Health in Bethesda, Maryland, the unaltered enzyme was infused directly into the venous circulation ; at CityThe needed conceptual breakthrough was provided by the identification of a mannose receptor on macrophages and the suggestion that this might prove useful in replacement therapy for Gaucher disease . This leThe first study of commercially produced mannose-enriched glucocerebrosidase was carried out in Bethesda, Maryland, on only 12 patients, presumably because of a limited supply of the enzyme . Given tThe enzyme was promptly approved and marketed. Since only a single dose had been tested, this was the dose that most physicians administered in clinical practice. But the preparation was extremely costly—about US$4.00 per unit. At the dose used in the pivotal trial, a 70-kg patient would receive enzyme costing US$16,800 every two weeks.But was the large dose given actually the dose required? There were no data, and many physicians were unwilling to give less than the dose that had been used in the pivotal trial. Moreover, since most physicians took care of only one or at most two patients with the disease, they were not in a position to perform a dose-ranging study. And industry had no interest in supporting studies to show that a lower dose yielded equivalent results.But clinical trials carried out in our National Institutes of Health–sponsored General Clinical Research Center quickly established that a quarter of the dose given at more frequent intervals was fully effective . By 2000Recent “consensus recommendations,” which were supported in part by the Genzyme Corporation, the manufacturers of recombinant human glucocerebrosidase , suggest that children be given an initial dose of 30 to 60 units every two weeks . But theIt is often assumed that patients with severe disease require larger doses of enzyme than those with mild disease, but a meta-analysis based on liver size or spleen size made it clear that this is not the case  12]. La. La12]. The response of enlarged viscera to enzyme infusion is much more rapid than the response of bones. In one early study, the large dose used in the pivotal trial was given for up to four years to patients with bone disease, and although the response was slow, gradual improvement occurred . Strangec.1226 C → G (N370S) mutation never come to medical attention [The severity of Gaucher disease is very variable. We have estimated that some 60% of patients homozygous for the common ttention . Accordittention ,17. WhatEvaluating dose–response relationships in patients with Gaucher disease has been difficult for several reasons. The number of new patients requiring therapy is relatively small, and the Genzyme Corporation has done little to encourage the performance of dose–response studies, making it difficult to enroll patients. But beyond that, the response of patients to any dose is variable. Some authors have suggested that this may be due to individual differences in dose requirements—that some patients are relatively resistant and require a large dose, while others do well on a small dose . This isThe quality of life for patients with Gaucher disease has been greatly improved by the development of enzyme replacement therapy. Manufacturing and selling the enzyme has also been enormously profitable for industry. This profitability has served as a stimulus for the development of enzyme replacement treatments for diseases less common and generally less responsive to treatment than Gaucher disease. Given the small target population, these treatments are enormously costly on a per-patient basis. Treatments for Fabry disease and Hurler-Scheie disease  are already licensed, and others are on the way ,21,22. T


Dementia is a chronic illness associated with a progressive loss of cognitive and intellectual abilities, such as memory, judgment and abstract thinking.The objective of this study was to assess the health utilities of patients with dementia in Europe and identify the key factors influencing their Health-Related Quality of Life (HRQol).This study used cross-sectional data from the Odense study; a Danish cohort of patients aged 65–84 living in Odense, Denmark. A total of 244 patients with mild to severe dementia were interviewed together with a caregiver about their health status and activities of daily living (ADL). Alzheimer's disease was diagnosed according to the NINCDS-ADRDA criteria for probable dementia. Vascular dementia and other types of dementia were diagnosed according to the DSM-IIIR criteria. Severity of dementia was defined by score intervals on the Mini Mental State Examination score: mild (MMSE 20–30), moderate (MMSE 10–19), and severe (MMSE 0–9). Based on the ADL information, the patients' dependency level was defined as either dependent or independent. Questions from the Odense Study were mapped into each of the five dimensions of the EQ-5D in order to assess patients' HRQol. Danish EQ-5D social tariffs were used to value patients' HRQol.A regression analysis of EQ-5D values was conducted with backward selection on gender, age, severity, ADL level and setting in order to determine the main factor influencing HRQoL.The EQ-5D weight in patients independent upon others in ADL was 0.641 (95% CI: [0.612–0.669]), and in those dependent upon others was 0.343 (95% CI: [0.251–0.436]).Dependency upon others to perform ADL was the main factor affecting HRQoL. Dementia is a chronic illness associated with a progressive loss of cognitive and intellectual abilities, such as memory, judgment and abstract thinking. Cognitive disabilities are those that impact an individual's ability to access, process, or remember information. People with profound cognitive disability will need assistance with nearly every aspect of daily living. The most visible manifestation of dementia is the progressive inability – proportional to the severity of the disease – to perform activities of daily living (ADL) and the subsequent loss of independence . ProgresMeasuring the QoL of patients suffering from dementia can take several forms. Firstly, QoL can be measured using generic health indices like the other disease specific measures. Recently several scales have been developed and validated specifically for dementia patients such as the Quality of Life-Alzheimer's Disease (QOL-AD). Another alternative to assess QoL is to use utility measurements, which are preference-based . PrefereHealth utilities have already been measured in AD in the US and Canada using the Health Utility Index (HUI) . In the Based on data collected alongside an epidemiological study conducted in Odense Denmark, we attempted to assess the health utilities of patients with dementia and identify the key factors influencing their HRQoL.Data were derived from the Odense study, an epidemiological survey in which the objective was to estimate the prevalence and incidence of dementia in Denmark ,11 In thDemented patients were classified by type of dementia and by severity of dementia. Alzheimer's disease (AD) was diagnosed according to the NINCDS-ADRDA criteria for probable dementia . VasculaAll interviews were conducted by a certified nurse in the patient's home., Patient's and caregiver's socio-economic and socio-demographic status as well patients' health status and ADL were recorded. In the event that a relative was not present during the interview, a professional caregiver verified information provided by the patient.Each interview included the following information:- sociodemographic questions .- activity of daily living (ADL) questionnaire using 7 items describing patients' ability to perform physical activities  and psychosocial activities (activities in the home and hobbies inside and outside of the home). Each activity was scored using a four-point Likert scale anchored at the ends with 1 = "Unable to perform the activity" and 4 = "Perform the activity without help from others". The physical ADL ranged between 4 (worst state) and 16 (best state), while the psychosocial ADL scored between 3 (worst state) and 12 (best state) ). Severity of dementia and setting has no statistically significant impact on QoL.For the first time, this study provides health utilities for patients with dementia in Denmark. This study has shown that the factor that most affects the HRQoL of a patient with dementia is their dependency status as defined based by their ability to perform activities of daily living. The type of dementia doesn't seem to have a great an influence on patient's HRQoL, and severity does not appear to discriminate significantly between health utilities. However, due to missing data – particularly among patients with severe dementia – caution must be exercised when interpreting the results.In the utility results previously measured by Neumann et al.  using thAs the EQ-5D values were estimated based on mapped questions, it raises the possibility of quotation bias and goodness of fit. Also, both patients and caregivers answered questions. Yet, with AD – and especially when patients are severely demented – it is impossible to collect non-proxy measurements in the later stages. The same methodology was performed in previous evaluations without knowing the impact of the difference between caregivers' and patients' perceptions.A particular strength of this study was that all data have been collected in conjunction with an epidemiological study wherein patients with dementia had been examined carefully and dementia criteria were explicitly stated.Measuring HRQoL is as important as measuring disease severity, progression, symptom response, cognition and behavioural disturbance when assessing the impact of disease and determining proper intervention in the treatment and management of dementia. However, HRQoL is difficult to assess in a disease such as dementia for which patients suffer from cognitive disabilities. Based on study results and as previously shown by Kurz et al. , dependeADL Activities of Daily LivingEQ-5D EuroQol – Five Dimension ScaleEuroQol EuroQol Scalerd editionDSM-IIIR Diagnostic and Statistical Manual of Mental Disorders 3HRQol Health-Related Quality of LifeMMSE Mini Mental State ExaminationQoL Quality of LifeSAS Statistical Analytical SoftwareCKA is principal author and responsible for quality control. K W-J provided mapping of the questions from the Odense Study into the five dimensions of the EQ-5D. AL and KA provided data analysis and data analyses of the Odense database. P K-S provided access to the Odense database.


Celiac artery stenosis (CAS) may be caused by atherosclerotic degeneration or compression exerted by the arched ligament of the diaphragm. Mitral valve prolapse (MVP) is the most common valvular disorder. There are no reports on an association between CAS and MVP.1560 (41%) out of 3780 consecutive patients undergoing echocardiographic assessment of MVP, had Doppler sonography of the celiac tract to detect CAS.CAS was found in 57 (3.7%) subjects  none of whom complained of symptoms related to visceral ischemia. MVP was observed in 47 (82.4%) subjects with and 118 (7.9%) without CAS (p < 0.001). The agreement between MVP and CAS was 39% (95% CI 32–49%). PSV (Peak Systolic Velocity) was the only predictor of CAS in MPV patients  as selected in a multivariate logistic model.CAS and MVP seem to be significantly associated in patients undergoing consecutive ultrasonographic screening. Celiac artery stenosis (CAS) may be caused by atherosclerotic degeneration, as observed in different vascular districts, or by extrinsic compression (ECCA) usually exerted by an abnormally developed arched ligament of the diaphragm -7. ECCA Mitral Valve Prolapse (MVP) is the most common valvular disorder, with a prevalence of about 6–7% in the general population ,9. It coThe association between MVP and CAS has not been extensively investigated. Accordingly, this study was aimed at verifying this hypothesis based on a possible common origin of the two conditions, i.e. the abnormal development of connective tissue causing both ECCA exerted by the arched ligament of the diaphragm and exuberance of valvular mitral tissue.The study population consisted of 1560 out of 3780 (41%) consecutive patients undergoing echocardiographic assessment of MVP, who also had celiac artery Doppler ultrasound performed between November 1999 to September 2004.MVP was defined as clear-cut billowing of one or both mitral leaflets across the mitral annular plane in 2-dimensional parasternal long axis recording or >2 mm late systolic posterior displacement of mitral leaflets by M-mode. A >4 mm displacement defined a moderate-severe prolapse.Ultrasonography  was performed without intestinal preparation to limit meteorism. The digestive phase was taken into account, because it may influence visceral arterial flow. The evaluation of arterial flow in the celiac tract was recorded by continuous Doppler (CW) and, whenever possible, with the aid of the color-Doppler signal. No corrections of flow velocity by evaluation of the cosine of the insonorisation axis and the axis of the vessel were used: in this way, flow velocity can never be overestimated. The Doppler signal was collected in apnea during inspiration. During expiration, an increase in the compression of the celiac tripod by the arched ligament occurs in the cases of CAS caused by ECCA, with concurrent increase in flow velocity with respect to the velocity during inspiration . An exam45678CAS was defined as severe in case of Peak Systolic Velocity (PSV) flow velocity in the upper celiac tract greater than 2.0 metres/second ,16 and EContinuous variables are expressed as means ± 1 standard deviation (SD). Differences between groups were compared using Student's t-test and chi-square test, as appropriate. Association between CAS and MPV was performed using the Kappa statistics, estimated with 95% confidence interval.Univariate odds ratio (OR) along with their corresponding 95% confidence intervals were computed for describing association of clinical variables with MPV in CAS patients, using a logistic regression model. Selection of variables significantly associated with MPV in CAS patients was made using a multivariate logistic model. Selection criterion was the Akaike Information Criterion, applied backward to the multivariate logistic model. The statistical significance was settled at a p value < 0.05. The R (release 1.9) statistical package and the Harrell's Design and Hmisc libraries were used for analysis.The incidence of MVP in patients with (10.6%) and without (9.3%) assessment of CAS did not differ significantly.Among those undergoing both examinations, CAS was found in 57/1560 (3.6%) patients. The clinical characteristics of patients with and without CAS are reported in Table No patient with CAS was complaining of symptoms related to visceral ischemia, i.e. abdominal or gastric pain in concurrence with food ingestion.Thirteen (22.8 %) of the 57 subjects with CAS were suffering from cardiovascular disorder. In particular, a macrovascular atherosclerotic disorder  was ascertained in 7 subjects , whilst no sign of macrovascular atherosclerosis was found in the remaining 50 subjects .In patients with CAS, factors associated with MPV are age, sex, PSV and BMI. At multivariate analysis only PSV resulted as an independent factor associated with MPV (OR 0.24 95% CI 0.08 to 0.69).The incidence of CAS is not clearly established, the majority of the literature consisting of isolated reports -21. SurpThe incidence of CAS and MVP in the present study dealing with an unselected population was 3.7% and 10.6%, respectively. In addition, this is the first report, to our knowledge, to demonstrate a strong association between the two conditions.CAS may be caused by ECCA or atherosclerosis. Unfortunately, the ultrasound technique has not enough resolution to allow an etiological discrimination that may be difficult to get even with angiographic examination . HoweverThe results of this study demonstrate that CAS is a relatively frequent finding among patients undergoing Doppler sonography of the celiac artery and is frequently associated to MVP. On the basis of these findings, further investigations are warranted aimed at determining the exact incidence of ECCA associated with MVP, the family distribution of the association between MVP and ECCA, or the prognostic implication of ECCA in subjects with MVP. Additionally, genetic studies could be advisable in subjects presenting with MVP associated to ECCA.CAS Celiac Artery StenosisMVP Mitral Valve ProlapseECCA Extrinsic Compression of Celiac ArteryCACS Celiac Artery Compression SyndromePSV Peak Systolic VelocityEDV End Diastolic Velocity


Yukio Mano, MD, PhD (1943–2004)Associate Editor, Journal of NeuroEngineering and Rehabilitation I was terribly shocked to hear of the tragic and sudden passing of Yukio Mano on November 7, 2004, at the age of 61. He had not been well this past year but had been working continuously until just ten days before his death.Yukio Mano Figure  was bornHis research interest was rehabilitation medicine, especially brain plasticity. He was the first Japanese developer of an apparatus that could deliver transcranial magnetic stimulation. Using this apparatus, he analyzed changes in the central nervous system resulting from various diseases, including brain plasticity after anastomosis of the musculocutaneous and intercostal nerves following cervical root avulsion, and cortical reorganization in training. The knowledge resulting from his research proved beneficial in the rehabilitation of disabled patients. He also emphasized a multidisciplinary approach to rehabilitation medicine and adopted new techniques from engineering. He received the Best Paper Award from ANNIE  in 2000 for his work entitled, "Adaptive FES Switching System for Hemiplegics".Yukio Mano served as a council member of the International Society of Electrophysiology and Kinesiology (ISEK) and, in 2000, he organized the XIII Congress of the ISEK in Sapporo, Japan. He was also a member of the editorial board of the Journal of Electromyography and Kinesiology. He served as a council member of many Japanese societies and organized nationwide congresses in Japan even in the year he died. He welcomed the launch of the new Journal of NeuroEngineering and Rehabilitation (JNER) and was honored to be asked to join the Editorial Board as an Associate Editor. He was indeed fully active to his last day.We cannot help praising him for all that he has accomplished in the fields of rehabilitation medicine, neurophysiology and kinesiology. We must also not forget that this remarkable scientist was also a caring family man. He always showed his love for his family as well as for his colleagues and friends. The loss of such an outstanding personality has been met with great sorrow by his family and the international scientific community. We will always remember him with great affection.


Birds, for example, know instinctively what type of nest to build for their offspring; salamanders don't need lessons to swim. But when it comes to primates—including humans—a good deal of behavior is learned. Primates exhibit a wide range of behaviors, not just among species but also among populations and even individuals. Yet the nature versus nurture debate still rages, particularly when it comes to understanding the roots of aggression. While bonobos are famous for using sex to resolve disputes, aggression is far more common in most primate species—again humans included. Our closest relative, the chimpanzee, has a reputation for being among the most belligerent, with rhesus monkeys and baboons not far behind. For many of these species, bouts of violence are often followed by gestures of reconciliation, such as grooming or, in the case of chimps, kissing. Since most primates live in social groups, it may be that such conciliatory measures serve to maintain some semblance of social structure, offsetting the disruptive effects of aggression.  but are also passed on to succeeding generations. Such cultural traditions have been documented in African chimp populations, which display over 39 behaviors related to “technology” (such as using stones to crack nuts), grooming, and courtship. While most of these cases involve either tools, foraging, or communication, Robert Sapolsky and Lisa Share report evidence of a higher order cultural tradition in wild baboons in Kenya. Rooted in field observations of a group of olive baboons  since 1978, Sapolsky and Share document the emergence of a unique culture affecting the “overall structure and social atmosphere” of the troop.A Primate's Memoir, Sapolsky studied the activities and lifestyle of the Forest Troop to explore the relationship between stress and disease. In typical baboon fashion, the males behaved badly, angling either to assume or maintain dominance with higher ranking males or engaging in bloody battles with lower ranking males, which often tried to overthrow the top baboon by striking tentative alliances with fellow underlings. Females were often harassed and attacked. Internecine feuds were routine. Through a heartbreaking twist of fate, the most aggressive males in the Forest Troop were wiped out. The males, which had taken to foraging in an open garbage pit adjacent to a tourist lodge, had contracted bovine tuberculosis, and most died between 1983 and 1986. Their deaths drastically changed the gender composition of the troop, more than doubling the ratio of females to males, and by 1986 troop behavior had changed considerably as well; males were significantly less aggressive.In his book After the deaths, Sapolsky stopped observing the Forest Troop until 1993. Surprisingly, even though no adult males from the 1983–1986 period remained in the Forest Troop in 1993 , the new males exhibited the less aggressive behavior of their predecessors. Around this time, Sapolsky and Share also began observing another troop, called the Talek Troop. The Talek Troop, along with the pre-TB Forest Troop, served as controls for comparing the behavior of the post-1993 Forest Troop. The authors found that while in some respects male to male dominance behaviors and patterns of aggression were similar in both the Forest and control troops, there were differences that significantly reduced stress for low ranking males, which were far better tolerated by dominant males than were their counterparts in the control troops. The males in the Forest Troop also displayed more grooming behavior, an activity that's decidedly less stressful than fighting. Analyzing blood samples from the different troops, Sapolsky and Share found that the Forest Troop males lacked the distinctive physiological markers of stress, such as elevated levels of stress-induced hormones, seen in the control troops.In light of these observations, the authors investigated various models that might explain how the Forest Troop preserved this (relatively) peaceful lifestyle, complete with underlying physiological changes. One model suggests that nonhuman primates acquire cultural traits through observation. Young chimps may learn how to crack nuts with stones by watching their elders, for example. In this case, the young baboon transplants might learn that it pays to be nice by watching the interactions of older males in their new troop. Or it could be that proximity to such behavior increases the likelihood that the new males will adopt the behavior. Yet another explanation could be that males in troops with such a high proportion of females become less aggressive because they don't need to fight as much for female attention and are perhaps rewarded for good behavior. But it could be that the females had a more direct impact: new male transfers in the Forest Troop were far better received by resident females than new males in the other troops.Sapolsky and Share conclude that the method of transmission is likely either one or a combination of these models, though teasing out the mechanisms for such complex behaviors will require future study. But if aggressive behavior in baboons does have a cultural rather than a biological foundation, perhaps there's hope for us as well.


Oryza sativa L.), barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) were linkage maps based on cDNA-RFLP markers. The low number of polymorphic RFLP markers has limited the development of dense genetic maps in wheat and the number of available anchor points in comparative maps. Higher density comparative maps using PCR-based anchor markers are necessary to better estimate the conservation of colinearity among cereal genomes. The purposes of this study were to characterize the proportion of transcribed DNA sequences containing simple sequence repeats (SSR or microsatellites) by length and motif for wheat, barley and rice and to determine in-silico rice genome locations for primer sets developed for wheat and barley Expressed Sequence Tags.Earlier comparative maps between the genomes of rice  and motifs varied with the length of the SSRs within and among the three species, with trinucleotide SSRs being the most frequent. Distributions of genomic microsatellites (gSSRs), EST-derived microsatellites (EST-SSRs), and transcribed regions in the contiguous sequence of rice chromosome 1 were highly correlated. More than 13,000 primer pairs were developed for use by the cereal research community as potential markers in wheat, barley and rice.Trinucleotide SSRs were the most common type in each of the species; however, the relative proportions of SSR types and motifs differed among rice, wheat, and barley. Genomic microsatellites were found to be primarily located in gene-rich regions of the rice genome. Microsatellite markers derived from the use of non-redundant EST-SSRs are an economic and efficient alternative to RFLP for comparative mapping in cereals. The genetic maps of grass species have been constructed using a variety of marker types. Most of the older species-specific molecular maps were constructed with RFLP markers, but in recent times there has been increased utilization of PCR-based markers because of accessibility and higher throughput. Conservation of gene content and order has been detected among grass genomes through the use of comparative maps ,2. The aGenomic SSR (gSSR) markers are biased towards genome specificity ,5 and geRecently, several researchers -17 have When compared to gSSRs, EST derived SSRs (EST-SSRs) were less polymorphic in a study in hexaploid wheat  with onlin-silico to the rice genome. SSR-containing transcripts derived from different species, sharing a pre-determined threshold of similarity and matching the same location in rice were considered putative orthologs that may be used as anchors in comparative mapping studies.We used the rice genome sequence generated by the International Rice Genome Sequence Project (IRGSP)  to identThis paper describes a methodology for developing EST-SSR markers from wheat, barley and rice as markers for developing independent species maps as well as for homologous anchor markers for comparative maps. Over 13,000 untested PCR primer pairs for EST-SSRs were generated from the three gene indices and made available to the research community interested in grass genomes. Researchers are encouraged to evaluate a subset of primer pairs and send feedback regarding their utility to the GrainGenes  databaseBased on combinations of all four nucleotides, the canonical set of SSR motifs is represented by four different duplets , 10 different triplets, 33 different quadruplets and 102 different quintuplet motifs. In the source sequences, all these basic nucleotide motifs can be represented in variant forms of the same basic set or by their reverse complements but to keep a consistency in the database for estimating frequencies, they were transformed into the canonical motifs. Reverse complements and variants would include, for example, CT for AG and GAG for AGG. Sets of unigene sequences such as the TIGR gene indices have the advantage of built-in elimination of redundant SSR counts allowing for more precise estimates of EST-SSR frequency. The rice genomic SSR (gSSR) counts were processed a-posteriori to eliminate redundancy due to BAC/PAC clone overlaps (see methods). Mononucleotide repeats are common in genomic DNA and some are known to be polymorphic but these were deliberately avoided in the unigene database, because they are usually added by the RNA polymerase and are not present in the template DNA (e.g. poly A tails).Table The abundance of SSRs (perfect and imperfect) in the unigenes can range from one in every 100 to one in every two unigenes depending on the minimum length Table .Wheat unigenes contained a larger number of SSRs for all repeat length categories, followed by rice and barley unigenes. This is probably because wheat has more than twice the number of unigenes than rice or barley with 109,782 for wheat, 51,569 for rice and 48,159 for barley. The larger number of unigenes in hexaploid wheat may result from divergence of the genes in the three genomes, but also from a relatively larger EST dataset, i.e., more ESTs have been sequenced for wheat, with a sequence redundancy of 3.8×, versus 6× and 2.7× for barley and rice, respectively (see methods).The number of the ten most frequent motifs was tabulated for different minimal SSR lengths Table . The relp > 0.01).The longest SSRs were genomic microsatellites (as long as 726 bp). Unigenes had few SSRs longer than 40 bp, up to 333 bp in wheat ESTs. Often these were not useful for developing SSR markers because no flanking sequence was available to design primers. The overall mean length for rice gSSRs equal to or longer than 12 bp was 16.5 (s.d = 12.7), with no significant differences in mean gSSR lengths among the chromosomes in rice, while the mean length for EST-SSRs was 15.3 (s.d = 6) with no significant differences among the three species gene indices , and for anchoring rice, wheat and barley unigenes associated with microsatellites (by sequence similarity). Best similarity matches between the rice genome and 8,259 barley, 16,917 rice and 13,565 wheat EST-SSR unigenes were identified using BLASTN. From these, a total of 6,373 EST-SSRs mapped to R1 pseudomolecules including 1,104 from barley, 3,568 from rice and 1,701 from wheat with 88.8%, 98.4% and 89.1% average sequence similarity respectively.The contiguity of the pseudomolecule sequence of rice chromosome 1 (R1) , with only eight gaps for the whole chromosome, provided a convenient framework of coordinates for calculating density estimates of gSSRs features  the density of all rice unigenes mapped to R1, and b) the density of EST-SSRs (a subset from the unigenes) mapped to R1 provided an estimate of the relationship between gSSRs and gene regions in the rice genome. There was a striking resemblance in the patterns of the plots for the density of gSSRs and the density of unigene-derived EST-SSRs in R1 pseudomolecules Figure . The simA decomposition of the set of stringent gSSRs (see the methods section for criteria defining the "stringent gSSRs") by types in R1 Figure  showed tWe designed primer pairs for 5,425 wheat, 3,036 barley and 4,726 rice EST-SSRs conforming to the stringent restrictions described in the methods. The average product size expected from the set of designed primers was 217 bp for rice EST-SSRs, 213 for wheat and 218.9 for barley.in-silico to the rice genome. The Of those EST-SSRs, 42% of the wheat and 56% of the barley were mapped r2 = 0.94, p < 0.006) between genomic microsatellite frequency and the percentage of single copy DNA in several plant species with a wide range of genome sizes. Estimates of repetitive and non-repetitive single-copy DNA fractions were based on reviews of the literature describing renaturation kinetics experiments for each of the species. Plant species that have gone through genome expansion due to retrotransposon amplification, such as maize and wheat, had a lower genomic microsatellite frequency indicating that SSR frequency is not a function of overall genome size but rather the relative proportion of single-copy DNA.Morgante and colleagues  reportedr = 0.45, p < 1 × 10-5 and r = 0.62, p < 1 × 10-10, respectively) . The density pattern of transcribed regions (unigenes in R1) and of SSRs within transcribed regions (unigenes in R1 with SSRs or EST-SSRs) followed closely the density pattern of gSSRs in rice chromosome 1  of gSSRs along R1 was higher than both the density of transcribed regions and the density of EST-SSRs. A large number of gSSRs that are not already included in the set of EST-SSRs could still be associated with genes because, as Figure Other reports have documented a role for SSRs that are associated with genes in the control of gene expression. For example, several human diseases have been linked with events of triplet expansions in the past . ChromatIn rice, although the presence of a single-base mutation breaking an intron splice site is more directly responsible for the difference in phenotypes of the waxy gene, polymorphism due to SSR expansion has been associated with variation of expression levels in different japonica and indica varieties ,29. The One strategy to better exploit a database of EST-SSRs in order to find polymorphic markers is to first sample the longest SSRs ≥ 30 bp), favoring dinucleotide repeats, then follow with trinucleotide, tetranucleotide and pentanucleotide repeats . After e bp, favoOne percent of unigenes from the three species examined in this study have SSRs starting with a minimum of 30 bp Table . OverallAfter relaxing the microsatellite length constraint to a minimum of 20 bp, the overall number of SSRs in the unigenes increased to around 5%. An additional 3,195, 2,703 and 1,991 EST-SSRs in wheat, rice and barley become available for primer design. Acceptable primer pairs were designed for 2,622 wheat, 2,183 rice and 1,476 barley EST-SSRs in this category (which included the set mentioned previously).The set of EST-SSRs with acceptable primer pairs  were selThe relative proportions of di-, tri-, tetra-, and penta nucleotide repeats and motifs varied widely depending on length and were not consistent among the species examined. We have shown that ESTs are a good source of SSRs that can be exploited to develop microsatellite markers for wheat, barley and rice. The advantage to this approach is that the sequences are already available resulting in a lower cost than designing and testing microsatellites from anonymous genomic libraries, even if the polymorphism rate for EST-derived markers is lower.EST-SSRs are useful for enhancing individual species maps, but can be used as anchor probes for creating links between maps in comparative studies when designed from sets of orthologous genes, as demonstrated by Yu et al . The annUsing a different methodology, our results substantiated the report by Morgante et al  suggestiTIGR's non-redundant gene indices  from wheAll analyses of the rice genome used the version released in December 2002 by the International Rice Genome Sequencing Consortium  and consThe TIGR gene indices and the genome of rice were scanned with a modified version of Sputnik  availablIn order to eliminate the problem of counting the same microsatellites several times in the rice genome due to the redundancy created by overlapping regions between contiguous BAC/PACs in chromosomes 2 to 12, the gSSRs were annotated as redundant or not, according to their location in the tiling path. When located to a region in the BAC/PAC that overlapped with a neighbor clone :a) The SSRs are dinucleotides or trinucleotides of length equal or larger than 18 bp, tetranucleotides equal or larger than 16 bp or pentanucleotides equal or larger than 20 bp.b) The imperfect SSRs have less than 10% mismatches or gaps relative to a perfect SSR of the same length and motif.c) There is a minimum of 50 bp surrounding the SSR edges in the source sequence to allow for possible primer design.The parameters used for the Primer3 program specified an optimal Tm of 60°C with a minimum and maximum of 57°C and 65°C, respectively, and a 30% to 70% GC content with a low chance of dimer or hair-loop formation. The range for PCR product length was set to be between 100 and 300 bp.EST: Expressed Sequence Tag. SSR: Simple Sequence Repeat. gSSR: genomic SSR. EST-SSR: EST-derived SSR. UTR: Untranslated region flanking a coding region in DNA and messenger RNA. IRGSP: International Rice Genome Sequence Project. R1: rice chromosome 1. BAC/PAC: Bacterial artificial chromosomes or bacteriophage P1 artificial chromosomes (for cloning of large DNA fragments). MITE: Miniature Inverted-repeat Transposable Element.ML did all programming and design of computational experiments and databases. RVK contributed in the first database design. JY did wet-lab testing of a subset of primer pairs.ML and MES drafted the manuscript. All authors read and approved the final manuscript.EST-SSR designed primers Table listing the stringent subset of SSRs  found in rice, barley and wheat gene indices for which primer sequences were designed, the source sequences, the primers, their in-silico mapping in the rice genome (to BAC/PAC clones or pseudo-molecule) and relevant metadata. File is a spreadsheet table, compressed with the zip program. The file (14 Mb uncompressed) is also available at the GrainGenes Triticeae EST-SSR Coordination page Click here for fileModified Sputnik source code and executable This is the source code with modifications, to the microsatellite searching program "Sputnik", originally written by Chris Abajian from the University of Washington at Seattle. The set of files are compressed using the zip program. File is also available at Click here for filePerl script to design primers in batch The script uses the Bioperl perl modules to control the Primer3 program in order to design primers from a microsatellite database stored in a MySQL database. The script can be modified to accommodate similar schemas on any database engine supported by the perl DBI module. File is also available at Click here for file


In view of the issues surrounding physical restraint use, it is important to have a method of measurement as valid and reliable as possible. We determined the sensitivity and specificity of physical restraint use a) reported by nursing staff and b) reviewed from medical and nursing records in nursing home settings, by comparing these methods with direct observation.We sampled eight care units in skilled nursing homes, seven care units in nursing homes and one long-term care unit in a hospital, from eight facilities which included 28 nurses and 377 residents. Physical restraint use was assessed the day following three periods of direct observation by two different means: interview with one or several members of the regular nursing staff, and review of medical and nursing records. Sensitivity and specificity values were calculated according to 2-by-2 contingency tables. Differences between the methods were assessed using the phi coefficient. Other information collected included: demographic characteristics, disruptive behaviors, body alignment problems, cognitive and functional skills.Compared to direct observation (gold standard), reported restraint use by nursing staff yielded a sensitivity of 87.4% at a specificity of 93.7% (phi = 0.84). When data was reviewed from subjects' medical and nursing records, sensitivity was reduced to 74.8%, and specificity to 86.3% (phi = 0.54). Justifications for restraint use including risk for falls, agitation, body alignment problems and aggressiveness were associated with the use of physical restraints.The interview of nursing staff and the review of medical and nursing records are both valid and reliable techniques for measuring physical restraint use among nursing home residents. Higher sensitivity and specificity values were achieved when nursing staff was interviewed as compared to reviewing medical records. This study suggests that the interview of nursing staff is a more reliable method of data collection. Nursing homes have the mandate to offer care settings to frail dependent older individuals. However, a renewed emphasis has emerged over the past decades to become more than just a home for older people . A growiAlthough predominantly intended as protective devices, physical restraints in nursing homes are being denunciated as measures that go conversely with the aforementioned principles . JustifiAccording to the literature, the overall prevalence of restraint use in nursing homes ranges between 4 and 68% ,20. ThisThe objective of this study was to determine the sensitivity and specificity of the measurement of physical restraint use reported by members of the nursing staff and reviewed from medical and nursing records among nursing home residents, compared to direct observation. Since underreporting is much more susceptible to be problematic than overreporting, another objective of this study was to compare the sensitivity of the information reported by one nurse with that reported by two nurses or more questioned together. Our research hypothesis was that sensitivity of the interview is highest when the information is collected from more than one nurse.The study was conducted in eight facilities representing a convenience sample of the long-term care facilities in the Quebec City area, Canada. These institutions were carefully selected in order to include a mix of characteristics in size , geographic location , university affiliation and vocation (units associated with psychiatric or rehabilitation team). Selection was made after discussion with nursing direction of each setting to gather units of different practice such as regular units and specialized units for residents with dementia or severe behavioral problems. Twenty-five subjects were randomly chosen from each unit; if an unit comprised less than 25 residents, all of its residents were included. This study was approved by the ethics committee at Laval University. Data collection took place between January and June 1992.A physical restraint was defined as a mechanical means applied on a resident in order to interfere with his/her mobility, including: vest, waist, wrist or ankle restraints, geriatric chair or wheelchair with fixed tray table, or any other type of locally designed devices . RestricPhysical restraint use was measured according to three methods: direct observation, interview with members of the nursing staff including licensed practical as well as registered nurses , and review of medical and nursing notes.Direct observation of restraints on care units were made independently by two trained research assistants using a pre-tested questionnaire. For practical reasons, observations were made before the chart reviews and the nurses' interviews on three occasions  on one day. These specific times were selected as being representative of periods of different nurse staffing, and of overloaded periods during morning and afternoon.In order to reduce the occurrence of an information bias, the nursing staff was blinded to the main objective of the research project. Structured interviews were carried out the day following direct observation by one of the authors (PJD), who was unaware of the observations. Interviews with the nurse in charge of each unit were scheduled, although he/she had the liberty to be represented or assisted by other members of the nursing staff. Physical restraint use on each subject was identified for every hour during the last 24 hours, without knowledge of the times that direct observation was made, by means of a pre-tested questionnaire. The questionnaire covered questions about types of physical restraints , reasons for use  and the duration including hours and minutes. Other information collected during the interview included: gross cognitive and functional information, risks for falls, history of falls during the last month, agitation, wandering, aggressive behavior and body alignment problems. Cognitive status was evaluated according to five items: recall, speech, and orientation to time, space and people. Three aspects of the functional status were assessed: urinary incontinence, fecal incontinence, and ability to transfer. Respondents could refer to subjects' clinical records at any time during the interview.Restraint use from subjects' medical charts and nursing orders for the last six months was reviewed with a pre-tested questionnaire by a research assistant who was blinded to the observations. Additional information taken into consideration comprised: demographic characteristics, prescriptions for restraints, methods of resident supervision, and psychotropic medications administered in the last 48 hours.Sociodemographic characteristics of the study sample as well as physical restraint use by methods of data collection were examined using descriptive analysis. Interrater reliability between the two research assistants was tested using the kappa statistic. Direct observation served as the gold standard . To be dData collection was carried out in 16 nursing units. Of these units, eight depicted skilled nursing home care units, seven nursing home care units, and one long-term care unit within a short-term care hospital. Information was collected for 377 residents with the help of 28 nurses. Residents' age ranged from 32 to 102 years, with a median of 80 years. The sample was 62% female, and median length of stay was 45 months (0 to 720 months). Benzodiazepines and neuroleptics were administered to 35% and 25% of the subjects, respectively.A total of 6,744 observations over a possibility of 6,786 were made (377 residents by three direct observations and six types of restraints). Prevalence results on physical restraint use according to direct observations : 0.73–0.97)), interviews with nursing staff and reviews of clinical records are summarized in Table The interview with nursing staff and the review of medical and nursing orders were both highly associated with the observation data Table . The intp = 0.03), agitated behavior (p = 0.04), body alignment problems (p < 0.001) and aggressive behavior (p = 0.01), and reported restraint use by nursing staff were observed. No association was observed for residents' age and sex, number of nurses interviewed, history of falls, wandering problem, disorientation to time, space or people, recall troubles, speech troubles, urinary and fecal incontinence, and ability to transfer.Sensitivity values according to specific residents' characteristics and other reported variables are given in Table The measurement of physical restraint use according to interview with members of the nursing staff and review of medical charts and nursing orders both reflect accurately the reality observed in long-term care setting residents. Our study has also shown that sensitivity and specificity values of the reported measurement are higher than those calculated from medical charts and nursing orders. This phenomenon is not surprising considering that the keeping of medical and nursing orders in nursing homes isn't usually done on a daily basis , as oppoThe current investigation was carried out in units of diverse facilities. The selection of these facilities was intended to allow the participation of subjects and care units of various characteristics as compared to other studies usually designed . The samLimitations of the current study must be taken into account when interpreting these findings. First, data were collected in 1992. Due to the implementation of the OBRA act, it is probable that the prevalence figures given in the current study are overestimations of those that would be observed in 2004. On the other hand, the province of Quebec just recently launched its first comprehensive policy on physical restraint use . FurtherIt is well known that the prevalence of residents with physical restraints is usually underreported since a social desirability bias tends to affect the validity of the information when the nursing staff has to declare the use of restraints . DespiteEven though other studies have observed an association between residents' characteristics and the risk of being restrained ,17,30,31Compared to review of clinical records, reported physical restraint use by interviewing nursing staff is a simple, efficient, and valid technique of collection of data regarding their use in nursing homes. No severe information bias was observed even though the use of physical restraints may be associated with poor quality of care. This method of measurement appears to be reliable and valid for research purposes. Moreover, our study provides support to the American initiative in regard to the monitoring of several outcomes in nursing homes through nursing staff reports -35. AccoThe authors declare that they have no competing interests.DL participated in the second line of statistical analyses, and drafted the manuscript. PV drafted parts of the document and contributed to the editing. RV contributed to the editing of the manuscript. PJD served as the Principal Investigator, designed the study, participated and oversaw field activity, revised and edited the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: