In our work, we have introduced an ant colony optimisation (ACO) algorithm to address the non-deterministic polynomial-time hard (NP-hard) combinatorial problem of predicting a protein's conformation from its amino acid sequence under a widely studied, conceptually simple model – the 2-dimensional (2D) and 3-dimensional (3D) hydrophobic-polar (HP) model.The protein folding problem is a fundamental problems in computational molecular biology and biochemical physics. Various optimisation methods have been applied to formulations of the We present an improvement of our previous ACO algorithm for the 2D HP model and its extension to the 3D HP model. We show that this new algorithm, dubbed ACO-HPPFP-3, performs better than previous state-of-the-art algorithms on sequences whose native conformations do not contain structural nuclei  at the ends, but rather in the middle of the sequence, and that it generally finds a more diverse set of native conformations.The application of ACO to this bioinformatics problem compares favourably with specialised, state-of-the-art methods for the 2D and 3D HP protein folding problem; our empirical results indicate that our rather simple ACO algorithm scales worse with sequence length but usually finds a more diverse ensemble of native states. Therefore the development of ACO algorithms for more complex and realistic models of protein structure holds significant promise. An example of stigmergy is the communication of ants during the foraging process: ants indirectly communicate with each other by depositing pheromone trails on the ground and thereby influencing the decision processes of other ants. This simple form of communication between individual ants gives rise to complex behaviours and capabilities of the colony as a whole.Ant Colony Optimisation (ACO) is a population-based stochastic search method for solving a wide range of combinatorial optimisation problems. ACO is based on the concept of et al. [e.g., [From the computational point of view, ACO is an iterative construction search method in which a population of simple agents ('ants') repeatedly constructs candidate solutions to a given problem; this construction process is probabilistically guided by heuristic information on the given problem instance as well as by a shared memory containing experience gathered by the ants in previous iterations ('pheromone trails'). Following the seminal work by Dorigo et al. ,2, ACO a. [e.g., -5).ab-initio protein folding under a widely studied abstract model – the hydrophobic polar (HP) model. In particular, we extend our previous ACO algorithm to the 3D HP model and improve its performance by modifying the subsidiary local search procedure.The research presented in this paper builds on an ACO algorithm first proposed in  (and latNP-hard) [ab-initio protein folding problem can be broken down into three sub-problems: 1) design of a model (with a desired level of accuracy); 2) definition of an energy function that can effectively discriminate between native and non-native states; and 3) design of a search algorithm that can efficiently find minimal-energy conformations. A number of search (or sampling) methods have been proposed in the literature to solve the protein folding problem, including Monte Carlo algorithms, Evolutionary Algorithms, Tabu Search and hybrid approaches. ACO, which has been very successfully applied to other combinatorial problems, appears to be a very attractive computational method for solving the protein folding problem, since it combines aspects of chain-growth and permutation-based search with ideas closely related to reinforcement learning. These concepts and ideas apply rather naturally to protein folding: By folding from multiple initial folding points, guided by the energy function and experience from previous iterations of the algorithm, an ensemble of promising, low-energy complete conformations is obtained. These conformations are further improved by a subsidiary local search procedure and then evaluated to update the accumulated pheromone values that are used to bias the generation of conformations in future iterations of the algorithm.The protein folding problem is one of the most challenging problems in computational biology, molecular biology, biochemistry and physics. Even under simplified lattice models, the protein folding problem is non-deterministic polynomial-time hard (NP-hard) . The ab-ab-initio protein folding problem under the 2D and 3D HP models? How does its performance scale with sequence length? What is the role of the parameters of the ACO algorithm for the efficiency of the optimisation process? Which classes of structures (if any) are solved more efficiently by ACO than by any other known algorithms? Finally, it should be noted that our ACO algorithm for this problem is based on very simple design choices, in particular with respect to the solution components reinforced in the pheromone matrix and of the subsidiary local search procedure. We discuss which of the many design choices underlying our algorithm should be reconsidered in order to achieve further performance improvements.In this paper, we ask and address the following questions: Is ACO a competitive method for solving the Due to the complexity of the protein folding problem, simplified models such as Dill's hydrophobic-polar (HP) model have become one of the major tools for studying protein structure . The HP c with exactly n such H-H contacts has energy E(c) = n·(-1); for example, the 2D HP conformation shown in Figure In the HP model, the primary amino acid sequence of a protein  is abstracted to a sequence of hydrophobic (H) and polar (P) residues that is represented as a string over the letters H and P. The conformations of such an HP sequence are restricted to self-avoiding walks on a lattice. For the 2D HP model, a 2-dimensional square lattice is typically used, and the 3D HP model is generally based on a 3-dimensional cubic lattice. An example of a protein conformation under the 2D HP model is shown in Figure (c) = n·-; for exas = s1 s2...sn, find an energy-minimising conformation of s, i.e., find c* ∈ C(s) such that E(c*) = min{E(c) | c ∈ C}, where C(s) is the set of all valid conformations for s. It has been proved recently that this problem and several variations of it are NP-hard [The HP Protein Folding Problem can be formally defined as follows: Given an HP sequence  NP-hard .A number of well-known heuristic optimisation methods have been applied to the 2D and 3D HP Protein Folding Problem, including Evolutionary Algorithms (EAs) -15 and MBesides general optimisation methods, there are other heuristic methods that rely on specific heuristics that are based on intuitions or assumptions about the folding process, such as co-operativity of folding or the existence of a hydrophobic core. Co-operativity is believed to arise from local conformational choices that result in a globally optimal state without exhaustive search . Among tet al. is based on the hypothesis that once a hydrophobic contact is formed it cannot be broken, and other contacts are formed in accordance with already folded parts of the chain (co-operativity of folding) [i.e., always guaranteed to find a global optimum; it attempts to find the hydrophobic core with the minimal possible surface area by systematically introducing geometric constraints and by pruning branches of a conformational search tree. A similar, but more efficient complete constraint satisfaction search method has been proposed by Backofen et al. [The hydrophobic zipper (HZ) strategy developed by Dill folding) . The confolding)  combinesfolding)  biases cfolding)  is compln et al.  for the et al. [An early application of Evolutionary Algorithms to protein structure prediction was presented by Unger and Moult ,15. Theiet al. ,18. PERMet al. [Other methods for solving protein folding problems include the dynamic Monte Carlo algorithm by Ramakrishnan et al. , which iet al. , which wet al. introduced the multi-self-overlap ensemble (MSOE) Monte Carlo method [Finally, Chikenji o method , which cOther Monte Carlo methods that have been particularly useful in off-lattice protein folding include generalised ensemble methods, such as umbrella sampling   based on the energies of the conformations obtained after the construction and the local search phases. A general outline of ACO is shown in Figure As usual, the ants in our ACO algorithm iteratively undergo three phases: the straight (S), left (L), right (R) in 2D, and straight (S), left (L), right (R), up (U), down (D) in 3D, which for each amino acid indicate its position on the 2D or 3D lattice relative to its direct predecessors in the given sequence .The solution components used during the construction process, the local search phase and the pheromone update are local structure motifs (or relative folding directions) n by a sequence of local structure motifs of length n - 2. For example, the conformation of Sequence S1-1 shown in Figure Since conformations are rotationally invariant, the position of the first two amino acids can be fixed without loss of generality. Hence, we represent candidate conformations for a protein sequence of length τ  and heuristic function values η ; details of this process are given in the 'Methods' section. The relative importance of τ and η is determined by parameters α and β, respectively, whose settings are detailed in the 'Discussion' section. Similar to other ACO algorithms known from the literature, our algorithm for the HP Protein Folding Problem incorporates a local search phase that takes the initially built protein conformation and attempts to optimise its energy further, using probabilistic long-range moves that are described in detail in the 'Methods' section.During the construction phase, ants fold a protein from an initial folding point by probabilistically adding one amino acid at a time based on the two sources of information: pheromone matrix values ρ (where 0 <ρ ≤ 1), and pheromone reinforcement is achieved by increasing the pheromone levels associated with the local folding motifs used in a fraction of the best conformations  obtained during the preceding construction and local search phase. Furthermore, to prevent search stagnation when all of the pheromone is accumulated on very few structural motifs, we introduce an additional renormalisation mechanism for the pheromone levels .Finally, the pheromone update procedure is based on two mechanisms: Uniform pheromone evaporation is modelled by decreasing all pheromone levels by a constant factor Different from our previous ACO algorithms for the HP Protein Folding Problem, our new algorithm, ACO-HPPFP-3, supports the 3D HP cubic lattice model in addition to the 2D HP square lattice model. Furthermore, it uses a different iterative improvement strategy, a modified long-range move operator and a less restrictive termination criterion in its local search phase. ACO-HPPFP-3 was used in all ACO experiments described in the following.To compare ACO-HPPFP-3 with algorithms for the 2D and 3D HP Protein Folding Problem described in the literature, we tested it on a number of standard benchmark instances as well as on two newly created data sets, one of which was obtained by randomly generating amino acid sequences with hydrophobicity value characteristic of globular proteins, while the other consists of biological sequences that were translated into HP strings using a standard hydrophobicity scale. (These new data sets will be described in more detail later in this section.)n ≤ 50, 100 runs for 50 <n ≤ 64, and 20 runs for n >64; in 3D: 100 runs for each sequence). Unless explicitly indicated otherwise, we used the following parameter settings for all experiments: α: = 1, β: = 2, ρ: = 0.8 and θ: = 0.05. Furthermore, all pheromone values were initialised to 1/3 in 2D and to 1/5 in 3D, and a population of 100 ants was used, 50% of which were allowed to perform local search. The local search procedure was terminated when no improvement in energy had been obtained after between 1 000 (for n ≤ 50) and 10 000 (for n > 50) scans through the protein sequence. We used an elitist pheromone updating scheme in which only the best 1% of all ants was allowed to perform pheromone updates. The probability  of keeping the previous direction when feasible during the long-range mutation move was set to 0.5 (see 'Methods' section). These settings were determined in a series of experiments in which we studied the influence of different parameter settings and will be further discussed later. All experiments were performed on PCs with 2.4 GHz Pentium IV CPUs, 256 Kb cache and 1 MB RAM, running Redhat Linux (our reference machine), and run-time was measured in terms of CPU time.The 21 standard benchmark instances for 2D- and 3D-HP protein folding shown in Table N- or C-terminus of the given HP sequence, and in many cases, this results in substantial differences in the performance of the algorithm. To capture this effect, we always ran PERM in both directions, and in addition to the respective average run-times, t1 and t2, we report the expected time for solving a given problem instance when performing both runs concurrently, texp = 2·(1/t1 + 1/t2)-1. For all runs of PERM, the following parameter settings were used: inverse temperature γ: = 26 and q: = 0.2.Most studies of EA and MC methods in the literature, including ,14,15,19The results obtained on standard 2D benchmark instances  and CHCC consistently outperform ACO-HPPFP-3 on these standard 3D HP benchmark instances, and CG reaches best known solution qualities substantially faster in many cases. We note that for Sequence S2-3 and S2-7, PERM'S performance is greatly dependent on the folding direction.In case of the 3D HP Protein Folding Problem (see Table H at each position of a sequence was chosen to be 0.45, and in the remaining cases (i.e., with probability 0.55), we generated a P.To thoroughly test the performance of ACO-HPPFP-3, we created two new data sets of random and biological sequences of length ≈ 30 and ≈ 50 amino acids  in order to obtain a non-redundant representative set of proteins. These protein sequences were translated into HP strings using the hydrophobicity scale classification of RASMOL , accordivs PERM in terms of mean CPU time over 10 runs per instance and algorithm; for practical reasons, each run was restricted to 1 CPU hour on our reference machine, and the lowest energies obtained in these runs : ACO-HPPFP-3 reaches the same energies as PERM, but on some instances, particularly of length 50, requires more run-time. In 3D, ACO-HPPFP-3 generally requires a comparable amount of run-time on sequences of length 30 and outperforms PERM on one random sequences of length 30, but performs noticeably worse on sequences of length 50 and in some cases does not reach the same energy. We also generated longer sequences of length 75; for these, ACO-HPPFP-3 failed to reach the minimal energy values obtained by PERM in a number of cases. The run-times for both algorithms are reported in detail in As can be seen from these results, in 2D, ACO-HPPFP-3 performs roughly comparably to PERM (PERM'S n ≤ 30), but by using parameter settings different from the ones specified earlier, the performance of both algorithms can be significantly improved in this case.In summary, the performance of ACO-HPPFP-3 is comparable with that of PERM  on biological and random sequences of length 30–50, but worse on longer sequences. This scaling effect is significantly more pronounced in 3D than in 2D. We note that neither ACO-HPPFP-3 nor PERM were optimised for short sequences , while using the same cut-off time and machine, ACO-HPPFP-3 found the optimum in an average run-time of 130 CPU seconds.The left side of Figure C-terminus. When testing the performance of PERM and ACO-HPPFP-3 on these sequences, we found that on D-1, ACO-HPPFP-3 requires a mean run-time of 236 CPU seconds, compared to t1 = 3 795, t2 = 1, texp = 2 CPU seconds for PERM . When this sequence was reversed, PERM started folding the sequence from the structural nucleus, and its mean run-time dropped to 1 CPU second. A result similar to that for sequence B50-7 was obtained for Sequence D-2, which has no structural nuclei at the ends, but a native state in which the ends interact with each other. Here, ACO-HPPFP-3 was found to require a mean run-time of 951 CPU seconds (again, mean run-times were obtained from 100 successful runs), compared to t1 = 9 257, t2 = 19 356, texp = 12 525 CPU seconds for PERM; as expected, in this case, reversing the folding order of the sequence did not cause a decrease in PERM'S run-time.We also designed two additional sequences, D-1 and D-2, of length 50 and 60, respectively, that have a unique native state in which both ends of the sequence interact with each other  is shown in the right panel of Figure t1 = 5, t2 = 118, texp = 9 CPU seconds, while the average run-time for ACO-HPPFP-3 is 820 CPU seconds. Our ACO algorithm generally performs worse than PERM on sequences that have structural nuclei at the ends, because it tends to spend substantial amounts of time compacting local regions in the interior of the sequence, while PERM folds more systematically from one end. These observations also hold in 3D, as seen from two random sequences folded in 3D . Relatil is the number of H-H contacts, n is the number of H residues in the sequence, and i and j are interacting H residues that are not neighbours in the chain. Intuitively, COH-H specifies the average sequence separation between H-H residues in contact per H in the sequence.where COH-H = 0.324 for Sequence S1-7 in 2D and relative COH-H = 0.75 for Sequence S2-5 in 3D are not found by PERM; similar results were obtained for other sequences), which further supports our hypothesis that both, in 2D and 3D, PERM is biased toward a more restricted set of native conformations. We performed analogous experiments for the case where PERM is allowed to keep certain statistics from one run to another as in [Figure er as in  (runs arSAH-H: = ∑hEh, where Eh is the number of unoccupied lattice sites around H residue h), the number of H-H contacts, and the H-H contact order as a function of the length of the sequence prefix (starting from the N-terminus of the sequence – where PERM starts folding). In this analysis, we calculated the properties of interest mentioned above for the native conformations found in 100 independent runs by ACO-HPPFP-3 and PERM, and plotted the mean values of the respective quantities as functions of the sequence prefix length  on prefix length, we furthermore observed that different from PERM, ACO-HPPFP-3 realises the bulk of its local H-H interactions in the middle of the given sequence  in 2D, as well as Sequence S2-5 of length 48 in 3D ; these sequences were chosen because the CPU time required to find the best known solutions was sufficiently small to perform a large number of runs (100–200 per instance).Following the methodology of Hoos and Stützle , we measα; the relative weight of the heuristic information, β; and the pheromone persistence, ρ .Two important components of any ACO algorithm are the heuristic function, which indicates the desirability of using particular solution components during the construction phase, and the pheromone values, which represent information learned over multiple iterations of the algorithm. Three parameters control the influence of the pheromone information versus heuristic information on the construction of candidate solutions: the relative weight of the pheromone information, α) and heuristic information (β), and their relative importance for the performance of our ACO algorithm. As can be seen from the results shown in Figure α: = 0 or β: = 0, respectively), the algorithm performs worse, particularly for longer 2D sequences . The optimal settings for α and β for most problem instances seem to be around α = 1 and β = 2, as shown in Figure In the first experiment, we investigated the impact of pheromone , but also to weaken its impact over time (i.e., to use ρ < 1). At the same time, closer examination revealed that for ρ > 0, attrition, i.e., the construction of inextensible partial conformations, is a major problem, which is a result of the accumulation of pheromone from multiple conformations. This is why the backtracking mechanism described in the 'Methods' section is extremely important for the performance of our algorithm in 2D. In 3D, for the previously stated reasons and because of the fact that the attrition problem is much less severe, the impact of the persistence parameter is generally smaller than in 2D.The goal of the next experiment was to further explore the role of experience accumulated over previous iterations in the form of pheromone values. To this end, we varied the pheromone persistence, i.e., the number of ants used in each iteration) and the duration of local search  are correlated and significantly affect its performance. To further investigate this phenomenon, we conducted additional experiments in which we fixed the ant colony size and varied the maximal number of non-improving steps during local search, and vice versa. In this series of experiments, different colony sizes were considered, from a single ant up to a population of 5 000 ants, and the number of non-improving steps in local search was varied from 100 to 10 000. The results, shown in Figure During the initial empirical evaluation of our algorithm, we observed that ant colony size (n ≤ 50) and around 10 000 for long 2D sequences (n > 50); the latter value also appeared to be optimal for all 3D sequences considered here. This observation follows the intuition that more degrees of freedom, as present for longer sequences and in higher dimensions, require more time for local optimisation, since for any conformation, improving neighbours tend to be rarer and hence harder to find.Our results also indicate that the performance of ACO-HPPFP-3 is more sensitive to the number of non-improving steps than to ant colony size. The optimal value for the maximum number of non-improving steps tolerated (per ant) before the local search phase terminates was found to be around 1 000 for short 2D sequences , but performance deteriorates markedly when all ants in the colony are allowed to update the pheromone matrix.i.e., of the probability  of retaining (feasible) previous relative directions during long-range mutation moves. As can be seen in Figure  values between 0.3 and 0.7. Both extreme cases,  = 0, which corresponds to an extremely H-contact greedy mutation operator, and  = 1, in which refolding always follows previous directions when feasible, result in a substantial decrease in performance. When  = 0, the decrease of performance in 3D is smaller than in 2D, because there is no severe attrition as in 2D, where greedy placement of H residues leads to early formation of very compact partial conformations, which often cannot be extended into valid complete conformations. The performance decrease for high  values is due to insufficient ability of the chain to fold into a new conformation that accommodates well the local change in structure which triggered the refolding.In a final experiment, we studied the impact of the persistence of local search, In this work, we have shown that ant colony optimisation (ACO) can be applied in a rather straight-forward way to the 2D and 3D HP Protein Folding Problems. Even though our ACO-HPPFP-3 algorithm is based on very simple structure components (single relative directions) and a simple subsidiary local search procedure (iterative first improvement), it performs fairly well compared to other algorithms and specialised heuristics on the benchmark instances considered here, particularly in 2D. The only non-specialised algorithm that typically performs better than our ACO algorithm, both in 2D and 3D, is PERM. We observed that, particularly in 3D, the run-time required by ACO-HPPFP-3 for finding minimum (or best known) energy conformations scales worse with sequence length than PERM. However, our results show that our ACO algorithm finds a different ensemble of native conformations compared to PERM, and has less difficulty folding sequences whose native states contain structural nuclei located in the middle rather than at the ends of a given sequence, as well as sequences with structures in which the ends interact. We found that two major components of ACO-HPPFP-3 – the pheromone values, which capture experience accumulated over multiple iterations of the search process and from multiple conformations, as well as the heuristic information that provides myopic guidance to the folding process – play a significant role for longer 2D sequences and, to a lesser extent, for 3D sequences, which suggests that in 3D, it may be preferable to associate pheromone values with more complex solution components.We also found that the subsidiary local search procedure is crucial for the performance of the algorithm; in particular, to ensure that high-quality conformations are obtained, it is very important to allow the local search procedure to run sufficiently long. In an earlier version of our algorithm , we usedi.e., fraction of elitist ants).We have shown that all components of our ACO algorithms contribute to its performance. In particular, its performance is affected by the following components and parameters (listed in the order of decreasing impact): pheromone values, termination criterion for local search, persistence of long-range moves, ant colony size, pheromone persistence, heuristic function, selectivity of local search, and selectivity of pheromone update  energy conformations, our new ACO algorithm can greatly facilitate investigations of the topology and location of structural nuclei, which we plan to undertake in future work. Finally, while HP protein folding problems are of considerable interest because of their conceptual simplicity, ultimately, most applications of protein folding algorithms require the use of more realistic models of protein structure. Since it does not rely on heuristics and properties that are specific to the HP model and yet performs very well on this restrictive, but not entirely unrealistic abstract model, we believe that relatively straight-forward extensions of our ACO algorithm to more complex and realistic models of protein structure hold significant promise.Our new ACO algorithm, ACO-HPPFP-3, iterates construction, local search, and pheromone update phases until a termination condition is satisfied; in the context of this work, we mostly terminated the algorithm upon reaching a given energy threshold. In the following, we describe the three search phases in detail.During the construction phase of ACO-HPPFP-3, each ant first determines a starting point within the given protein sequence; this is done by uniform random choice. From this starting point, the sequence is folded in both directions, adding one residue at a time. Each ant performs probabilistic chain-growth construction of the protein conformation, where in every step, the structure is extended either to the left or to the right, such that the ratio of unfolded residues at each end of the protein remains (roughly) unchanged.S, L, R} instead of five {S, L, R, U, D}, see also [ηi,d and pheromone values τi,d, according to the formula:Here, we assume that folding is performed in 3D . The reτi,d indicate the desirability of using the local structure motif with relative direction d ∈ {S, L, R, U, D} at sequence position i. Initially, all τi,d are equal, such that local structure motifs are chosen in an unbiased way; but throughout the search process, the pheromone values are updated to bias folding towards the use of local motifs that occur in low-energy structures (the updating mechanism will be described in more detail later). The heuristic values ηi,d are based on the energy function E. They are defined according to the Boltzman distribution as ηi,d: = , where γ is a parameter called the inverse temperature , and hiattrition, and our algorithm overcomes it as follows: First, starting at the end at which attrition occurred, half of the sequence that has been folded up to this point is unfolded. Then, this segment of the chain is refolded; the first residue (i.e., the last one that was unfolded) is placed such that its relative direction differs from what it had been when attrition occurred, while all of the subsequent residues are folded in a feasible direction that is chosen uniformly at random. This backtracking mechanism is particularly important for longer protein sequences in 2D, where infeasible conformations are frequently encountered during the construction phase.During construction, it may happen that the chain cannot be extended without running into itself. This situation is called i.e., the end, crankshaft and corner moves [The local search phase is based on a long-range mutation move that has been designed to avoid infeasible conformations. It also has a number of important advantages over the more commonly used point mutation moves or Monte Carlo moves (er moves ): It is er moves . (0 ≤  ≤ 1) its previous relative direction, if it is still feasible, is left unchanged, and otherwise (i.e., with probability 1 - , or if the previous direction has become infeasible), a different relative direction is chosen, where the probability for each direction d is proportional to the corresponding heuristic value ηi,d. Formally, this can be written as follows:From studies of protein folding dynamics, it is known that proteins display a broad range of motions that range from localised motions to slow large-scale movements . InspireP[di : = ] is the probability of choosing direction  as the relative direction di at sequence position i. Unlike in our previous implementation [c' improves over the current solution quality (energy) of c. This search process is greedy in the sense that it does not allow worsening steps, and it is terminated when no improving steps have been found after a specific number of scans through the chain . Since this local search procedure has a relatively high time-complexity, in each iteration of ACO-HPPFP-3 it is only applied to a certain fraction of the highest-quality conformations constructed by the ants in the preceding construction phase.where entation , the locAfter each construction and local search phase pheromones are updated according toτi,d:= ρ·τi,d,     (4)ρ ≤ 1 is the pheromone persistence, a parameter that determines how much of the information gathered in previous iterations is retained. Subsequently, selected ants with low-energy conformations update the pheromone values according towhere 0 <τi,d:= τi,d + Δi,d,c,     (5)i,d,c is the relative solution quality of the given ant's candidate conformation c if that conformation contains local structure motif d at sequence position i, and zero otherwise.where ΔMAX - MIN Ant System [τ values are normalised such that ∑d∈{S,L,R,U,D} τi,d = 1 for every residue i; additionally, whenever for a given sequence position i the minimal normalised pheromone value (mind∈{S,L,R,U,D} τi,d)/(∑d∈{S,L,R,U,Dr} τi,d) falls below a threshold θ , the minimal τi,d value is set to θ, while the maximal τi,d value is decreased by θ - mind∈{S,L,R,U,D} τi,d.  This guarantees that the probability of selecting an arbitrary local structure motif for the corresponding sequence position does not become arbitrarily small, and hence ensures the probabilistic approximate completeness of our algorithm ..ACO-HPPFP-3 has been implemented in C++ and compiled using gcc (version 3.3.3) for the Linux operating system; a Linux executable is available from Both authors contributed to the development of ideas, design of experiments, analysis and interpretation of results, and the writing of the paper. AS implemented the proposed method and performed the computational experiments.Additional information on biological and randomly generated HP sequences. This file (in .pdf format) contains tables providing additional information on our new test sets of biological and randomly generated HP sequences and the results from our computational experiment with ACO and PERM.Click here for file


It's a common runner's complaint. Just when you've built up enough strength and endurance to make running fun, those niggling aches and pains won't go away. Every time your foot hits the ground, a force equal to about twice your weight shoots through your body, eventually chipping away at bones, cartilage, muscles, tendons, ligaments, and joints. For those lucky souls who can take the pounding, the main limitation to running performance stems from muscle fatigue. Now, Randall Johnson and colleagues report that a protein found in skeletal muscle profoundly influences muscle endurance.Running, like any sustained skeletal muscle activity, consumes large quantities of adenosine triphosphate (ATP), a molecule that fuels many essential cell processes. A number of metabolic pathways supply muscle tissue with the ATP needed to power muscle contraction and sustain ongoing exercise. Which pathway predominates depends on factors like speed, duration, and type of activity, as well as on the availability of oxygen, which fluctuates during activity. (For more on muscle cell type and endurance, see the synopsis titled “Gene Targeting Turns Mice Into Long-Distance Runners.”)Say you start a half-hour run with a sprint. Within a few seconds, your body uses up the oxygen in its muscles and has to switch to anaerobic pathways, which metabolize sugars and fats to regenerate ATP. Aerobic pathways operate inside mitochondria, the cell's major power generators. Anaerobic pathways like glycolysis function in the cytoplasm.Hypoxia  governs how ATP is recycled and which energy-producing substrates (for example, glucose or fatty acids) are used; it also generates metabolic by-products, like lactic acid, during strenuous exercise. (Runners know the “lactic acid burn” associated with reduced blood pH.) Glycolysis—the primary source of anaerobic energy in animals—uses glucose, stored as glycogen in muscle cells, to produce ATP. When blood oxygen levels drop, the gene transcription factor hypoxia-inducible factor 1α (HIF-1α) triggers the glycolytic pathway.To understand how HIF-1α regulates skeletal muscle function, Johnson's team generated mice that couldn't express HIF-1α in skeletal muscle. Normal and mutant mice went through exercise routines that included swimming and running on treadmills. After exercise, the normal mice had increased levels of gene transcripts and enzymes involved in glucose transport and metabolism. In the mutant mice, expression of these glycolysis-associated genes and enzymes was significantly lower. The mutants' ATP levels, however, were normal. Without the molecular machinery to engage anaerobic metabolism, their muscles switched to aerobic pathways. The presence of enzymes that respond to reduced ATP levels by increasing mitochondrial ATP production, combined with low levels of lactic acid, confirmed the switch.During endurance tests, the mutants could swim and run uphill (on treadmills tilted upward) longer than the normal mice, but when it came to running downhill, the normal mice prevailed. Downhill running, it turns out, favors glycolytic metabolism; uphill running and swimming favor oxidative pathways, which the mutants were predisposed toward. But their inappropriate use of this pathway came at a cost. By the final day of a four-day exercise routine, the mutants' run time was significantly shorter and their muscles were clearly damaged.The mutants displayed a number of the trademark muscle defects seen in human patients with glycolytic processing disorders. These patients often have reduced lactate levels and elevated levels of mitochondrial enzymes, which apparently can cause a second wind and enhance endurance. This inappropriate use of oxidative pathways—which compensates for the inability to trigger glycolysis—may account for the exercise-induced muscle damage associated with these diseases.These results demonstrate that losing the molecular wherewithal to engage hypoxia response pathways has serious consequences for muscle function during exercise; it can give increased endurance, but at a high price. The mouse model presented here will help researchers explore how muscles normally function in response to low oxygen and how metabolic deficiencies cause debilitating muscle disease.


The function of esophagus is to move food by peristaltic motion which is the result of the interaction of the tissue forces in the esophageal wall and the hydrodynamic forces in the food bolus. The structure of the esophagus is layered. In this paper, the esophagus is treated as a two-layered structure consisting of an inner collagen-rich submucosa layer and an outer muscle layer. We developed a model and experimental setup for determination of elastic moduli in the two layers in circumferential direction and related the measured elastic modulus of the intact esophagus to the elastic modulus computed from the elastic moduli of the two layers.in vivo length and pressure-diameters relations were recorded for the rat esophagus. Furthermore, the zero-stress state was taken into consideration.Inflation experiments were done at 2O the muscle layer had a larger radius and strain than the mucosa-submucosa layer. The strain for the intact esophagus and for the muscle layer was negative at low pressures indicating the presence of residual strains in the tissue. The stress-strain curve for the submucosa-mucosa layer was shifted to the left of the curves for the muscle layer and for the intact esophagus at strains higher than 0.3. The tangent modulus was highest in the submucosa-mucosa layer, indicating that the submucosa-mucosa has the highest stiffness. A good agreement was found between the measured elastic modulus of the intact esophagus and the elastic modulus computed from the elastic moduli of the two separated layers.The radius and the strain increased as function of pressure in the intact as well as in the individual layers of the esophagus. At pressures higher than 1.5 cmH The majority of previous mechanical studies on visceral organs, including the blood vessels, have considered them as homogenous tubes; i.e., a single layer structure. Most visceral organs are, however, multilayered, e.g. the arteries consist of intima, media and adventitia and the gastrointestinal tract has circumferential and longitudinal muscle layers, submucosa and mucosa layers.The esophagus represents a very interesting biomechanical model since it is the only organ that can be separated into two layers without damage to either layer. Hence, the muscle layers can be separated from the mucosa-submucosa layer by dissection, leaving two intact tubes. Separation experiments of the esophagus in guinea pigs and rabbits showed that the submucosa-mucosa layer had larger residual strains and opening angles than the muscle layer -3. ConsiIn this study we recognize that the esophagus consists of mucosa-submucosa and muscle layers. We analyze these layers as elastic shells. Each layer has its own zero-stress state, and its own elastic constants. We will determine the material properties of each layer separately. Specifically, the material properties in the individual layers will be computed from the pressure-diameter relation and zero-stress state with the method of analysis presented below. We will then propose a simple model to combine the two layers to predict the overall behavior of the esophagus under certain hypotheses. The limitations and implications of the model will be discussed.-1 ip). Papaverine (15 mg) was injected into the tail vein to relax the visceral muscles and to euthanize the rat. The cervical segment of the esophagus was dissected free from its adjacent tissue. Next, the thoracic and abdominal cavities were opened. After pouring cold Krebs solution into the thoracic cavity, the esophagus was quickly dissected free from adjacent tissues and its in situ length was measured. A 2-cm-long segment from the middle part, intended for the distension test, was marked. The length of this segment and that of the entire esophagus was measured. The entire esophagus was then cut at the proximal and distal ends including the very first part of the stomach, and immediately placed in calcium-free Krebs solution containing 6% dextran and 0.25% ethylene glycol-bis (β-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA). The solution was aerated with a gas mixture of 95% O2-5% CO2 at pH of 7.4. After careful removal of all extra-esophageal tissue, the length was measured in vitro.Eight male Wistar rats, weighing 380–420 grams, were used in the study. Approval of the protocol was obtained from the Danish Animal Experiment Committee. The animals were anesthetized with sodium pentobarbital  monitored the changes in diameter and length during the distension and images were grabbed by a PC.The middle part of the intact esophagus was mounted in an organ bath containing the Ca2O.After the test of the intact esophagus, the muscle and submucosa-mucosa layers were gently separated into two tubes. The tubes were studied separately using the same procedure outlined above. The only difference was that the maximum pressure was set to 6 cmHThe zero-stress state of the esophagus was obtained in accordance with the method used for blood vessels . Briefly2O increment. The outer diameter was measured at each pressure level and averaged over three locations. At the no-load and zero-stress states, the inner and outer circumferential lengths were measured along with the thickness and area of the wall and layers . The opening angle was defined as the angle subtended between two radii drawn from the midpoint of the inner wall to the tips of the inner wall of the open sector.The morphometric measurements were made using SigmaScan Pro image analysis software (Jandel Scientific, Germany). The data were obtained from the images of the tubes in the distended state, rings in the no-load state and sectors in the zero-stress state. In the distended state for both the intact esophagus and the separated tubes, images were analyzed for each 0.5 cmHi-p), the wall thickness (Hp), the mid-wall circumference (Cm-p) at a given pressure were computed as ri-p = [(r2o-p - An/πλ1)]1/2, Hp = ro-p - ri-p, and Cm-p = 2π(ri-p + Hp/2). The outer radius (ro-p) of the intestine was computed according to the outer diameter (Do). The circumferential Green's strains and Kirchhoff's stress were computed according to the equations:The stresses and strains of the esophagus and its sublayers in the pressurized state were determined under the assumption that the geometric configuration of the lumen is cylindrical, the wall of the esophagus is incompressible, and the material in each layer is homogenous. Based on the above measurements and assumptions, parameters of the esophagus such as the luminal radius  corresponds to Young's modulus in the linear stress-strain regime.We assume that the circumferential stress-strain relationships for the inflation experiment obey Hooke's law for each layerσθθsm) (= Eθθsm)(eθθsm) (σθθm) (= Eθθm)(eθθm) (    (4)σ, E and e indicate the Cauchy stress, Tangent modulus and Green strain, respectively. θθ, sm and m indicate the circumferential direction and the submucosal and muscle layers, respectively.where θ the circumferential axis and z the radial coordinate. Nθ denotes the tensile membrane stress resultant in θ direction. The displacement in the x, θ, and z directions of a point on the neutral axis surface are denoted by u, v, and w, respectively. The displacements of any point, A, is denoted by uA, vA, wA as follows: 1) uA = displacement along the generator, positive in the direction of increasing x; 2) vA = displacement along a circle of radius a + z, positive in the direction of increasing θ and 3) wA = radial displacement, positive outward.In general, the circumferential stress is a function of circumferential and longitudinal strain. In the present analysis, we assume that the cross-modulus is small such that the longitudinal term is negligible in comparison with the circumferential term and hence eq. (4). We further assume that the esophagus is a circular cylinder. The basic equations of equilibrium and deformation are given in Flugge . Let x dAccording to the Bernoulli-Kirchhoff hypothesis , we havewhere a indicate the neutral axis.The circumferential strain which is assumed to be small is given byθ is given byThe circumferential membrane stress resultant Nθ and w does not change with x, we obtainBy substitution of Eqs. (4), (5), and (6) into Eq. (7) and noting that in the inflation experiments u, v, and w do not change with Eq. (8) can be integrated to yieldI, asm and am are the neutral axes for the submucosa (1.35) and muscle (1.15). Equation (9) can be solved in terms of EI aswhere I stands for intact esophagus; aI from Eq. (3) with EI computed from Esm and Em as given by Eq. (9b).Hence, we can compare measured EThe data were assumed to be representative of a normal distribution. The results are expressed as means ± SE. Student's t test and analysis of variance were used to detect possible differences between curves obtained from the intact esophagus and the two sublayers. The results were regarded as significant if P < 0.05.in vivo length before the mechanical tests. The length was fixed during the distension protocol.The esophagi shortened by approximately 30% after excision. However, the segments were stretched to the 2O the muscle layer had a higher radius and strain than the mucosa-submucosa layer. The strain for the intact esophagus and for the muscle layer was negative at low pressures indicating the presence of residual strains in the tissue. The thickness of the intact wall and the separated layers decreased as function of pressure. The mucosa-submucosa was the thinnest layer.The outer radius, wall thickness, and circumferential Green's strain as function of pressure is shown in figure Cross-sectional views of the esophagus and its two layers were obtained at the no-load state and zero-stress state. Upon reducing the no-load state to the zero-stress state by cutting the ring radially, the opened ring expanded itself into a sector with an opening angle of about 140° for the intact esophagus figure , top. SeThe stress-strain data are depicted in figure 2O) where the assumption of linear stress-strain relationship is most justified.Figure The peristaltic transport of swallowed material by the esophagus to the stomach is a neuromuscular function affected by a number of neuromuscular factors -14. The The unique structure of the esophagus as a composite of submucosa and muscle layers allows the separation of the two layers and an experimental determination of the constitutive properties of each layer. This is difficult to carry out in the blood vessels or in other organs. The coronary arteries can be separated at the external elastic laminae but only by tearing the adventitial layer . The resA major difference between the structures of the esophagus and blood vessels is the ease with which the wall can be separated into layers. This fact allowed us to obtain the stress-strain relation and the zero-stress state of the esophageal tissue layers, as reported above. In contrast, in spite of the extensive effort on theoretical and experimental bilayer models of arteries by Berry et al , DemirayThe opening angle of the inner submucosa layer is larger than that of the outer muscle layer which agrees with our previous report ,3 and laα, can be used as a measure of stiffness [The stress-strain curve for the submucosa-mucosa layer was shifted to the left of the curves for the muscle layer and for the intact esophagus at strains higher than 0.3, indicating that the submucosa-mucosa has the highest stiffness figure . This cotiffness . The dattiffness ,23,25,26A limitation of the study is that only uni-axial data were obtained in this study. Intuitively the circumferential direction seems to be the most important for cylindrical organs. Since longitudinal changes may also be important for esophageal function, future studies should implement bi- or tri-axial data. Another limitation is that the analytical model is restricted to the linear stress-strain regime. Furthermore, the esophagus and its layers are assumed to be cylindrical tubes, and that the esophageal tissue is incompressible. The last assumption is possibly true in the pressure range studied and it is also known from yet unpublished studies that the esophagus attains circular geometry both at the inner and outer surfaces even at low pressures. The linearity assumption needs to be generalized. We also assumed that each layer was homogeneous, though it is well known that the muscle layer is composed of longitudinal and circumferential muscle bundles. Hence, the muscle layer can be modeled into further sublayers.We have developed an analytical tool that can be used to analyze the mechanics of bilayered organs. The model was used to study the esophagus. The model may be useful for studying the mechanical properties of other organs that can be separated into layers. There are two immediate implications of the results in this study for the understanding of esophageal function and for clinical practice. It is well known that pain may arise from the esophagus and that the receptors involved in the mechanotransduction are located at different positions in the wall. Detailed information about the stress and strain distributions in the layers is therefore important for the interpretation of receptor-mediated responses. Furthermore, the stress reduction during loading  may serve as a mechanism to reduce damage to the esophagus during excessive loadings caused by swallowing of large objects or by acute esophageal obstruction.For the esophagus the model may be applied to the study of remodeling of the individual layers in response to disease. For example, in systemic sclerosis the muscle layers in esophagus are slowly replaced by fibrotic tissue, creating a passive conduit . It is already known that the esophageal stiffness increase in patients with systemic sclerosis ,28 but wYanhua Fan carried out the experimental work, the measurements, and read and approved the final manuscript. Hans Gregersen designed the study, partly analyzed the data, and drafted the manuscript. Ghassan S. Kassab suggested the analysis, analyzed the data, drafted and approved the final manuscript.


Design and test the reliability of a web-based system for multicenter, real-time collection of data in the emergency department (ED), under waiver of authorization, in compliance with HIPAA.This was a phase I, two-hospital study of patients undergoing evaluation for possible pulmonary embolism. Data were collected by on-duty clinicians on an HTML data collection form (prospective e-form), populated using either a personal digital assistant (PDA) or personal computer (PC). Data forms were uploaded to a central, offsite server using secure socket protocol transfer. Each form was assigned a unique identifier, and all PHI data were encrypted, but were password-accessible by authorized research personnel to complete a follow-up e-form.From April 15, 2003-April 15 2004, 1022 prospective e-forms and 605 follow-up e-forms were uploaded. Complexities of PDA use compelled clinicians to use PCs in the ED for data entry for most forms. No data were lost and server log query revealed no unauthorized entry. Prospectively obtained PHI data, encrypted upon server upload, were successfully decrypted using password-protected access to allow follow-up without difficulty in 605 cases. Non-PHI data from prospective and follow-up forms were available to the study investigators via standard file transfer protocol.Data can be accurately collected from on-duty clinicians in the ED using real-time, PC-Internet data entry in compliance with the Privacy Rule. Deidentification-reidentification of PHI was successfully accomplished by a password-protected encryption-deencryption mechanism to permit follow-up by approved research personnel. The ability of medical researchers to obtain and store electronic clinical data was complicated by requirements of the Patient Privacy Rule of the Health Insurance Portability and Accountability Act ("HIPAA") of 1996 in title 45 of the Federal Register, parts 160, subparts A and E of part 164 . The HIP® do not mandate user identification, do not partition and encrypt sensitive data, and do not maintain a record and audit trail of use. Accordingly, we developed a comprehensive electronic system for clinicians to capture clinical research data from the bedside using commercially available hardware and data upload over the Internet. The system was programmed with multiple security steps and authentication procedures to maintain data security and privacy.Existing electronic data collection methods are limited in their ability to centralize data in a fashion that expedites data sharing while remaining in compliance with HIPAA. For example, commercial spreadsheets that run on WindowsWe tested the hypothesis that real-time clinical data can be obtained from clinicians in multiple hospitals using electronic data collection stored in an off-site server, under waiver of Authorization, while remaining in compliance with the Privacy Rule. This study represents the development and implementation phase of an ongoing multicenter study to collect prospective and follow-up clinical data from patients undergoing evaluation for pulmonary embolism in the emergency department. The specific aims of this study were to: 1. Test the feasibility of real-time, electronic data collection on personal digital assistants and personal computers in the emergency department setting in two hospitals. 2. Test if the system would correctly upload protected health information (PHI) in a secure and encrypted fashion, but allow follow-up to be performed by selected individuals using password-protected access to PHI.Patients were enrolled from two hospitals in Charlotte, NC: Carolinas Medical Center Main and Carolinas Medical Center University. The clinical protocol was approved under waiver of informed consent and waiver of authorization by the Carolinas HealthCare Institutional Review Board and the Institutional Privacy Board in accordance with the published guidelines of the Department of Health and Human Services (DHHS) , which wBecause of institutional sensitivity about maintaining compliance with the privacy rule, this project required intensive planning and due diligence. Over a 6-month period, the authors scheduled several meetings with the Director of Privacy in Clinical Research, the hospital's Assistant Vice President of Privacy, and the Director of Information Security to discuss the protocol and methods. These individuals had oversight for privacy issues for both hospitals. Then, to facilitate the process of gaining assistance and approval from the Information Systems Department in implementing the technical aspects  at both hospitals, we obtained a letter of approval from each of these individuals to physically show to technical support personnel.All patients in this study underwent evaluation for pulmonary embolism. The method of selection and diagnosis have been described previously . For eacThis system was designed to allow data to be transferred from multiple sites and stored on one server using technical requirements described in part 160 and 164 of the Privacy Rule. Figure The trigger for data entry was the decision to order a diagnostic test to rule out pulmonary embolism in a symptomatic emergency department patient. Patient data were entered on the prospective e-form. The e-forms were programmed using hypertext markup language (HTML) in conjunction with active server pages (ASP) and Standard Query Language (SQL). The prospective and follow-up e-forms are shown in Figures To test for data validity in the prospective forms uploaded by clinicians, two authors independently examined each of 70 fields for all patients uploaded. We evaluated for blank cells, nonsensical character entry, or numeric entry that fell outside the predefined ranges.® operating system followed by synchronized upload to the hosted server. All clinicians owned a compatible PDA. The authors and staff assistants provided technical support to assist clinicians in the process of downloading the prospective e-form to their PDAs and uploading completed e-forms to the study's central server using commercial software (Microsoft ActiveSynch® v. 3.5). The clinicians were shown that prospective data entry forms could also be accessed through a URL hyperlink that was posted on the desktop of all Internet-connected computers in both emergency departments. When the user clicked the hyperlink, this action routed the user through the firewall directly to the hosted server for this study. All computers ran Windows 95 or higher, with ethernet connection to a T3 44.736 Mbps channel.Forms were completed by attending physicians (N = 22), resident physicians (N = 20), and physician assistants (N = 6) in two emergency departments while the patient of interest was still in the emergency department. Prior to study deployment, each clinician was individually trained in a 10-minute session by the study principal investigator using a pre-defined protocol, and each clinician received a follow-up letter that summarized the training session. Data forms could be accessed in the emergency department using designated Internet-connected personal computers within patient care areas, or could be completed using individually owned personal digital assistants using the Pocket PCUpon opening the first web page, the user viewed a list of clinician names Figure . The cli®, Seattle WA). However, all PHI data fields were remained encrypted.Multiple methods were used to ensure that protected health information would not be subject to unauthorized access, viewing or hijacking. When clinicians entered data in the emergency department, the server polled the form for inactivity exceeding 30 minutes, at which time the page would automatically close without being uploaded. We anticipated scenarios where a clinician would enter data on a prospective form that would need to be revised as a result of updated information . To allow for such editing, the clinician could re-access any prospective form for a period of 60 minutes after initial upload, provided that the Internet provider address of the computer was the same as the computer from which the form originated. After 60 minutes, the prospective e-form could be altered only by a study administrator. All data were transferred using secure socket link (SSL) protocol. The central server (Win2000 OS) was located off-site at a large commercial web hosting facility (NTT/Verio Inc). Upon upload, all fields containing any of the 18 elements of that constitute PHI were subjected to 128 key-bit encryption. Data were stored in relational tables. To allow data analysis for research purposes, the study PI could access stored by file transfer protocol and exported into a format compatible with commercial software  to determine if the patient had any of the outcomes of interest in the follow-up form. If no follow-up data were available within the hospital system to prove the patient was alive at 45 days, then the follow-up protocol required query of the public social security master death index to determine if a death certificate had been filed for the patient. Finally, if no valid follow-up were documented by electronic database search, we then attempted to contact the patient through a previously described, stepwise procedure, consisting of a mailed questionnaire, followed by a telephone call, if necessary .When all of the data required for the follow-up form were obtained and input into the form, the research coordinator would complete the form and press the "check form for completeness" button. This would activate a system to ensure that all necessary follow-up data were entered. For example, every patient had to have a valid 45-day follow-up, either in the form of a documented follow-up to a clinic, telephone follow-up with the patient, or confirmation of patient death within 45 days.The system was fully implemented on April 15, 2003. As of April 15, 2004, prospective data forms were uploaded from 1022 patients evaluated for acute pulmonary embolism. Prospective data have been entered by 42 clinicians and 6 physician assistants from two hospitals in Charlotte, NC. The primary technical barrier to implementation was the process of loading and using the prospective e-forms on a personal digital assistant. All 48 clinicians required individual help and training, of over one hour each to show them how to install the e-form on their PDAs. This impedance was compounded by real-time difficulties associated with stylus use on a small PDA screen, followed by difficulties with uploading to the website from the PDA led to abandonment of this method of data entry. Out of 48 clinicians, only 6 successfully uploaded more than one e-form from the PDA. Only 12 of 1022 uploaded e-forms originated from a PDA.The primary technical barrier to implementation on the personal computers included maintaining the URL icon on desktops in the ED . This problem was solved by the permanent link on the hospital's intranet home page. In two separate instances, clinicians reported that they had populated the e-form, attempted to submit, and for unknown reasons, were unable to upload the e-form, and they had to reenter the data and resubmit the e-form. The server has maintained a log of all successive e-forms uploaded by each clinician. No uploaded prospective forms have been lost or deleted. The side-server system was designed to prevent e-form upload with missing or erroneous data.To examine if this system properly, two observers reviewed the eight parametric field entries (age, heart rate, respiratory rate, systolic blood pressure, pulse oximetry, height, weight and temperature) that were key-entered by clinicians for 1022 patients. Validity required that both observers agree that the entry was a real number within the prespecified range of the parameter. In 44/8176 fields of 12 patients (0.6%), two observers deemed the parametric field entry to be useless for statistical analysis. Stated another way, these data would be coded as missing after data cleaning were completed. However, no categorical data were missing or erroneous. As a result, 1010/1022 (98.8%) of prospective e-forms had usable data in all 70 fields.Ninety-four percent of all 1022 patients have reported the site hospital to be their hospital of choice. Follow-up forms Figure  have beeFollow-up has revealed that all prospective e-forms were authentic, and each was completed on an emergency department patient who underwent at least one clinical test for pulmonary embolism. No bogus forms were detected during follow-up to date. This demonstrated a low likelihood of an unauthorized person generating a spoofed form on one of the designated computers in the ED treatment area, given that the automatic control system would not allow a form to be uploaded until all 70 fields are completed.All uploaded prospective and follow-up data were obtained by the central PI using file transfer protocol and were inputted into a spreadsheet without difficulty. Figure ® Server 2003 operating system. The only means of electronic access to the server is via hypertext transfer protocol (HTTP) and file transfer protocol (FTP). Both of these system services log all requests made to their ports. An example log entry is shown in the appendix.Query of the server log revealed no evidence of website hijacking or other intrusion. The server computer which houses the study database and runs the web application uses the WindowsThe step of obtaining written Authorization to comply with HIPAA can impart a selection bias in registries intended to study acute disease processes . In sectThe first research objective was to determine if PDAs would represent an efficient and secure mechanism for clinicians to record real-time data at the bedside in the emergency department setting. Our experience in this phase I, two-hospital study demonstrated that PDAs presented unexpected complexities that eroded our enthusiasm. The clients were clinicians with variable levels of technical sophistication. Despite our use of a relatively standard process, clinicians found it difficult to download the e-form from the website onto their PDAs, and many needed help from the study authors. Clinicians frequently forgot to bring their PDA devices to work, and during the one-year course of this study, 10 of 48 clinicians bought new PDA devices. Clinicians consistently reported difficulty with the small screen size and data entry with a stylus. Unfortunately, we did not quantify this opinion using a structured survey. We believe this represents the first published experience at using PDAs to collect research data in the emergency department setting. Our results are somewhat less positive than other studies that have reported the use of hand-held computers to maintain clinical databases . HoweverThe second objective was a relatively complex task intended to determine if the system would allow prospective and follow-up data collection over the Internet in compliance with the requirements of the Privacy Rule. From a functional standpoint, we sought to determine if the system would allow us to protect the data fields that needed to be protected, but allow the non-sensitive data to be accessed by study personnel who did not have local IRB approval. This was accomplished while maintaining strict security standards at each step of data transfer . Accordingly, the data collection instrument must accurately capture the information that the clinician uses to motivate his or her test ordering behavior, rather than to collect data after the test results have returned, and more complete medical records may have arrived. This report represents a phase I study. For the second phase, we will deploy this system to 10 US hospitals to allow collection of data from 5000 patients. The ultimate goal of this project is to collect a large, multicenter database, as the substrate for a mathematical model to generate a pretest probability of pulmonary embolism based upon beliefs of many clinicians.Research data can be successfully collected, entered and uploaded to a hosted server by emergency physicians working in different emergency departments, and in compliance with the Privacy Rule. Use of server side controls to test for data validity ensured that 98.8% of uploaded forms contained complete data usable for statistical analysis. The PHI data were successfully encrypted and deencrypted using password access to allow follow-up at a later date. Server log query demonstrated no evidence of intrusion or data loss, suggesting that data were securely stored.ASP – Active server pagesED – Emergency departmentFTP – File transfer protocolDHHS – Department of Health and Human ServicesHIPAA – Health Insurance Portability and Accountablity ActHTML – Hypertext markup languageHTTP – Hypertext transfer protocolIRB – Institutional Review BoardPDA – Personal digital assistantPHI – Protected health identifiersPI – Principal investigatorSQL – Standard query languageRUL – Uniform resource locatorJeffrey A. Kline is primary author on a US copyright certificate "Electronic Form ("e-form") For Secure Collection of Clinical Data Via the Internet" and inventor on a US patent (pending) that describes the present work. Jeffrey A. Kline and Charles L. Johnson own stock in BreathQuant Medical Systems.Example of a log entry for the HTTP system service" "Mozilla/4.0 "208.10.156.69 – [02/Mar/2004:23:55:55 +0000] "POST /pestudy/peadmin.asp HTTP/1.1" 302 1349 "208.10.156.69 and the resource requested is the ASP file . For the FTP system service this would be a typical log entryThe IP address of the HTTP requesting browser is 208.10.156.69 – generic [06/Aug/2003:17:00:14 +0000] " [22786]USER generic FTP" 331 0 "-" "-"208.10.156.69 and the USER identity that was logged in was "generic". Both these log files are scanned once a week to look for any suspicious requests. To date there have been no identified intrusion or hijack attempts on the specific study database. As an HTTP responding server on the public Internet, the web (HTTP) server program does receive many well identified "virus spreading" requests which it denies and whose denials are logged. One such "virus related" request common to all web logs today isThe IP address of the HTTP requesting browser is 404 380661.100.6.181 – [01/May/2004:11:30:52 +0000] "GET /scripts/nsiislog.dll HTTP/1.1" 404 code denotes that the request was denied. As security updates from Microsoft become available the study web server is updated. At present there are no known HTTP vulnerabilities which would allow an unauthorized user to gain access to files on the server.This example virus was developed to attack a vulnerability which existed in Microsoft Web Servers but was eliminated by a security update for their web (HTTP) server. The highlighted JAK conceived the study, obtained funding and participated in the system design, data collection and drafted the manuscript. CLJ carried out all technical programming, participated in system design and drafting the manuscript. WBW participated in idea conception, system design, data collection and drafting the manuscript. MSR participated in data collection and drafting the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Human metastatic cutaneous melanoma has gained a well deserved reputation for its immune responsiveness. The reason(s) remain(s) unknown. We attempted previously to characterize several variables that may affect the relationship between tumor and host immune cells but, taken one at the time, none yielded a convincing explanation. With explorative purposes, high-throughput technology was applied here to portray transcriptional characteristics unique to metastatic cutaneous melanoma that may or may not be relevant to its immunogenic potential. Several functional signatures could be identified descriptive of immune or other biological functions. In addition, the transcriptional profile of metastatic melanoma was compared with that of primary renal cell cancers (RCC) identifying several genes co-coordinately expressed by the two tumor types. Since RCC is another immune responsive tumor, commonalities between RCC and melanoma may help untangle the enigma of their potential immune responsiveness. This purely descriptive study provides, therefore, a map for the investigation of metastatic melanoma in future clinical trials and at the same time may invite consideration of novel therapeutic targets. Human metastatic cutaneous melanoma relative to other common solid tumors shares with renal cell cancer (RCC) the well deserved reputation of being responsive to immune manipulation ,2. HowevWe have previously shown that the microenvironment of a subgroup of melanoma metastases expresses at the transcriptional level an array of biologically active factors that may influence both the innate and the adaptive arm of the immune system . We have2-value ≤ 0.001 (unpaired two-tailed Student t test). This test identified 4,658 cDNA clones differentially expressed between melanoma metastases and tumors of other histology  which is a previously described serum marker of RCC also associated with renal carcinogenesis [-7 and 1 × 10-6 for two clones representing this gene . Overall, this cluster was enriched of genes associated with active cellular metabolism and included only few genes of previous known relevance to melanoma with the exception of a member of the melanoma antigen family D (MAGED2, Table Cluster b included 91 clones (72 named representing 65 distinct genes) predominantly associated with growth regulation and apoptosis. Among the genes included in this cluster was BNIP3L  that we have previously reported to be associated with the immune responsiveness of melanoma metastases [cluster c and d) included 262 and 613 clones, respectively (112 and 296 named corresponding respectively to 110 and 289 genes). These clusters were characterized by a high density of unnamed clones and by relatively low Cy5/Cy3 ratios. However, it should be noted that these clusters may be of particular interest because the gene expression profile was similar between melanoma and RCC tumors suggesting that some of these genes may conceal the enigma of immune responsiveness. In particular, a relatively sizable sub-cluster was noted with genes predominantly up-regulated in RCC but also expressed by melanoma lesions compared with other tumors  that was previously also reported in association with melanoma immune responsiveness to interleukin-2-based immunotherapy [JAK-1 was recently linked to the apoptotic role that interleukin-24 (melanoma differentiation associated gene-7: MDA 7) may exert on melanoma cells [cluster e) included 208 clones (151 named representing 143 different genes) predominantly associated with immune function. This immune signature was underlined by the high prevalence of expression of these genes in samples obtained from lymph node metastases whether from melanoma or colorectal primaries. Cluster f integrated 129 clones (91 named representing 87 distinct genes) including a mixture of genes with disparate functions difficult to categorize into a predominant pattern. This group also included APPBP1  which was previously reported in association with melanoma immune responsiveness [APPBP1 is a recently discovered epidermal growth factor that regulates dendrite motility and melanin release in epidermal melanocytes and melanoma cells [Cluster g included genes preferentially up-regulated in subcutaneous melanoma metastases known to be more responsive to immunotherapy with interleukin-2 [PRAME and tyrosine-related protein-1; TRP-1). In a small proportion, this cluster included a group of genes only over-expressed in fine needle aspirates (FNA) and generally expressed by circulating cells revealing blood contamination of FNA material . Cluster I included 222 clones (171 named identifying 155 genes) with most of the melanoma differentiation antigens (MDA) clustering in close proximity with the exception of the TRP-1 already discussed in cluster g. Interestingly, this cluster was also highly enriched of genes associated with ribosomal function and active translation. Furthermore, it included the melanocyte master regulator MITF  which has been shown to modulate lineage survival and melanoma cell viability through interaction with the anti-apoptotic protein BCL2 [MITF was coordinately expressed with several genes associated with calcium and other solute metabolism including cytochrome p450  solute carrier family 7 , G protein coupled receptor 56  and calpain 3 , a calcium-regulated gene found to be highly expressed in melanoma cells [cluster J included 88 clones of which the 62 named identified 58 genes.Several signatures representing genes preferentially expressed by melanomas were identified that could be partially linked to specific gene functions. Those signatures were segregated according to unsupervised gene rearrangement based on the Eisen's clustering method. The first cluster  and the tyrosine-related protein-1 (TRP-1). When the melanoma associated genes were studied alone, PRAME clustered close to the other MDA believe to be involved in the pigmentation process (tyrosinase, MART-1/Melan and gp100/PMel17). This is of particular interest because PRAME has been also reported to be highly expressed in other cancers of ectodermic origin such as medulloblastoma and neuroblastoma suggesting a link between ectoderm and pigmentation [SILV) in melanoma metastases while genes of the MAGE family manifested an independent behavior [Genes previously described to be preferentially expressed by melanoma lesions were confirmed to be so at a very high level of significance Figure . Excepti d Table . Clusterentation ,20. The entation -25, we ncluster e. These genes appeared up-regulated in lymph node metastases of melanoma as well as those from colorectal primaries suggesting that their expression results from lymphoid cell infiltration  natural killer cell-related genes suggests potent chemo-attraction toward natural killer cells by the tumor micro-environment of subcutaneous and cutaneous melanoma metastases. This is also emphasized by the high expression of interleukin-21 receptor which is usually expressed by natural killer cells and stimulates their cytolytic activity upon ligation with interleukin-21 produced by activated T cells [IRF)-7 implicated in the amplification of the innate immune response [The large majority of genes associated with immune function were included in n Figure . The samer cells , was coner cells  and to bin vitro ,29. Of itivation ,31. The d CXC3L1  may be ad CXC3L1 . Overall T cells ,35. The response  through response  may leadresponse . More puresponse  and CD4+response ; a relatclusters f to j. These genes had an expression profile opposite to the immune-related genes seen included in cluster e and appeared over-expressed in subcutaneous compared to lymph node metastases. Two granzyme-related genes were found strongly up-regulated in cluster e including granzyme A and M. This observation contrasted with the increased expression of cathepsin F and L in cluster f to j suggesting an opposite regulation of these genes involved in cell death or survival.A second and smaller group of immunologically-related genes was identified and included genes that segregated separately in clusters g - j appeared to demonstrate a preferential expression of genes in subcutaneous metastases independent of the technique used for biopsy (excision versus FNA). In particular, cluster g contained a small node of 47 clones highly expressed in subcutaneous metastases that included PRAME and TRP-1. This cluster also included the renal tumor antigen RAGE which has been previously shown to be highly expressed by melanomas [PRAME was the pattern of expression of the serine/threonine-specific protein kinase B-RAF. This gene is mutated in approximately 70 % of melanomas and it is often over-expressed [It has been reported that subcutaneous metastases of melanoma are more responsive to immunotherapy with interleukin-2 than lymph nodal and visceral metastases . Therefoelanomas  and melaelanomas . Interesxpressed . Althougt test and identified 4,221 genes at a cut off p2-value of ≤ 0.001. The data set was then filtered using the Cluster Program (Stanford, CA) selecting genes that were expressed in at least 80 % of the experiments and for which a Cy5/Cy3 log2 ratio ≥ 2 was present in at least one experiment. Two-thousand eight hundred and forty-three genes resulted from this filter. Because of the predominance of melanoma lesions (69 melanoma lesions compared to 14 RCC) the genes identified strongly represent differences between melanomas and other tumors. Therefore, we identified among them those that were not differentially expressed between melanoma and RCC samples to identify those genes that are truly uniquely expressed by the two immune responsive cancers. Two-thousand three hundred and fifty-eight genes were expressed similarly between the two types of cancer . A significant number of genes commonly expressed by melanoma and RCC and not by other tumors had no known function (681 genes). The remaining 1, 677 genes were further analyzed by separating those up-regulated from those down-regulated in melanoma and RCC compared with other tumors. The genes up-regulated in RCC and melanoma were considered those with a median LogRatio above 0.3 in either RCC or melanomas  which is a modulator of cell cycle progression and angiogenesis in melanoma [MIF has modulatory properties on natural killer cell mediated lysis of cancer cells contributing, therefore, to an immune privileged microenvironment in uveal melanoma [LCAM) and melanoma cell adhesion molecule (MCAM) were also up-regulated in both lesions and may play an important role in mediating migration of immune cells to the tumor deposits [We then pooled together melanoma and RCC samples data to identify genes commonly expressed by these tumors and not by tumors of other histology. Significance was assessed by a two-tailed unpaired student's ure MITF ,44. thature MITF  was founure MITF . It is pure MITF . In addimelanoma ,48. was melanoma . Two gendeposits . FinallyThis is a descriptive study where genes specifically expressed by melanoma metastases were identified comparing a large collection of samples from patients with metastatic cutaneous melanoma with other primary tumors and lymph nodal metastases. A limitation of the study is the lack of other samples including visceral metastases of melanoma and metastases from tumors of other histology. Nevertheless, we considered useful to compile a list of genes characteristically expressed by subcutaneous and lymph nodal lesions of melanoma for reference purposes and we are willing to provide full information about these genes upon request. In spite of the limitations of this study, few general conclusions could be drawn.Fourteen primary renal cell carcinoma (RCC) specimens were collected at the Department of Urology of The Johannes Gutenberg-University, Mainz, Germany; one primary melanoma, three in transit metastases, seven cutaneous metastases, thirty-five lymph nodal metastases and two visceral metastases of cutaneous melanoma were collected at the Department of Surgical Sciences, University of Padua, Italy; twenty-one fine needle aspirates of cutaneous melanoma metastases were obtained at the Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD; seventeen primary epithelial ovarian cancer (EOC) specimens were obtained at the Department of Gynecologic Oncology, MD Anderson Cancer Center, TX; three primary sarcoma, one primary endometrial cancer, one primary laryngeal cancer, two primary breast cancers and one primary colon adeno-carcinoma were obtained from the Tissue Network (Philadelphia, PA); twelve primary carcinomas of the esophageal junction were obtained from the NCI (Division of Cancer Treatment and Diagnosis); thirty-five primary, 16 lymph node metastases and one hepatic metastasis from colorectal adeno-carcinomas were obtained from the Department of Pathology of the University of Pisa, Italy. Specimens were collected as the result of routine operative procedures and portions were frozen for subsequent analysis while the remnant tissue was used for pathological confirmation. Tissue procurement followed standard ethical procedure according to institutional policy. A summary of the specimens studied is presented in Table Total RNA was extracted from frozen material using Trizol reagent according to manufacturer's instructions (Invitrogen, CA) and amplified into anti-sense RNA (aRNA) as previously described ,27,51,52. Micro-arrays were printed at the Immunogenetics Section, DTM, CC, NIH with a configuration of 32 × 24 × 23 and contained 17,500 elements. Clones used for printing included a combination of the Research Genetics RG_HsKG_031901 8 k clone set and 9,000 clones selected from the RG_Hs_seq_ver_070700 40 k clone set. The 17,500 spots included 12,072 uniquely named genes, 875 duplicated genes and about 4,000 expression sequence tags.Total RNA from peripheral blood mononuclear cells pooled from six normal donor was extracted and amplified to serve as constant reference as previously described ,27,51,522-based ratios normalizing the medial log2 ratio value across the array equal to zero. Validation and reproducibility were performed using our internal reference concordance system as previously described [t test. The same analyses were performed using un-paired Wilcoxon's non-parametric assessment and provided the same conclusions (not shown). Details of each analysis are presented in the results section.All statistical analyses were performed using the logescribed . Unsuperescribed  and visuescribed . DetailsAVE Ratio = average Log2 CY5/Cy3 ratio between test and reference sample. The t test p2-value refers to a two-tailed unpaired analysis between the samples mentioned below. RCC = renal cell cancer; MEL = melanoma; Other = tumors other than RCC and melanoma.Click here for file