Algorithms and information, fundamental to technological and biological organization, are also an essential aspect of many elementary physical phenomena, such as molecular self-assembly. Here we report the molecular realization, using two-dimensional self-assembly of DNA tiles, of a cellular automaton whose update rule computes the binary function XOR and thus fabricates a fractal pattern—a Sierpinski triangle—as it grows. To achieve this, abstract tiles were translated into DNA tiles based on double-crossover motifs. Serving as input for the computation, long single-stranded DNA molecules were used to nucleate growth of tiles into algorithmic crystals. For both of two independent molecular realizations, atomic force microscopy revealed recognizable Sierpinski triangles containing 100–200 correct tiles. Error rates during assembly appear to range from 1% to 10%. Although imperfect, the growth of Sierpinski triangles demonstrates all the necessary mechanisms for the molecular implementation of arbitrary cellular automata. This shows that engineered DNA self-assembly can be treated as a Turing-universal biomolecular system, capable of implementing any desired algorithm for computation or construction tasks. Engineered DNA self-assembly to produce a fractal pattern demonstrates all the necessary mechanisms for the molecular implementation of arbitrary cellular automata This organization is information-based: DNA sequences refined by evolution encode both the components and the processes that guide their development into an organism—the developmental program. For a language to describe this carefully orchestrated organization, it is tempting to turn to computer science, where the concepts of programming languages, data structures, and algorithms are used to specify complex organization of information and behavior. Indeed, the importance of universal computation for autonomous fabrication tasks was recognized in von Neumann's seminal work on self-reproducing automata, where he postulated a y object . If algoany algorithm can in principle be embedded in, and guide, a potentially aperiodic crystallization process. In this “algorithmic self-assembly” paradigm, a set of molecular Wang tiles is viewed as the program for a particular computation or molecular fabrication task  function: at each time step, each cell is computed as the XOR of its two neighbors. Beginning with a row of all ‘0's punctuated by a single central ‘1,' snapshots of the cellular automaton's state at successive time steps may be stacked one on top of the other to produce a space–time history identical to Pascal's triangle  modulo 2Whereas execution of a cellular automaton occurs perfectly and synchronously, molecular self-assembly is asynchronous and may have many types of errors. To be successful, an implementation of cellular automata by molecular tiling must address four challenges: (1) The abstract tiles must be translated into molecules (molecular tiles) that readily form 2D crystals. (2) Molecular tiles must be programmed with specific binding domains that match the logic of the chosen abstract tiles. (3) The binding of molecular tiles must be sufficiently cooperative to enforce the correct order of assembly and prevent errors. (4) Assembly of molecular tiles must occur on a specified nucleating structure, and spurious nucleation must be suppressed. These properties are necessary and sufficient for implementing not only the XOR cellular automaton, but also any other 1D cellular automaton. All four have been shown individually: several types of DNA Wang tiles have been designed and shown to grow into micron-scale 2D periodic crystals ; the intPreventing the types of errors mentioned above may seem impossible. For example, if a single binding domain is strong enough to hold a tile in place  may associate at a given site at a rate,  to zero . For randization , this modization  predictsdization B.The effects of non-idealities can also be explored in this model. For example, mcG ≈ 2seG, which corresponds to the melting temperature of the crystals, such untemplated nucleation is inhibited by a kinetic barrier—the existence of a critical nucleus size  in principle can be controlled by slowing down the growth processes, making experimental investigations the appropriate next step.Abstract Wang tiles are implemented as DNA tiles according to the scheme described earlier : each moSince untemplated crystals were not expected to produce recognizable Sierpinski triangles, it was necessary to create a proper nucleating structure to provide the initial input for the algorithmic self-assembly. Previous work using DNA tiles to self-assemble an initial boundary had proven to be difficult , so in tThe DAO-E Sierpinski tile set B consistIn order for the nucleating structure for the DAO-E lattice to assemble onto a long PCR-generated nucleating strand, the tiles on the input row must be of the DAE-O variant. Further, we simplified the construction so that all nucleating strands contain the same repetitive sequence, but the input tile strands are doped with a fraction of strands containing a ‘1' sticky-end, and again the nucleating structure contains a few randomly located sites from which a Sierpinski triangle should grow.In principle, two approaches can be taken for initiating algorithmic self-assembly of DNA tiles. In the preformed tile approach, each tile is prepared separately by mixing a stoichiometric amount of each component strand in the hybridization buffer and then annealing from 90 °C to room temperature over the course of several hours. The nucleating structure is similarly prepared by annealing the nucleating strand with input tile and capping strands. Then the rule tiles and nucleating structure are mixed together at a temperature appropriate for crystal growth. In the bulk annealing approach, the nucleating strand, the capping and input tile strands, and the strands for all rule tiles are initially mixed together and then annealed. Since, at the concentrations we use, the tiles themselves have melting temperatures between roughly 60 °C and 70 °C while the crystals have a melting temperature within a few degrees of 40 °C , during Results for the DAE-E tile set are shown in Shown in S of possible states for the memory cells and an update function f : S × S → S, one can create a set of |S|2tiles according to the scheme of S|) to about 20 for the DAO-E and DAE-E tile designs used here, but this is already sufficient to implement several known universal Turing machines and cellular automata  A rigid or quickly straightening nucleating structure was simulated by setting iS = 4, so that near the crystal melting temperature where mcG ≈ 2seG, the border growth is strongly favorable. This was used for iS = 0.25 for the border tiles; in this case, near the melting temperature border growth requires stabilization by growth of rule tiles, resulting in faceted crystals. In combination with doubled concentrations of T-00 and T-11 (iS = 2), this case was used for The xgrow program simulates the kTAM for a set of square Wang tiles see , beginniThe strong effect of these variations may be seen in not seen for the T-11 tile, despite its increased concentration. This is because, regardless of what information is presented on the facet, there is no way to create a layer containing more than 50% T-11 tiles and no mismatches; T-01 or T-10 tiles must intervene. Thus the nucleation rate is substantially reduced, relative to T-00 nucleation on all-zero facets. This can be assessed in simulations by measuring the probability, p(L), that a T-00 tile will be found after L layers of growth from a facet. Simulations with parameters similar to p(L) ≈ 0.66−L/27e + 0.34 for all-zero facets, indicating strongly preferential nucleation, but for all other facets p(L) relaxes quickly to the asymptotic distribution. Simulations with parameters similar to p(L) relaxes to the asymptotic distribution immediately for every facet type investigated.Simulations confirm the preferential nucleation of T-00 tiles on all-zero facets when T-00 and T-11 concentrations are doubled . In contDesign of DNA Wang tiles occurs in three steps. First, the tile and lattice geometry must be determined. Here, the sizes (number of base-pairs) of each double-helical domain and sticky end, and other structural adornments such as contrast hairpins, are decided. These decisions impact the stability of each tile molecule, as the natural geometry of the DNA double-helix (10.5 bp for a full turn of B-form DNA)  constraiAt the second level, specific sequences must be chosen. The issue here is that we wish to prevent undesired associations between strands that might inhibit formation of the correct molecular structure. We used the heuristic principle of sequence symmetry minimization , 1990 toThe third level of design concerns variations. We conceptualize DNA Wang tiles as consisting of three modules: the sticky ends, the core helical regions, and adornments such as the hairpin structures that provide contrast for AFM imaging. A given double-crossover core can be given different sticky ends (reprogrammed) by replacing just one or two strands, thus allowing reuse of core designs to implement different tile sets. In our experience, the structural and thermodynamic stability of a given core is not significantly affected by changes in the sticky-end sequences. Similarly, using additional strands, a given core can be used with or without the hairpin adornments, which can be inserted at various locations. Although the hairpin adornments can affect the integrity of a DNA tile (e.g., strand dimers or other high molecular weight species), we have seldom found the undesired products to exceed 20% of the material.The core sequences for R-00 and S-00 are identical to the A and B tiles from a previous study . We usua2O (purified by a Milli-Q system, Millipore, Bedford, Massachusetts, United States) based on extinction coefficients estimated using a nearest-neighbor model (2+ buffer, as described in ±10%. This suggests that purification of tile complexes could result in cleaner self-assembly reactions and lower error rates.All oligonucleotides were synthesized by standard methods (Integrated DNA Technologies), PAGE purified, and quantitated by UV absorption at 260 nm in Hor model . DNA tilThe single-stranded nucleating strands were synthesized using a procedure based on Stemmer's assembly PCR . In asse+)*. The fraction of NRE subsequences is controlled by the amount of SplintNREUE2 and SplintNUERE2, which mediate the transitions into and out of the NRE sequence. (Here, we used these splints at one-fifth the concentration of other splints.) The NRE input tile outputs ‘0' and ‘1,' while the NUE input tile outputs ‘0' and ‘0'. To generate a different language, or a different distribution of sequences in the same language, a new assembly PCR must be run. Assembly reactions for DAO-E and DAE-E nucleating strands were designed using slightly different principles. The improved design used for DAO-E nucleating strands is simpler: A single periodic sequence is generated. The fraction of ‘1' sites is determined by the stoichiometry of input tile strands used in subsequent self-assembly reactions—strands A4SV and A4-S00 both assemble in the input tile in the same place, but one carries a ‘1' sticky end while the other carries a ‘0' sticky end. The approach used for DAE-E nucleating strands is more complex but more powerful for generating non-trivial input patterns. By having multiple splints that can overlap a given sequence, the assembly can be directed to non-deterministically choose one of several ways to extend a sequence. Thus, assembly PCR can be used to generate any regular language . In thisN types) is prepared without polymerase . To avoid mispriming events, the splints are annealed in the reaction mixture at 37 °C for 5 min. The polymerase (0.4 μl) is added and the reaction is subjected to an initial 72 °C extension step, followed by 40 cycles . In stage 2, 40 μl of new PCR Mix B (Mix A minus the splints, plus 0.4 μl of polymerase, water adjusted to 20 μl), is added to the first reaction volume and the reaction cycled for an additional 25 cycles . In stage 3, the 60 μl reaction volume is split into three 20 μl volumes, an additional 40 μl of Mix B is added to each and an additional 20 cycles  are performed. At this point, long double-stranded product should be formed. (We have observed that such products remain in the well of an agarose gel long after a 20 kb marker has entered the gel.) Also the dNTPs in the mixture are presumably nearly exhausted—specifically there is little dGTP left. (Any remaining dGTP will be used up early in stage 4.) In stage 4, to create single-stranded nucleating strands, 5 μl of the stage 3 product are mixed with 55 μl of fresh PCR mixture  for additional 60 cycles of the stage 3 program (94 °C for 15 s, 40 °C for 30 s, 72 °C for 70 s + 1 s/cycle). While addition of asymmetric primers at this stage might yield more single-stranded product, a satisfactory yield of single-stranded product results without doing so. After the final PCR, the reaction mixture is extracted with phenol:chloroform:isoamyl alcohol (Sigma, St. Louis, Missouri, United States), ethanol precipitated, and resuspended in purified water; the yield was estimated by UV absorbance. Typically, three 60 μl tubes of stage 4 product were pooled in a single recovery step and DNA was resuspended in 200 μl of water. Absorbance measurements of freshly resuspended material appeared unstable, perhaps because clumps of nucleating material scatter light. Long single-stranded DNAs may be prone to hydrolysis in water or strand-breakage upon freeze–thaw. However, after storage in water at 4 °C for a year, the nucleating structure still works well  MX 4000 real-time PCR instrument using a Perkin-Elmer  GeneAMP XL kit that uses rTth polymerase. In stage 1, a 20 μl reaction mixture containing 1 pmol total of splints , and began with preannealed samples at 15 °C, increasing to 80 °C over the course of several hours. Single-tile melts were superimposable with the reanneal from 80 °C back to 15 °C, indicating that equilibrium values were measured. Raw absorbance values were normalized. Whereas S-00 has a sharp melting transition  near 65 °C, the R-00-23J tile has a somewhat more gradual transition, which we attribute to the presence of the hairpin. Above 40 °C, the absorbance of the mixture equals the average absorbance of the individual tiles, indicating that crystals have completely melted by that point. Prior to the crystal melting transition between 36 °C and 40 °C, there is significant noise in the measurement, presumably due to light scattering.Melting temperatures for tiles and crystals were estimated based on UVth tiles . These t260 melts of all tiles; however, several other DAO-E and DAE-E tiles have similar transitions between 50 °C and 70 °C. Therefore we assume that the templated and untemplated Sierpinski crystals also melt at approximately 40 °C and that at that temperature, the DNA tiles are reasonably well formed.We have not performed UV2+ buffer (40 mM Tris–acetate [pH 8.0], 2 mM EDTA, 12.5 mM Mg2+), annealing from 90 °C to 20 °C at a rate of 1 °C/min (taking about 1 h). Longer annealing schedules  did not seem to decrease the error rate or the number of untemplated tubes or crystals.Self-assembly was performed by bulk annealing of all relevant rule tile, input tile, capping, and nucleating strands in a  50 μl volume of 1× TAE/MgDAO-E reactions contained nucleating strands sufficient to bind 0.004 μM of input tile (as estimated from binding capacity gels), 0.2 μM of each capping and input tile strand (A1S, A2, A3-nick, A4-S00, cpBr1, and 1/100 as much A4SV), and 0.2 μM of each rule tile strand (for each of the five or six tiles used). An excess of input tile strands was used to ensure complete coverage of the nucleating strand. The excess partial input tiles appeared not to significantly interfere with the self-assembly of algorithmic crystals.DAE-E reactions contained nucleating strands sufficient to bind 0.002–0.008 μM of input tile (as inferred from the estimated yield of the PCR), 0.2 μM of each capping and input tile strand (NRE1 to NRE4, NUE1 to NUE4, CapNRERE and CapNUERE), and 0.2 μM of each tile strand (for each of the four tiles used). Again, an excess of input tile strands was used to ensure complete coverage of the nucleating strand.2+ buffer on a Digital Instruments Nanoscope III (Veeco Metrology, Woodbury, New York, United States) equipped with a nano-Analytics Q-control III  and a vertical engage J-scanner, using the roughly 9.4 kHz resonance of the narrow 100 μM, 0.38 N/m force constant cantilever of an NP-S oxide-sharpened silicon nitride tip (Veeco Metrology). After self-assembly is complete, samples were prepared for AFM imaging by deposition of 5 μl onto a freshly cleaved mica surface (Ted Pella) attached by hot melt glue to a 15 mm metal puck; an additional 30 μl of buffer was added to both sample and cantilever (mounted in the standard tapping mode fluid cell) before the sample and fluid cell were positioned in the AFM head. The tapping amplitude setpoint, after engage, was typically 0.2–0.4 V, the drive amplitude was typically 100–150 mV, scan rates ranged from 2 to 5 Hz. Individual tiles are most clearly resolved for low amplitude setpoint and high drive amplitude values. However, under such conditions, the greatest damage is done to the sample and the hairpin labels are less distinct, sometimes disappearing entirely. Thus, to prevent damage to samples, amplitude setpoint was maximized and/or drive amplitude minimized subject to the constraint that tiles and their hairpin labels be visible.AFM imaging was performed in tapping mode under TAE/MgAfter acquisition, most images were flattened by subtracting a low-order polynomial from each scan line, or by adjusting each scan line to match intensity histograms. For some images see , bottom,Figure S1(57 KB PDF).Click here for additional data file.Figure S2(160 KB PDF).Click here for additional data file.Figure S3(126 KB PDF).Click here for additional data file.Figure S4(16 KB PDF).Click here for additional data file.Figure S5(21 KB PDF).Click here for additional data file.Figure S6(16 KB PDF).Click here for additional data file.Figure S7(22 KB PDF).Click here for additional data file.Figure S8(244 KB PDF).Click here for additional data file.Figure S9(22 KB PDF).Click here for additional data file.Figure S10(25 KB PDF).Click here for additional data file.Figure S11(53 KB PDF).Click here for additional data file.Figure S12(33 KB PDF).Click here for additional data file.Figure S13(234 KB PDF).Click here for additional data file.Figure S14(203 KB PDF).Click here for additional data file.Figure S15(226 KB PDF).Click here for additional data file.Figure S16(256 KB PDF).Click here for additional data file.Figure S17(234 KB PDF).Click here for additional data file.Figure S18(146 KB PDF).Click here for additional data file.Figure S19. Compiled Figures S1–S18(1.7 MB PDF).Click here for additional data file.Video S1Each frame is an average of three raw images. At the center is an amalgamation of many individual algorithmic crystals, each with its own characteristic pattern of tiles . While no large undamaged Sierpinski triangles were seen in this series of images, in some frames it is possible to see both double-helices within the tiles, as well as the major and minor grooves within the helices.(17.8 MB MPG).Click here for additional data file.


The prediction of ancestral protein sequences from multiple sequence alignments is useful for many bioinformatics analyses. Predicting ancestral sequences is not a simple procedure and relies on accurate alignments and phylogenies. Several algorithms exist based on Maximum Parsimony or Maximum Likelihood methods but many current implementations are unable to process residues with gaps, which may represent insertion/deletion (indel) events or sequence fragments.Here we present a new algorithm, GASP , for predicting ancestral sequences from phylogenetic trees and the corresponding multiple sequence alignments. Alignments may be of any size and contain gaps. GASP first assigns the positions of gaps in the phylogeny before using a likelihood-based approach centred on amino acid substitution matrices to assign ancestral amino acids. Important outgroup information is used by first working down from the tips of the tree to the root, using descendant data only to assign probabilities, and then working back up from the root to the tips using descendant and outgroup data to make predictions. GASP was tested on a number of simulated datasets based on real phylogenies. Prediction accuracy for ungapped data was similar to three alternative algorithms tested, with GASP performing better in some cases and worse in others. Adding simple insertions and deletions to the simulated data did not have a detrimental effect on GASP accuracy.GASP  will predict ancestral sequences from multiple protein alignments of any size. Although not as accurate in all cases as some of the more sophisticated maximum likelihood approaches, it can process a wide range of input phylogenies and will predict ancestral sequences for gapped and ungapped residues alike. Many eve.g. ,3). In oe.g. [Nevertheless, predicting ancestral sequences is not a simple procedure. It relies on a quality alignment plus an accurate – and correctly rooted – phylogenetic tree. Strict consensus methods are quick but can suffer from over-representation of larger clades of related sequences, which contribute more sequences to the consensus than more sparsely populated clades. Maximum Parsimony (MP) methods  overcomee.g. -8) and ie.g.  and FASTe.g.  provide e.g. .GASP  is an ancestral sequence prediction algorithm that is designed to handle gapped alignments of any size using a combination of MP and likelihood methods. Although probably not as accurate as some of the more sophisticated maximum likelihood approaches, it permits the estimation of ancestral states at residues that are gapped in any sequences of the alignment with comparable accuracy to that of ungapped residues.et al. 1992 [.GASP uses input from three sources: a multiple sequence alignment (MSA); an accompanying phylogenetic tree in Newick format ; and a Pal. 1992 . SequencGASP outputs an alignment in fasta format with both input terminal sequences and predicted ancestral node sequences. Ancestral sequences can either be grouped together at the end of the file or interspersed throughout the terminal sequences to reflect the tree topology Figure . Three tpX is the likelihood for a PAM distance of X , i is the ancestral amino acid at position r,j is the descendant amino acid at position r, pijX is the substitution probability of i to j in a PAMX matrix, and N is the number of residues in the alignment. Substitutions involving gaps are ignored in this calculation.where This allows a visual comparison between the branch lengths of the input phylogeny and the predicted branch lengths given the ancestral sequence predictions.r, GASP starts at the tips and works deeper into the tree, assigning a probability of a gap at each node n, which is equal to the mean probability of a gap at the descendant nodes:If the MSA has gaps, GASP will first assign gap status to every residue at every node. Insertions and deletions are assumed to be equally likely, although a gap is assigned in the case of a tied probability (below). For each residue p is the gap probability for residue r at node n. p1 and p2 are the gap probabilities for r at the two descendant nodes.where r is fixed as a gap, otherwise r is fixed as a 'non-gap'. GASP then works back up the tree from the root, this time using the new ancestral gap probability and both descendant gap probabilities to recalculate the gap probability:Terminal branches are given a probability of 1 if a gap is present or 0 if not. Once the root is reached, the gap status is fixed for the root. If the probability of a gap is greater than or equal to 0.5, residue p0 is the gap probability for r at the ancestral node.where r is fixed as a gap if p ≥ 0.5. This continues until all nodes are assigned as 'gap' or 'non-gap'.As with the root, r is assigned a probability for each amino acid at each node n. At the tips, r has a probability of 1 for the amino acid that is present in the MSA. GASP then works down the tree assigning probabilities based on the descendant nodes, branch lengths and a substitution matrix. By default, the PAM matrix of Jones et al. 1992 [1 matrix, which represents the probability that a given amino acid will be substituted by each other amino acid when the mean substitution rate is 1/100 residues. To make a PAMX matrix, which represents a length of evolutionary time where a sequence will have undergone X substitutions per 100 residues, the PAM1 matrix is multiplied by itself X-1 times:Once gaps are assigned, ancestral sequences are predicted in a similar fashion. Each residue al. 1992  is used.i is the ancestral amino acid,j is the descendant amino acid, k is the 20 possible transitory amino acids, pijX is the substitution probability of i to j in a PAMX matrix, pik(X-1) is the substitution probability of i to k in a PAM(X-1) matrix and pkj1 is the substitution probability of j to k in a PAM1 matrix.where r, the ancestral probabilities for each amino acid are calculated for the two descendant branches individually, using a PAMX matrix, where X is 100 times the branch length as substitutions per site, i.e. a branch of 0.1 substitutions per site would use a PAM10 matrix:Unless the ancestral node has a gap  at position pi is the probability of amino acid i at residue r of node n, pijX1 and pijX2 are the probabilities of substitution from amino acid i to each amino acid j in the appropriate PAM matrix for the two descendant branches, pdj1 and pdj2 are the probabilities of amino acid j being at position r at the two descendant nodes.where Once the root is reached, the most probable amino acid is fixed as the ancestral sequence. As with gaps, GASP then works back up the tree, using the fixed ancestral node amino acid and the descendant node probabilities to give new probabilities for each amino acid. The most probable amino acid is then fixed and the process continues until all residues and all nodes have a fixed sequence.GASP is primarily designed for reasonably small trees (6–30 sequences), although there is no limit on input tree size. For larger trees, probabilities for each amino acid get very small near the root, which can lead to a heavy bias towards the fixed ancestral amino acid when GASP works back up the tree. To counter this GASP arbitrarily reduces any probabilities below a certain exclusion threshold (0.05 by default) to zero, thus reducing the slow accumulation of very unlikely amino acids.To optimise the PAM matrices used for probability calculations, GASP uses the variable branch lengths read from the input phylogeny. There is also an option to fix the PAM distance used for all branches, which would allow the use of trees without branch lengths.n and its direct ancestor n0. This ancestor itself is heavily influenced by the descendants of n but also by the 'outgroup' to n, namely those sequences that are descendant to n0 but not to n. The outgroup information contained by the ancestral node n0 can be vital in determining the correct sequence for n when the descendants of n are variable. For this reason, the GASP algorithm will, by default, fix ancestral sequences as it moves back 'up' the tree from the root, increasing the relative weighting of the outgroup to the two descendants. Because there is a chance of the wrong amino acid sweeping back up the tree , there is an option to use amino acid probabilities from the ancestral node in the last stage of GASP rather than giving the fixed amino acid an ancestral probability of 1. This option should be used with caution.Assignment of ancestral amino acids with the GASP algorithm is achieved by combining data from the descendants of a given node To test the GASP algorithm, a number of artificial phylogenies were simulated. Because there is a practically limitless number of possible tree sizes (in both numbers of sequences and branch lengths) and phylogenies, it was decided to test the algorithm on a set of simulated phylogenies based on real phylogenies that formed a subset of those for which the algorithm was originally written. This set comprised 94 neighbour-joining trees of protein families. Each tree contained at least two subfamilies of at least 3 members each, giving in total between 6 and 127 sequences. Simulations started by creating a random protein sequence 100 amino acids long. Each residue was assigned an amino acid randomly as determined by the amino acid frequencies in all the human sequences of SwissProt-TrEMBL (Release 42) . Sequencet al. 1992 [Three substitution methods were used. In the first 'PAM Equal Rates' model, the PAM1 matrix of Jones al. 1992  was usedBecause one of the main advantages of GASP is its ability to deal with gaps, a second test dataset was generated from the 'PAM Equal Rates' set of trees, this time with gaps added. The addition of gaps was kept simple so that the exact same trees could be used for the gap analysis, allowing direct comparison of the results with gaps and without. (See Testing the GASP Algorithm, below.) To do this, gaps were limited to single insertion/deletion ('indel') events per column of the MSA, allowing them to overlay onto the existing simulated 'PAM Equal Rates' data. In addition, no indels occurring next to root were allowed as it is impossible to judge without an outgroup whether such an event would be an insertion or deletion.r of the simulated sequences was considered in turn and had a probability of 50% of containing an indel. Gaps were all of length 1 . Although unrealistic for testing multiple alignment or phylogeny reconstruction programs, such a simplification is not a problem for ancestral sequence prediction as each residue is treated independently. The short gaps meant that, for the same total number of gapped residues, there is a higher diversity in the phylogenetic positioning of the indels.To make the gaps, each residue T between the tip (age 0) and the oldest direct descendant node from the root. A random branch (not leading to root) is then selected for which the ancestral node is older than T and the descendant node is no older. This is the branch on which the indel occurred. The indel is randomly assigned as an insertion or deletion event with equal probability. If it is an insertion then the ancestral node plus all nodes outside the descendant clade have residue r replaced with a gap. If it is a deletion then the descendant node and all its descendants have residue r replaced with a gap.Indels were placed randomly with respect to evolutionary time. Each node in the simulated data has an 'age', which is the number of rounds of potential substitution it took to complete the simulation after that node was formed. Each indel occurs at a random age The simulated trees and alignments were run through the GASP algorithm. Because the 'real' sequence of each simulated node was known, it was then possible to determine the accuracy of GASP predictions. To test the different parts of the GASP algorithm, predictions were also made using modified GASP algorithms with parts of the model excluded.Because prediction for invariant sites is trivial for all methods, the expectation is that accuracy is inversely related to the number of variable sites. Therefore, comparisons of methods are presented as a percentage of the variable sites. In this context 'variable sites' are defined independently for each node as those sites for which not all descendant nodes (including termini) have the same sequence as the ancestral node.The simulated phylogenies are of different sizes. Considering all nodes of all trees would bias results towards the larger trees. To avoid this, each tree was arbitrarily reduced to four representative nodes:1. 'Root' = The root of the tree.2. 'Near Root' = A direct descendant node of the root.3. 'Mid Tree' = A random node approx. midway in the tree.4. 'Near Tip' = A direct ancestral node of a terminal sequence.et al. 1995 [et al. 2000 [To determine whether the GASP algorithm was useful its performance was compared to a crude consensus sequence at each node. Where two amino acids were present at equal frequencies in a column of the MSA, the most frequent amino acid in the total MSA was selected for the ancestral sequence. GASP may be considered crude compared to some existing Maximum Likelihood approaches and so its performance was also compared to that of both ML algorithms implemented by the CODEML program from the PAML package , namely al. 1995  and the al. 2000 . In addiThe GASP model marginally out-performs all methods tested for constructing the ancestral sequence at the root of the tree Figure . For allet al. 1995 and Pupko et al. 2000 performed better overall for internal nodes, this difference was not seen for every node of every tree. At each level, GASP is sometimes better and sometimes worse than all three other algorithms  using fixed PAM matrices rather than matrices derived from observed tree branch lengths.(b) fixing ancestral sequences on initial pass towards root without a second pass back up the tree.(c) no filtering of rare amino acid probabilities.(d) using ancestral probabilities when working back up the tree rather than fixed ancestral amino acids.Elements (a) and (b) were chosen for testing because they increase computational time, while (c) and (d) may not intuitively give the best results.For the phylogenies used in these simulations, all four variants performed worse than the standard GASP algorithm (data not shown). Using a fixed PAM distance for all branches rather than approximating the PAM distance using tree branch lengths (a) gives an unfair weighting to long branches and thus increases the probability of substitutions that are, in reality, unlikely. Fixing ancestral sequences on the way 'down' the tree to the root (b) does not use any outgroup information and is therefore significantly worse at distinguishing between two or more amino acids with similar ancestral probabilities. Less intuitive is the effect of reducing low amino acid probabilities to zero (c) and using fixed ancestral sequences when recalculating amino acid probabilities using all three connected nodes (d). Indeed, excluding these two elements have a much smaller effect but still reduce the overall accuracy of the algorithm (data not shown).i.e. combining (c) and (d)).Using fixed amino acids when working back up the tree increases the influence of the outgroup sequence. As was seen by the difference in accuracy between predictions at the root and nodes near the root Figure , outgroui.e. not correcting for multiple substitutions.) This is not testing the GASP algorithm per se but does provide information on the importance of using an accurate phylogeny construction algorithm. (The PAML package does not require pre-defined branch lengths and is therefore only affected by errors in supplied topology and not in branch lengths.) In many cases there was no difference. However, nearer to the root, using observed branch lengths rather than the real ones decreased prediction accuracy slightly. This decrease was correlated with total tree age (data not shown). This would imply that branch lengths corrected for multiple substitutions should be used for trees fed into the GASP algorithm, particularly with deep trees containing long branches.A final test was performed to compare the use of 'real' versus 'observed' branch lengths.  To analyse the effect of gaps on prediction accuracy, pairwise comparisons were made between the gapped datasets and the corresponding ungapped simulations Figure . As woulAlthough explicitly designed for use with protein sequence alignments and trees, it is relatively simple to convert GASP for use with nucleotide datasets. To do this, a new 'PAM matrix' should be created with substitutions probabilities for A, C, G and T only. This structure would allow the user to fit fairly complex substitution models, with different substitution probabilities for each pair of nucleotides. If the aligned sequence is coding DNA, however, it is highly recommended to use the protein sequences or a different algorithm such as those in the PAML package , as the We have presented an algorithm for predicting ancestral sequences in gapped datasets. At the root of the tree, GASP marginally outperforms three existing algorithms implemented in the PAML package. For other nodes of the tree, however, the ML algorithms of CODEML -7 generaFor real life datasets, as for all evolutionary studies, predictions are dependent on the quality of input alignments. Gapped residues are, by their nature, often located in regions of evolutionary instability and therefore the interpretations of predictions at such sites require extra care. In many scenarios, however, gaps are introduced into alignments by the missing termini of fragment sequences. In these situations, the complete sequences that form the rest of the alignment may be very well aligned and so it is highly desirable to have an algorithm that can process the gaps introduced by the truncated sequences.Project name: GASP Project home page: Operating system(s): Platform Independent. (Tested on PC (Windows 98/XP) and UNIX (Red Hat Linux 7.3))Programming language: Perl.Other requirements: None.License: None.Any restrictions to use by non-academics: Author's permission required.GASP. Gapped Ancestral Sequence Prediction.Indel. Insertion or deletion event.ML. Maximum Likelihood.MP. Maximum Parsimony.MSA. Multiple Sequence Alignment.PAM. Point Accepted Mutation.RE conceived the algorithm, coded the Perl script, designed and performed the accuracy tests and statistical analyses, designed the phylogeny simulation method, generated the simulated datasets and drafted the manuscript. DS helped in the design of test simulations and in drafting the manuscript.


Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or “noise.” Several recent studies in  Analysis of gene expression data for nearly every gene in yeast provides evidence that random variation in the production rate of proteins could significantly affect the fitness of an organism Stochasticity is a ubiquitous characteristic of life. Such apparent randomness, or “noise,” can be observed in a wide range of organisms, resulting in phenomena ranging from progressive loss of cell-cycle synchronization in an initially synchronized population of microbes to the pattern of hair coloration in female calico cats. An important source of stochasticity in biological systems is the random noise of transcription and translation, which can result in very different rates of synthesis of a specific protein in genetically identical cells in essentially identical environments .Understanding how stochasticity contributes to cellular phenotypes is important to developing a more complete picture of how cells work. Accordingly, noise in gene expression and other cellular processes has been a major focus of research for more than a decade. While several cases have been described where stochasticity is advantageous  maxTo test this prediction, we estimated protein production rates . Similar results were found when using different numbers of bins, when using halves or quartiles instead of thirds, or when separating bins by transcription rate instead of by number of translations per mRNA (data not shown). This result cannot be explained by the overall positive correlation between dispensability and rate of protein synthesis. In the first of these two methods, we binned yeast genes by their protein production rate, so that all genes in each of 15 bins had approximately equal levels of protein production see A. A Fishf, where f = 0 indicates no effect on growth when a gene is deleted, f = 1 indicates that a gene is essential, and 0 < f < 1 indicates a quantitative growth defect [f versus transcription [txn] rate | protein production rate, Spearman partial r = 0.282, n = 4,746, p = 10−87; f versus translations [tlns] per mRNA | protein production rate, Spearman partial r = −0.258, n = 4,746, p = 10−75). We also expected that the relationship between gene importance and implementation of the expression strategy that minimizes noise could additionally be seen by considering transcription rate and translation rate per mRNA together, as a ratio; a large ratio of transcription rate to translations per mRNA would indicate that transcripts are produced quickly but are translated slowly, corresponding to our expression strategy 1. Confirming this, the correlation between fitness effect and the ratio of transcription rate to translations per mRNA (controlling for protein production rate) is highly significant . Partial correlation analysis is thus in accordance with the trend illustrated in Because binning genes still allows for a small amount of variability in protein production within each bin see , we sough defect ) would cp ≤ 0.02) for all but one bin. As in In addition to essential genes, genes whose protein products participate in stable protein complexes (“complex subunits”) would also be expected to exhibit sensitivity to randomness in expression: producing too little or too much of a single protein complex subunit can compromise the proper assembly of the entire complex and waste the energy invested in the production of the other complex subunits. In support of this, it has been found that both under- and overexpression of complex subunits is more likely to result in a reduced growth rate or inviability of yeast than is misexpression of other genes, and also that complex subunits tend to be more precisely coexpressed with other genes than noncomplex subunits . Using dr = 0.203, n = 4,900, p = 10−46) and a low number of translations per mRNA . Using the ratio of transcription rate to translations per mRNA also yielded similar results . Thus, partial correlations confirm the finding illustrated in When we repeated the partial correlation analysis for complex subunits , we found similar results. When total protein synthesis was controlled for with the partial correlation, complex subunits were more likely to have a high transcription rate , suggesting that fitness effect and protein complex membership are independently associated with the expression strategy that minimizes stochastic fluctuation.  Repeating the partial correlations above with either transcription rate or translations per mRNA in place of their ratio gave significant partial correlations with both fitness effect and protein complex membership as well (data not shown).Since proteins that participate in many protein–protein interactions are more likely to be essential , it was r = −0.11, p = 10−9) between an mRNA's rate of decay and the evolutionary rate of the protein it encodes. This correlation was surprising, as it is precisely the opposite of what one would expect if the relationship between the rates of mRNA decay and protein evolution were mediated by the level of expression: slow decay would result in increased expression, which is known to be associated with slow evolution (p i= 10−36 by the Wilcoxon test [The hypothesis that genes of large fitness effect are under stronger selection to reduce stochastic fluctuation in expression provides an explanation for a previously observed, but as yet unexplained, correlate of protein evolutionary rate. volution . Thus, wvolution . (While xon test ).We found that noise in protein production is minimized in genes for which it is likely to be most harmful, specifically essential genes and genes encoding protein complex subunits. This finding supports the hypothesis that noise in gene expression is generally deleterious to yeast.Yeast appear to control the noise in their gene expression at both transcriptional and translational levels preferentially for some genes; however, this noise minimization is not without a cost, as the high transcription and high mRNA decay rates that are needed to minimize noise are energetically expensive and are thus expected to be advantageous only when the benefit of reducing noise in a particular gene's expression outweighs this cost . ProteinAs is the case with many genome-wide studies, it is possible that a hidden variable could bias our results. For example, it is possible that essential genes and genes encoding protein complex subunits tend to have high transcription and low translation for reasons unrelated to noise minimization. However, until such a reason is identified, the most parsimonious interpretation of our results is that yeast adaptively minimize noise in the expression of certain genes.Escherichia coli  * A/T, where R is transcription rate, A is mRNA abundance, and T is mRNA half-life. Translation rates per mRNA in rich glucose medium were calculated from ribosome occupancy data by Transcription rates were calculated from mRNA abundances and decay rates in log-phase yeast growing in rich glucose medium  accordinFitness effect ranks were calculated from 12 replicate growth experiments for all viable homozygous yeast deletion strains in rich glucose medium; growth experiments were conducted using the method described in ADH1*) was 10-fold weaker than the other two at full induction, but all three showed very similar relationships between noise strength and percent transcriptional induction. Since we do not have genome-wide data for the percent induction for genes in rich glucose medium (or any other environment), in our analysis we make the assumption that the promoters of more highly transcribed genes tend to be at higher percent induction levels. While this certainly does not hold for all genes, we believe that it is a reasonable approximation for most genes.XYr be the correlation coefficient between variables X and Y. To control for a third variable Z,Partial correlations were calculated as described by t distribution, by the equationTo assess the significance of the partial correlation, it is transformed to be distributed according to a Student's p-value can then be calculated according to where the t-value falls with respect to its expected distribution.The two-sided Figure S1–49).Fitness effect ranks are shown on the y-axis . Protein production rate (proteins/s) is shown on the x-axis. The Spearman rank correlation coefficient is r = –0.202 (p = 10(316 KB PPT).Click here for additional data file.Table S1(37 KB DOC).Click here for additional data file.


Relatively little is known about interest in pediatric pulmonology among pediatric residents. The purpose of this study, therefore, was to determine at this institution: 1) the level of pediatric resident interest in pursuing a pulmonary fellowship, 2) potential factors involved in development of such interest, 3) whether the presence of a pulmonary fellowship program affects such interest.A questionnaire was distributed to all 52 pediatric residents at this institution in 1992 and to all 59 pediatric residents and 14 combined internal medicine/pediatrics residents in 2002, following development of a pulmonary fellowship program.Response rates were 79% in 1992 and 86% in 2002. Eight of the 43 responders in 1992 (19%) had considered doing a pulmonary fellowship compared to 7 of 63 (11%) in 2002. The highest ranked factors given by the residents who had considered a fellowship included wanting to continue one's education after residency, enjoying caring for pulmonary patients, and liking pulmonary physiology and the pulmonary faculty. Major factors listed by residents who had not considered a pulmonary fellowship included not enjoying the tracheostomy/ventilator population and chronic pulmonary patients in general, and a desire to enter general pediatrics or another fellowship. Most residents during both survey periods believed that they would be in non-academic or academic general pediatrics in 5 years. Only 1 of the 106 responding residents (~1%) anticipated becoming a pediatric pulmonologist.Although many pediatric residents consider enrolling in a pulmonary fellowship (~10–20% here), few (~1% here) will actually pursue a career in pediatric pulmonology. The presence of a pulmonary fellowship program did not significantly alter resident interest, though other confounding factors may be involved. The specialty of pediatric pulmonology is relatively new, having been recognized as a pediatric sub-specialty by the American Board of Medical Sub-specialties in 1984. In 1997, there were approximately 500 board certified pediatric pulmonologists in the United States and Canada . It has This study involved the distribution of a questionnaire to all pediatric residents. The questionnaire was initially distributed in 1992 prior to institution of a pulmonary fellowship. The questionnaire was placed in the hospital mailbox of each resident. The study was repeated (and the questionnaire redistributed) in 2002 after the fellowship, which began in 1994, had been functioning for several years. To improve the response rate, the questionnaire was distributed twice, one month apart, during each time period. The questionnaire was a three-page, 18 question form that took approximately 15 minutes to complete (copy enclosed in Appendix see ). The quThe questionnaire was distributed to 52 pediatric residents (including 2 chief residents) in 1992 and to 59 pediatric residents (including 3 chief residents) and 14 medicine/pediatric residents (including 1 chief resident) in 2002. A medicine-pediatric residency program did not exist during the initial distribution period. To avoid compromising confidentiality in the relatively small medicine/pediatric group, residents were not asked to list their residency program in 2002 and consequently the 2 resident groups during that period were combined. Forty-three of the 52 residents completed the survey in 1992 (79%) compared to 63 of 73 (86%) during 2002 (p = NS).Of the 43 respondents in 1992, 30 (70%) had considered doing a fellowship in any pediatric subspecialty and of those, 15  believed they were "very likely" to do a fellowship. Of the 63 respondents in 2002, 40 (63%) had considered doing a pediatric fellowship and 16 of those  were "very likely" to continue with fellowship training. These numbers compare with 9 of the 52 residents in 1992 (17%) who actually completed fellowship training compared to 16 of the 47 graduating residents from the 2002 survey (34%) who began fellowship training in 2003 or 2004.Eight residents 19%) in 1992 had considered a pulmonary fellowship compared to 7 residents (11%) in 2002 (p = NS). Table 9% in 199Table The last question in the survey asked residents what they thought they would be doing 5 years in the future. These responses are shown in Table This study found that a significant percentage of pediatric residents considered doing a pulmonary fellowship after their residency training, ranging from 11% in the 2002 group to 19% in the 1992 group. However, these residents also viewed themselves as less likely to enter general pediatrics perhaps suggesting that they were simply considering several pediatric subspecialties at some time during their residency training. This seems likely, as the highest scored factor by the +PF residents was the desire to continue their education after residency. Despite fairly high percentages of residents considering a pulmonary fellowship, only 1 resident in the entire group (~1%) actually believed that they would be a pediatric pulmonologist 5 years after the survey was completed. This percentage is very similar to that of first-time takers of the 1995 General Pediatrics Certifying Examination who believed they would be a pediatric pulmonologist in the future (1.1%) . If one The majority of residents who considered doing a pulmonary fellowship (8 of 15) were in their first year of residency. From personal experience, residents often consider various practice options early on in their training and frequently do not tend to narrow their choices until their second or third year of residency. This observation should be kept in mind when trying to recruit residents for pulmonary fellowship positions by seeking out those residents potentially interested in Pulmonology early on in residency rather than later.The ranking of factors that may contribute to an interest in a pulmonary fellowship were remarkably similar during the 2 time periods. The only score that approached a statistically significant difference was the statement "I enjoy the tracheostomy/ventilator population" and this score tended to increase in 2002. However, a dislike for the tracheostomy/ventilator population also received the highest score among those residents not interested in a pulmonary fellowship and was even higher than both the desire to enter general pediatrics and another fellowship program. These data may simply be a center phenomenon but might suggest a more global "disinterest" in this patient population that may need to be further studied. Two scores in the -PF resident group decreased from 1992 to 2002: not enough pulmonary patient experience to decide on a pulmonary fellowship and too few perceived pulmonary job openings. The first may be a positive reflection on the local resident experience with pulmonary patients in recent years or on the fellowship program itself though this is only speculative. The second received very low scores during both periods and may not be very relevant. However, there has been some evidence of a reversal in the prior trend of residents entering general pediatrics in recent years, suggesting greater interest among residents in fellowships . On the This study has certain shortcomings. This study asked residents to score specific factors that may or may not have been relevant to an individual resident. Although residents were given the opportunity to add personal comments, few did. In addition, certain patient populations, e.g those with respiratory infections, were not included as options in the survey and these omissions could cause study bias. Other factors that may contribute to residents' decisions regarding fellowship training were not addressed in this study. These factors include resident teaching by the faculty, resident gender, spouse occupation, mentor encouragement or other personal reasons -12. In aAlthough many pediatric residents consider enrolling in a PF (~10–20% here), few (~1% here) will actually pursue a career in pediatric pulmonology. The presence of a PF program did not significantly alter resident interest, though other confounding factors may be involved.BPD: bronchopulmonary dysplasiaPF: pulmonary fellowship+PF: those residents who considered taking a pulmonary fellowship-PF: those residents who had not considered taking a pulmonary fellowshipNone declared.The pre-publication history for this paper can be accessed here:Appendix. Resident questionnaireClick here for file


The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation. Cycles of Tat acetylation and deacetylation, mediated by human sirtuin 1 (SIRT1), regulate HIV transcription suggesting that SIRT1 could be a therapeutic target The Tat protein of human immunodeficiency virus 1 (HIV-1) is essential for the transcriptional activation of the integrated HIV-1 provirus. Without Tat, HIV transcriptional elongation is inefficient and results in abortive transcripts that cannot support viral replication . Tat is trans-acting responsive element (TAR), an RNA stem-loop structure that forms at the 5′ end of all viral transcripts [Tat activates HIV transcription through the nscripts ,4. The Tnscripts .We and others have shown that Tat is acetylated at lysine 50 by the transcriptional coactivators p300 and human GCN5  ,8,9,10. Our current working model is that Tat acetylation disrupts the Tat/TAR/cyclinT1 complex and leads to the transfer of Tat and PCAF to the elongating polymerase. According to this model, both forms of Tat, unacetylated and acetylated, play distinct roles in the HIV promoter transcriptional cycle and lead to the sequential recruitment of the cofactors cyclinT1 and PCAF. Because of the limiting amounts of Tat protein in the early stages of HIV infection and the critical role of unacetylated Tat for pTEFb recruitment to TAR, the question arises whether Tat acetylation can be reverted via a cellular Tat deacetylase.There are three distinct classes of human histone deacetylases (HDACs) based on their homology with yeast transcriptional repressors. Class I and II HDACs are homologous to the yeast proteins Rpd3p (reduced potassium dependency 3) and Hda1p (histone deacetylase A1), respectively ,15. The +) as a cofactor [I68 (Tata box-binding protein-associated factor I of 68 kDa) [Class III HDACs, also named sirtuins (SIRTs), are homologous to the yeast transcriptional repressor silent information regulator 2p (Sir2p) . Sir2p icofactor ,18,19. Scofactor . Despitecofactor ,22,23, T 68 kDa) , PCAF an 68 kDa) , p300 [2 68 kDa)  and Fork 68 kDa) ,27, the  68 kDa) , and the 68 kDa) . The cyt 68 kDa) ,32.Here, we identify the class III HDAC SIRT1 as a specific Tat deacetylase and demonstrate that SIRT1 is a novel cofactor necessary for efficient Tat-mediated transactivation of the HIV promoter.To test the ability of SIRT1–7 to deacetylate Tat in vitro, we transfected HEK 293 cells with expression vectors for human SIRT1–7 and immunoprecipitated the FLAG-tagged proteins A. The im+ and completely inhibited by nicotinamide, an inhibitor for class III HDACs [SIRT2 and SIRT3 proteins are localized primarily in the cytoplasm and the mitochondria ,31, and II HDACs ,35. TSA,To test whether Tat and SIRT1 interact, Tat/FLAG and SIRT1/influenza hemagglutinin (HA) were overexpressed in HEK 293 cells, and cellular lysates subjected to coimmunoprecipitation assays. Tat was detected with an α-FLAG antiserum in material immunoprecipitated with SIRT1 by the α-HA antibody in cells transfected with SIRT1- and Tat expression vectors, but no signal was obtained when SIRT1 or Tat alone was expressed (IP: α-HA in To test whether Tat and SIRT1 interact directly, increasing amounts of biotinylated synthetic Tat (72 amino acids) were incubated with recombinant full-length SIRT1. After pulldown with streptavidin-conjugated agarose, SIRT1 coimmunoprecipitated with Tat in a dose-dependent manner D. RecombThe effects of SIRT1 on Tat function were assessed after transfection into HeLa cells. SIRT1 modestly, but reproducibly, enhanced Tat-mediated transactivation of an HIV promoter luciferase construct -mediated knockdown of SIRT1. HeLa cells were transfected with double-stranded RNA oligonucleotides directed against SIRT1 or against firefly luciferase expressed from the pGL3 vector as a control. All luciferase reporter constructs described in this study are based on the pGL2 vector, which is not affected by GL3 siRNAs (siRNAs directed against firefly luciferase expressed from the pGL3 vector) . Levels +/+ MEFs   derived from SIRT1 knockout mice . The HIV+/+ MEFs A. In con−/− MEFs A. Ectopi−/− MEFs B. In cond VP16]) C. These This model was further tested in nuclear microinjection experiments using synthetic full-length Tat and AcTat. Microinjection of increasing amounts of either Tat or AcTat proteins into HeLa cells caused a marked transactivation of the HIV LTR luciferase reporter in a dose-dependent manner (Wt TAR in According to our working model, AcTat represents a second step in the transactivation cycle . Since AS. cerevisiae Sir2p protein [50 (concentration causing 50% inhibition) of less than 5 μM  open reading frame in place of the viral nef gene  [env gene. Viral particles were produced by cotransfection with a construct expressing the glycoprotein of the vesicular stomatitis virus (VSV-G). Jurkat T cells were incubated with viral supernatant for at least 18 h, washed to remove extracellular virus, and treated with HR73 (1 μM) or dimethyl sulfoxide (DMSO), as a control. We observed that HIV gene expression was reduced 5-fold in cells treated with HR73 as measured by GFP expression (HIV GFP in To examine the effect of HR73 on HIV infection, we generated infectious HIV particles using a molecular clone of HIVisolate) . To rest+-dependent class III protein deacetylase, as a Tat deacetylase and regulator of Tat activity. Tat and SIRT1 coimmunoprecipitate and synergize to activate the HIV promoter. Conversely, knockdown of SIRT1 via siRNA inhibits Tat-mediated transactivation of the HIV LTR. Tat transactivation is defective in SIRT1-null MEFs and can be rescued by expression of SIRT1.Our previous work documented that Tat acetylation by p300 at lysine 50 is a necessary step in Tat-mediated transactivation and leads to the recruitment of PCAF to the elongating polymerase. We now report the identification of SIRT1, an NADRecent observations indicate that SIRT1 can regulate transcription via histone-dependent and -independent mechanisms . Since SSIRT1 also deacetylates nonhistone factors, including the tumor suppressor p53 ,22,23. DInterestingly, HDAC1 (a class I HDAC) has also been reported to deacetylate p53 , but theWe have observed that Tat acetylation at lysine 50 is not specifically enhanced in response to TSA . These rAnother system that offers intriguing parallels to our observation is the role of the SIRT1/MyoD/PCAF complex in myogenesis . In thisIn most identified sites of action, SIRT1 showed a negative activity on transcriptional regulatory mechanisms, resulting in an inhibition of target gene expression. In contrast, we have identified a positive regulatory activity of SIRT1 on Tat-mediated transactivation. According to our working model, Tat becomes acetylated at the level of the HIV promoter after binding to TAR. The acetylation of the Tat ARM can potentially affect other biological activities associated with this domain, including RNA binding, nuclear or nucleolar import, and protein stability. Tat acetylation could also be involved in the delivery of Tat to the transcription complex, and/or might moderate interactions with cyclinT1/CDK9 prior to transcription. However, we provided evidence that Tat acetylation leads to a dissociation of Tat from cyclinT1 and TAR and to its transfer to the elongating polymerase . AcetylaAn unresolved question raised by this model is the fate of acetylated Tat at the end of the transcription cycle. Since acetylated Tat cannot recruit cyclinT1 to the TAR element, a new transcription cycle would require either the synthesis of a new unacetylated Tat protein or the deacetylation of Tat. Tat concentrations are limiting early in the virus life cycle since Tat expression is under the control of the basal LTR. Tat recycling is therefore likely to represent a rate-limiting step in HIV transcription early during the replicative cycle of the virus. Experiments presented in this manuscript demonstrate that SIRT1 catalyzes this rate-limiting step in HIV transcription.+ as a cofactor. NAD+ and its reduced form NADH serve as cofactors in many metabolic and stress reactions involving oxidation and reduction. Changes in the cellular NAD+/NADH ratio may regulate SIRT1 enzymatic activity, Tat transcriptional activity, and HIV replication and pathogenesis.These observations raise a number of additional questions. The demonstration that Tat transactivating activity is controlled by SIRT1 connects HIV transcription with the metabolic state of the cell. The HDAC activity of SIRT1 requires NADThe site of Tat deacetylation is unknown at this point. We hypothesize that the deacetylation could be triggered by molecular events linked with the end of the transcriptional elongation process. Deacetylation of Tat by SIRT1 could lead to its dissociation from the elongating polymerase and from PCAF. The previous demonstration that PCAF and SIRT1 can be coimmunoprecipitated from cells supports this model .S. cerevisiae Sir2 enzymatic activity, did not inhibit the mammalian sirtuins, we screened several compounds structurally related to splitomicin for their ability to inhibit the mammalian sirtuins. This process led to the identification of HR73, an inhibitor that suppresses SIRT1 enzymatic activity in vitro at low micromolar concentrations. This inhibitor induced p53 hyperacetylation in vivo (unpublished data) and significantly decreased HIV transcription. These results validate SIRT1 as a novel therapeutic target for HIV infection. Future efforts will analyze in additional molecular detail the role of Tat acetylation and deacetylation in HIV transcription and replication.The identification of SIRT1 as an enzyme that catalyzes a rate-limiting step in HIV transcription suggests that it could be targeted therapeutically. Since our preliminary experiments indicated that splitomicin, an inhibitor of the ΔNF-κB-luciferase, the full-length (101 amino acid) CMV-Tat/FLAG and CMV-Tat/T7 expression vectors [HeLa, HEK 293, and Jurkat cells (obtained from the American Type Culture Collection, Manassas, Virginia, United States), and wild-type or Sir2α knockout MEFs  were mai vectors , the ful vectors , the ful vectors , the Gal vectors , the hum vectors , as well vectors  were pre223→A) forward, 5′-GGTTAGACCAGAACTGAGCCTGGGAGC-3′ and reverse, 5′-GCTCCCAGGCTCAGTTCTGGTCTAACC-3′. The TAR Δloop mutation (C230→A, T231→G, and G234→T) was generated with the following primers. Forward, 5′-GGTTAGACCAGATCTGAGCAGGGTAGCTCTCTGGCTAACTAGGG-3′ and reverse, 5′-CCCTAGTTAGCCAGAGAGCTACCCTGCTCAGATCTGGTCTAACC-3′.The SIRT1 cDNA was subcloned to generate a C-terminal HA-tagged fusion in a derivative pcDNA 3.1 (+) backbone (HA vector) by standard PCR-based strategies. The mutant CMV-Tat/FLAG expression vector TatK50R was generated by site-directed mutagenesis with the following primers. Forward, 5′-CCTATGGCAGGAGGAAGCGGAGACAGCG-3′ and reverse, 5′-CGCTGTCTCCGCTTCCTCCTGCCATAGG-3′. The mutant HIV LTR luciferase constructs (nucleotides 1–791) were generated by site-directed mutagenesis with the following primers. TAR Δbulge (TThe CMV-luciferase construct was generated by cloning the firefly luciferase gene as a HindIII/BamHI fragment obtained from pGL2 Basic (Promega, Madison, Wisconsin, United States) into pcDNA3.1 . The CMV-GFP expression plasmid is commercially available .Synthesis of 72-amino acid Tat proteins was as described ,13. HR732, and 0.2 mM DTT). The beads were resuspended in 100 μl of SIRT deacetylation buffer containing 1 μg synthetic Tat (72 amino acids) carrying an N-terminal biotin label and an acetyl group at position 50 [+ (1 mM), reactions were incubated for 2 h at room temperature with constant agitation. Reactions were stopped by the addition of SDS loading buffer, boiled, and after brief centrifugation, analyzed by WB with rabbit α-AcTat antibodies [Human SIRT1–7 FLAG-tagged plasmids were transfected in HEK 293 cells with Lipofectamine reagent (Invitrogen). Cells were lysed 24 h after transfection with lysis buffer (50 mM Tris-HCl [pH 7.5], 0.5 mM EDTA, 0.5% NP40, and 150 mM NaCl) in the presence of protease inhibitors . Equal amounts of total proteins were immunoprecipitated with α-FLAG M2 agarose (Sigma, St Louis, Missouri, United States), for 2 h at 4 °C. Immunoprecipitated material was washed twice with IP buffer and one time with SIRT deacetylation buffer (50 mM Tris-HCl [pH 9], 4 mM MgClition 50 . Reactiotibodies  or SA-HR+ (Sigma) and enzymatically [3H]-acetylated histone H3 peptide (amino acids 1–24) [+ for 10 min at room temperature prior to the initiation of the reaction by addition of NAD+ (1 mM).The histone deacetylation assay with recombinant SIRT1  was performed as described previously for SIRT2  in 100 μds 1–24) . Splitom2PO4 (pH 7.5), 5 mM EDTA, 30 mM sodium pyrophosphate, 10 mM NaF, and protease inhibitors . Duplicates were pooled, and 1 mg of lysate was immunoprecipitated either with monoclonal α-HA  together with protein G-Sepharose (Amersham Biosciences, Piscataway, New Jersey, United States) with α-FLAG M2 agarose (Sigma) or α-T7-agarose (Amersham Biosciences) for 2 h at 4 °C. Beads were washed three times in lysis buffer, boiled in SDS loading buffer, and analyzed by WB with polyclonal α-FLAG (Sigma), monoclonal α-HA (Roche), or monoclonal α-T7 (Novagen, Madison, Wisconsin, United States) antibodies. For the IP of Tat with endogenous SIRT1, HEK 293 cells were transfected only with CMV-Tat/FLAG or the CMV-empty vector using Lipofectamine reagent. Cell lysates were immunoprecipitated with rabbit α-SIRT1 antibodies (generated against amino acids 506–747) together with protein G-Sepharose (Amersham Biosciences). Immunoprecipitated material was analyzed by WB with the M2 α-FLAG antibody (Sigma) or rabbit α-SIRT1 antibodies.HEK 293 cells were cotransfected in duplicate with expression vectors for CMV-Tat/FLAG; CMV-Tat/T7 or CMV-TatK50R/FLAG; and the SIRT1/HA or SIRT1-, SIRT2-, and SIRT6-FLAG expression vectors or the respective empty vector controls using Lipofectamine reagent (Invitrogen). Cells were lysed after 24 h in 250 mM NaCl, 0.1% NP40, 20 mM NaHFor in vitro interactions, 10 U of recombinant SIRT1 (Biomol) was incubated with biotinylated synthetic Tat or acetylated Tat proteins (0, 0.25, 1, and 4 μg) together with streptavidin-Sepharose (Amersham Biosciences) in lysis buffer in the presence of 5 mM nicotinamide (Sigma) for 3 h at 4 °C. Pelleted beads were washed three times in lysis buffer, resuspended in SDS loading buffer, and analyzed by WB with polyclonal α-SIRT1 antibodies, rabbit α-AcARM, or SA-HRP (Jackson Immunoresearch Laboratories).Double-stranded siRNAs directed against nucleotides 408–428 in the SIRT1 mRNA or control GL3 siRNAs (both Dharmacon Research, Lafayette, Colorado, United States) were transfected into HeLa cells plated in six-well plates with Oligofectamine reagent according to the manufacturer's guidelines (Invitrogen). The mutant SIRT1 siRNA was identical to SIRT1 siRNA except for a two-nucleotide mismatch between the target mRNA for SIRT1 and the antisense strand of siRNA at nucleotides 418 and 419. After 48 h, cells were retransfected with the HIV LTR luciferase construct (200 ng) together with increasing amounts of CMV-Tat expression vectors  and corresponding amounts of empty pcDNA3.1 vector (Invitrogen). In the control experiment, CMV-Tat was replaced by the CMV-luciferase construct, and HIV LTR luciferase was replaced by an HIV LTR promoter construct driving the expression of chloramphenicol acetyl transferase (CAT) ). Cells ΔNF-κB-luciferase reporter (200 ng) and increasing amounts of RSV-Tat (0, 2, 20, and 200 ng) using the Lipofectamine reagent (Invitrogen). In the control experiment, RSV-Tat was replaced by RSV-luciferase (200 ng), and the HIV LTR luciferase construct was replaced by the HIV LTR CAT reporter. In transfections with HR73, HeLa cells were cotransfected with the HIV LTR luciferase reporter (200 ng) and RSV-Tat expression vectors (0, 20, and 200 ng) or the empty vector using Lipofectamine reagent. The RSV-luciferase construct was used as described above. After 4 h incubation with the DNA/Lipofectamine mix, the culture medium was changed and supplemented with indicated concentrations of HR73 dissolved in DMSO or DMSO alone. Cells were harvested 8 h later and processed for luciferase assays.In cotransfection experiments, human CMV-SIRT1 or CMV-SIRT1H363Y (600 ng) was cotransfected into HeLa cells plated in six-well plates with the HIV LTR luciferase reporter (200 ng) or the LTRSubconfluent MEFs (70%) were grown on Cellocate coverslips (Eppendorf, Westbury, New York, United States), and nuclear microinjections were performed at room temperature with an automated injection system (Eppendorf Micromanipulator 5171 together with Eppendorf Transjector 5246). Samples were prepared as a 20 μl injection mix containing the HIV LTR luciferase reporter or 5xUAS luciferase (each 100 ng/μl), RSV-Tat (10 ng/μl) or Gal4-VP16 (50 ng/μl), CMV-cyclinT1 (100 ng/μl), CMV-SIRT1 (100 or 300 ng/μl), together with CMV-GFP (50 ng/μl) in sterile water. At 6 h after microinjection, cells were examined under a Nikon Eclipse TE300 inverted fluorescent microscope (Nikon, Tokyo, Japan) to determine the number of GFP-positive cells, washed in cold phosphate buffer, and stored at −70 °C for luciferase assays (Promega). In HeLa cells, synthetic Tat or AcTat proteins (each 30 or 100 ng/μl) were coinjected with the wild-type or mutant HIV LTR luciferase reporters (each 100 ng/μl) together with CMV-GFP (50 ng/μl), and harvested 4 h after injection. Cells were treated immediately after injection with DRB , TSA (400 nM), or nicotinamide (5 mM). Microinjections in siRNA-treated cells were performed 48 h after siRNA transfection.−/GFP containing the GFP open reading frame in place of the nef gene and a frameshift mutation in the env gene, as well as the method to generate pseudotyped viral particles with VSV-G, were previously described [5 Jurkat cells with different amounts of viral suspension. The titer of the viral stock was measured by flow cytometric analysis of GFP expression 48 h after infection. The pHR′-EF-1α/GFP construct is a minimal nonreplicative HIV-1 genome containing a heterologous promoter, EF-1α, driving GFP expression [−/GFP or pHR′-EF-1α/GFP viral particles at a theoretical multiplicity of infection of 0.5 in 24-well plates. Cells were repeatedly washed and resuspended in fresh medium containing HR73 (1 μM) or DMSO alone. Viral infection was monitored 36 h later by flow cytometry analysis using a Calibur FACScan .The HIV molecular clone HIV-R7/Eescribed . The numpression . Viral ppression . All vec


Iron and copper play an important role in oxidative mechanisms, producing the deleterious hydroxyl radical (*OH) that peroxides lipid membranes and damages DNA. Myeloperoxidase (MPO) and nitric oxide (NO) are known sources of free radicals and induce reduction of ferritin-Fe3+ into free Fe2+, contributing to oxidative damage.The exogenous administration of Insulin-like Growth Factor-I (IGF-I) induces hepatoprotective and antifibrogenic actions in experimental liver cirrhosis. To better understand the possible pathways behind the beneficial effect of IGF-I, the aim of this work was to investigate severe parameters involved in oxidative damage in hepatic tissue from cirrhotic animals treated with IGF-I . Fe and Cu were assessed by atomic absoption spectrometry and iron content was also evaluated by Perls' staining. MPO was measured by ELISA and transferrin and ferritin by immunoturbidimetry. iNOS expression was studied by immuno-histochemistry.Liver cirrhosis was induced by CClLiver cirrhosis was histologically proven and ascites was observed in all cirrhotic rats. Compared to controls untreated cirrhotic rats showed increased hepatic levels of iron, ferritin, transferrin (p < 0.01), copper, MPO and iNOS expression (p < 0.01). However, IGF-treatment induced a significant reduction of all these parameters (p < 0.05).the hepatoprotective and antifibrogenic effects of IGF-I in cirrhosis are associated with a diminution of the hepatic contents of several factors all of them involved in oxidative damage. Insulin-like growth factor-I (IGF-I) is an anabolic hormone produced in different tissues in response to growth hormone (GH) stimulation . Liver sIn order to give a better insight into the pathways by which IGF-I seems to exert its the hepatoprotective and antifibrogenic actions, this study was aimed at analyze several parameters involved in oxidative stress or inflammation in the liver, such as metals ions (iron and copper), iron transport and store proteins (transferrin and ferritin) and enzymes (myeloperoxidase -MPO- and inducible nitric oxide synthase -iNOS-) both in IGF-I treated and untreated cirrhotic rats.3+ into free Fe2+ contributing to oxidative damage [Metal ions, such as iron and copper, exhibit the ability to produce reactive oxygen species, resulting in lipid peroxidation, DNA damage, depletion of sulfhydryls and altered calcium homeostasis -19. Irone damage ,23.4 inhalation (Merck, Darmstadt, Germany) twice a week for 11 weeks with a progressively increasing exposure time from 1 to 5 minutes. From that time until the 30th week rats were exposed to CCl4 once a week for 3 min. During the whole period of cirrhosis induction animals received Phenobarbital  in the drinking water (400 mg/L). Rats were housed in cages placed in a room with 12-hour light-dark cycle and constant humidity and temperature (20°C). Both food  and water were given ad libitum. Healthy, age and sex-matched control rats were maintained under the same conditions but receiving neither CCl4 nor Phenobarbital.Cirrhosis was induced as previously described ,12. BrieThe Guiding Principles for Research Involving Animals [All procedures were performed in conformity with  Animals .th -30th) of CCl4 exposure (from day 0 to day 22nd). In the morning of day 0, animals were weight and blood samples were drawn from the retroocular venous plexus from all rats with capillary tubes (Marienfeld, Germany) and stored at -20°C until used for analytical purposes. Cirrhotic rats were randomly assigned to receive either vehicle  (CI, n = 10) or recombinant human IGF-I (Pharmacia-Upshon, Sweden) (2 μg × 100 g bw -1 × day -1 in two divided doses, subcutaneous) (CI+IGF, n = 10) for three weeks. Control rats (CO, n = 10) received saline during the same period. The last dose of IGF-I was administrated the day 21st at 6 p.m.The treatment was administrated the last three weeks . The rest of liver samples were stored at -80°C.In the morning of the 22Bouin-fixed tissues were processed and sections (4-μm.) were stained with Haematoxylin and Eosin and Masson's trichrome. Liver cirrhosis was diagnosed according to the criteria previously described ,16. LiveImmunohistochemical staining of iNOS in paraffin sections (4 μm) was performed using an avidin-biotin peroxidase technique as described by Shu el al. , with soHepatic samples were homogenized in a Potter homogenizer in 7 volumes of cold buffer (0.1 M Tris-HCl, 0.25 M sucrose, pH= 7.4) containing 5 mM 2-mercaptoethanol, 0.5 μg/mL Leupeptin, 0.7 μg/mL pepstatin A and 100 μg/mL PMFS. Fibrous parts and unbroken cells debris were eliminated by centrifugion at 500 g for 5 min. Supernatans were used as the whole homogenate.® (OXIS Int. Portland, OR, USA). Transferrin and ferritin were determined by immunoturbidimetry, using a Hitachi 710 autoanalyzer (Roche Diagnostic, Basilea, Switzerland) and kits for clinical human, from the same laboratory. MDA was assessed after heating samples at 45°C for 60 minutes in acid medium. It was quantitated by a colorimetric assay using LPO-586 , which after reacting with MDA, generating a stable chromophore that can be measured at 586 nm . Total proteins were assessed by Bradford's method [MPO was measured by ELISA, using a commercial kit from BIOXYTECHs method .Representative samples (approximately 1 g. of each rat liver) were collected, weighed and later dried in stove (70°C) to constant weight. Iron and copper concentrations were determined by flame atomic absorption spectrophotometry (Perkin Elmer 460, Uberlingen, Germany) .P value < 0.05 was considered to be statistically significant. Calculations were performed with SPSS program version 6.0 (SPSS Inc., Chicago, IL).Data were expressed as mean ± SEM. To analyse the homogeneity among groups, Kruskall-Wallis test was used, followed by multiple post-hoc comparisons using Mann-Whitney U tests with Bonferroni adjustment. Any 4.Liver cirrhosis was histologically proven and ascites was observed in all rats treated with CClTable As shown in Figure Figure In order to find a relationship between the studied parameters and oxidative liver damage, MDA levels, an index of lipid peroxidation, were evaluated . Hepatic4-induced cirrhosis associated with iron and copper overload and an increase of myeloperoxidase and iNOS expression.These results show that the treatment with low doses of IGF-I induces a reduction of all studied parameters involved in oxidative damage mechanisms in this model of cirrhosis. These findings support the hepatoprotective and antifibrogenic effects previously reported ,16. ThisIt is well known that iron and copper promote oxidant forces ,18,21,302*- radicals [Free iron (or low molecular iron or chelatable iron pool) facilitates the decomposition of lipid hydroperoxides resulting in lipid peroxidation and induces the generation of OH radicals and also accelerates the nonenzymatic oxidation of glutathione to form Oradicals ,19,30,323+ per mole of protein [2*-, etc. [Most of the body's iron is tightly bound to transferrin, entering cells via receptor-mediated endocytosis. Transferrin avidly binds 2 moles of Fe protein . Normall protein ,30,31. I*-, etc. . EnhanceIn the present study, we have found that hepatic transferrin and ferritin levels increased in cirrhotic rats with a parallel rise in iron deposition, whereas in cirrhotic rats treated with IGF-I all the above-mentioned parameters appeared diminished  and secrete myeloperoxidase (MPO) [2*- formed during this respiratory burst is converted to the bactericidal oxidant hypochlorous acid (HOCl) via a series of reactions catalyzed by superoxide dismutase and MPO [After hepatic injury, several kinds of cells  are activated in the subsequent inflammatory response . Free rase (MPO) . The maj and MPO ,29. Nume and MPO . MPO has and MPO . The sam and MPO . In our Another result which deserves particular mention is that iNOS expression was significantly lower in cirrhotic rats treated with IGF-I compared to untreated cirrhotic animals. This finding is in accordance with those reported by other groups -53. Howe4-induced cirrhosis. The hepatic levels of both metals diminished in cirrhotic animals treated with IGF-I. MPO content, iNOS immunohistological expression and hepatic ferritin and transferrin levels were increased in untreated animals and returned to normal in cirrhotic animals treated with IGF-I.In conclusion, these results show that the hepatoprotective and antifibrogenic effect of IGF-I in rats with liver cirrhosis is associated with a significant reduction of the hepatic levels of several parameters such as Fe, Cu, MPO, iNOS, ferritin and transferring, all of them involved in oxidative damage. In this work, iron and copper overload have been demonstrated in the liver from rats with CClThe IGF-I effects described in the present study suggest that a therapeutical approach targeted at lowering oxidative stress marker levels could be effective in the chronic liver disease.2*-, superoxide radicals; MPO, myeloperoxidase; iNOS, inducible nitric oxide synthase; AU, arbitrary units.IGF-I, insulin-like growth factor-I; Fe, iron; Cu, cooper; MDA, malondialdehyde; CO, control healthy group; CI, untreated cirrhotic rats; CI + IGF, IGF-treated cirrhotic rats; OThe author(s) declare that they have no competing interests.MG: Analytical studies, hypothesis and paper elaboration.ICC: Experimental design and treatment (induction of liver cirrhosis and IGF-I administration), hypothesis, histopathological study and scores.MDS: Analytical studies and in vivo assay.IN: Atomic absorption spectrometry assay.JEP: In vivo assay.AC: Hypothesis and experimental design and revision.ADC: Experimental treatment and documentation.EC: Histopathological study and measurements.SGB: Revision.The pre-publication history for this paper can be accessed here:


The character of upper limb disorder in computer operators remains obscure and their treatment and prevention have had limited success. Symptoms tend to be mostly perceived as relating to pathology in muscles, tendons or insertions. However, the conception of a neuropathic disorder would be supported by objective findings reflecting the common complaints of pain, subjective weakness, and numbness/tingling. By examining characteristics in terms of symptoms, signs, and course, this study aimed at forming a hypothesis concerning the nature and consequences of the disorder.I have studied a consecutive series of 21 heavily exposed and severely handicapped computer-aided designers. Their history was recorded and questionnaire information was collected, encompassing their status 1/2 – 1 1/2 years after the initial clinical contact. The physical examination included an assessment of the following items: Isometric strength in ten upper limb muscles; sensibility in five homonymously innervated territories; and the presence of abnormal tenderness along nerve trunks at 14 locations.Rather uniform physical findings in all patients suggested a brachial plexus neuropathy combined with median and posterior interosseous neuropathy at elbow level. In spite of reduced symptoms at follow-up, the prognosis was serious in terms of work-status and persisting pain.This small-scale study of a clinical case series suggests the association of symptoms to focal neuropathy with specific locations. The inclusion of a detailed neurological examination would appear to be advantageous with upper limb symptoms in computer operators. Upper limb pain and dysfunction are frequent complaints associated with computer work. However, the responsible pathology and the pathophysiological mechanisms are insufficiently understood. In addition, there is no consensus with regard to physical findings that may reflect symptoms.The involvement of the nerves in "non-specific" upper limb disorder, e.g. in computer operators, is suggested by various observations: The demonstration of an elevated threshold to vibratory stimulation -3; abnorUpper limb pain in computer operators shares the features of a neuropathic pain: Common analgesics tend to be ineffective. Pain may be evoked spontaneously or may appear to constitute an abnormal response to stimuli with frequent occurrence of allodynia. In addition, there are often non-painful abnormal spontaneous or evoked sensory phenomena such as numbness/tingling. The common experience of weakness which may further deteriorate on use would also be compatible with an upper limb nerve affliction.A precise and accurate diagnosis is crucial for effective management and rehabilitation, and also for epidemiological studies concerning causation. In order to get a better understanding of the pathophysiological mechanisms, the injured tissue should be precisely located. This might not necessarily be where symptoms predominate.I have aimed at studying a clinical series of computer operators with upper limb complaints and dysfunction in terms of• exposure characteristics;• symptoms and past treatment;• physical findings which may reflect an affliction of the peripheral nerves;• prognosis with regard to symptoms and work-status.This study comprises a consecutive series of 21 computer-aided designers with pain and functional limitations in the dominant upper limb. All patients were referred to a department of occupational medicine for diagnostic and aetiological assessment and management. Three patients were males of median age 27 years (range 25–41) and 19 were females of median age 35 years (range 25–55).Patients were interviewed about the character, distribution, initial presentation and development of their symptoms. Special attention was given to the presence of upper limb pain, subjective weakness and numbness/tingling, and to other symptoms included in a standard protocol for work-related upper limb disorders .A subsequent physical examination included extracts of diagnostic criteria for selected clinical disorders  .Upper limb nerve afflictions were defined from an additional neurological examination consisting of the following components:• Manual assessment of the isometric strength in a selection of ten upper limb muscles Figure . Any red• The sensibility  was ass• The axillary nerve (the deltoid area);                                       • The musculocutaneous nerve ;                                       • The radial nerve ;                                       • The median nerve (the tip of the second finger);                                       • The ulnar nerves (the tip of the fifth finger).                                       • The perception of vibration .rk 256 Hz) was addAny sensory deviation from normal was registered as abnormal.• Assessment of tenderness with slight pressure at 14 locations along the course of nerves . Any mec• The brachial plexus ;                                       • The suprascapular nerve (suprascapular notch);                                       • The axillary nerve ;                                       • The musculocutaneous nerve ;                                       • The median nerve ;                                       • The radial nerve ;                                       • The posterior interosseous nerve at the arcade of Frohse (supinator tunnel);                                       • The ulnar nerve .                                       Assessments in patients with unilateral disorder were based on comparison to contra-lateral findings defined as normal. In patients with bilateral disorder, test-results were related to other findings in the same limb assumed to be normal, e.g., strength in adjacent muscles or sensibility in adjacent innervation territories .The definition and location of a nerve affliction ("neuropathy") was based on a traditional approach with a focus on the topography and innervation patterns of the upper limb nerves. Special consideration was given to the presence of normal strength in certain muscles and of reduced strength in others ,19, and I have operated with two sets of criteria for the definition of focal neuropathy assuming the second criterion to be more convincing:Criterion 1: The presence of a pattern of muscle-weakness suggesting a focal neuropathy at a defined location, at which mechanical allodynia with slight pressure at the nerve is present.• Criterion 2: Criterion 1 plus sensory deviations from normal in one or several sensory territories located peripherally to focal neuropathy.• In addition, double crush  at the aBrachial plexus neuropathy at chord level was defined with reduced strength in the deltoid, biceps, and radial flexor of the wrist muscles, when weaknesses were accompanied by brachial plexus tenderness at its passage behind the pectoral muscle. Depending on the extent of brachial plexus involvement, additional muscles may be weak and mechanical allodynia may extend in the proximal or medial direction.Median neuropathy at elbow level was defined with reduced strength in the radial flexor of the wrist muscle along with mechanical allodynia involving the median nerve at elbow level . With an isolated median neuropathy, the deltoid, biceps, and ulnar extensor of the wrist muscles must be intact.and the median nerve was defined in the following situations:Double crush involving the brachial plexus • Strength in the radial flexor of the wrist muscle was reduced as much as / more than it was in the deltoid or biceps muscles.• Mechanical allodynia was either the same or more conspicuous at the median nerve at elbow level than it was at plexus level.Posterior interosseous neuropathy was defined with reduced strength in the ulnar extensor of the wrist muscle along with tenderness at the nerve-passage below the arcade of Frohse in the dorsal proximal forearm. With an isolated posterior interosseous neuropathy, the deltoid, biceps, short radial extensor of the wrist, and radial flexor of the wrist muscles must be intact.and the posterior interosseous nerve was defined in the following situations:Double crush involving the brachial plexus • Strength in the ulnar extensor of the wrist muscle was reduced as much as / more than in the deltoid, biceps, or radial flexor of wrist muscles.• Mechanical allodynia was either the same or more conspicuous at the arcade of Frohse than it was at plexus level.Other potential focal neuropathy was defined according to similar criteria, e.g., an isolated carpal tunnel syndrome would require reduced strength in the short abductor of the wrist muscle but preserved strength in the radial flexor of the wrist muscle. An isolated ulnar neuropathy at elbow or wrist level would require reduced strength in the abductor of the fifth digit and intact proximal muscles. In addition, mechanical allodynia should be present at the appropriate locations along nerve trunks.Patients were recommended to freely move and use the symptomatic upper limb within the limits of immediate and subsequent pain aggravation. All patients were offered physiotherapy based on the concept of adverse neural tension ,24 and e1/2 – 1 1/2 years after the initial examination the patients responded to a questionnaire: The exposure characteristics; symptoms (pain, weakness/fatiguability, numbness/tingling); past treatment; pain intensity at the first encounter and at follow-up to be quantified on a VAS-scale from 0 (no pain) to 10 (extreme pain); and the present status with regard to functional limitations and work.The change of the level of reported pain between the first consultation at the department and at follow-up was assessed by Friedman's test.All 21 patients returned the questionnaire. The mean duration of work with computer-aided design was 95 months (16–260 months). The self-reported daily mean of time spent with computer work constituted 81% (50–100%) of the total working time. 86% of the respondents reported aggravating factors during the months prior to the onset of symptoms, including high work intensity, overwork or other work conditions causing an unusual strain.Pain in the dominant upper limb was common to all patients. It had a mean duration of 24 months (1–60 months) and was the main symptom in 13 patients. All but one patient had a subjective feeling of weakness/fatiguability. Five patients reported this to be the most disturbing symptom. 19 patients experienced numbness/tingling which constituted the main symptom in three of them. Five patients had bilateral symptoms.All patients had received treatment prior to admission: A limited and transitory effect of past physiotherapy was reported in four out of 17, of pain killers in one out of 10, and of local steroid injections in two out of three patients. For the remaining patients the past treatment had no effect.According to the defined criteria for work-related upper limb disorders , non-neuIn all 21 patients reduced strength was demonstrated in the following muscles: Deltoid, biceps, triceps, and the radial flexor, short radial extensor and ulnar extensor of the wrist. In a smaller number of patients there were additional strength-reductions in the pectoral, infraspinatus, latissimus and abductor of the fifth digit muscles  or unemployed (eight patients).2 = 8.0 and 9.0, p < 0.005 and 0.003, respectively). However, the pain persisted on a disturbing level in the majority of patients  were invariably involved. In a few limbs there was an additional involvement of the pectoral, small abductor of the fifth digit, latissimus dorsi, and infraspinatus muscles Figure . This isCaution should be exercised when drawing a comparison between the outcome of this study of patients, referred with a serious disorder, and studies of "healthy" computer operators in occupation. Still, it would be relevant to compare with upper limb findings in computer operators described by others. A study of 533 visual display terminal workers has suggested an array of upper limb disorders in 22%, dominated by tendon related conditions in 15% and probable nerve entrapment in 4% . In a reHowever, the diagnoses depend on the choice and validity of the clinical tests employed and on the diagnostic criteria applied. The "somatic shoulder/neck syndrome"  is charaMy findings are more in accordance with those of Pascarelli, who studied 485 upper limb patients out of which 70% were computer operators. A detailed and comprehensive physical examination demonstrated protracted shoulders in 78% and head forward position in 71%. This was also frequent in my study-patients but not systematically registered. A neurogenic thoracic outlet syndrome in 70% was suggested by tests stressing the brachial plexus and by the demonstration of mechanical allodynia . In a foThe limited success of the prevention and management of computer-related upper limb disorders demands new approaches to practice and research in the field. The inclusion in future studies of the presented systematic examination of the upper limb nerves may provide additional diagnostic information. This may lead to future improvement of the prevention and management of computer-related upper limb disorders.None declared.JRJ designed the study and conducted all the clinical examinations, and wrote the manuscript.The pre-publication history for this paper can be accessed here: