Caenorhabditis elegans, whose neuronal network is relatively small and stereotypical from animal to animal, published wiring diagrams are neither accurate nor complete and self-consistent. Using materials from White et al. and new electron micrographs we assemble whole, self-consistent gap junction and chemical synapse networks of hermaphrodite C. elegans. We propose a method to visualize the wiring diagram, which reflects network signal flow. We calculate statistical and topological properties of the network, such as degree distributions, synaptic multiplicities, and small-world properties, that help in understanding network signal propagation. We identify neurons that may play central roles in information processing, and network motifs that could serve as functional modules of the network. We explore propagation of neuronal activity in response to sensory or artificial stimulation using linear systems theory and find several activity patterns that could serve as substrates of previously described behaviors. Finally, we analyze the interaction between the gap junction and the chemical synapse networks. Since several statistical properties of the C. elegans network, such as multiplicity and motif distributions are similar to those found in mammalian neocortex, they likely point to general principles of neuronal networks. The wiring diagram reported here can help in understanding the mechanistic basis of behavior by generating predictions about future experiments involving genetic perturbations, laser ablations, or monitoring propagation of neuronal activity in response to stimulation.Despite recent interest in reconstructing neuronal networks, complete wiring diagrams on the level of individual synapses remain scarce and the insights into function they can provide remain unclear. Even for  Caenorhabditis elegans and present several analyses of its properties, finding many nonrandom features. We give novel visualizations and compute network statistics to enhance understanding of the reported data. We also use principled systems-theoretic methods to generate hypotheses on how biological function may arise from the reported neuronal network structure. The wiring diagram reported here can further be used to generate predictions about signal propagation in future perturbation, ablation, or artificial stimulation experiments.Connectomics, the generation and analysis of neuronal connectivity data, stands to revolutionize neurobiology just as genomics has revolutionized molecular biology. Indeed, since neuronal networks are the physical substrates upon which neural functions are carried out, their structural properties are intertwined with the organization and logic of function. In this paper, we report a near-complete wiring diagram of the nematode  Determining and examining base sequences in genomes Caenorhabditis elegans is a logical model system for advancing the connectomics program. It is sufficiently small that it can be reconstructed and analyzed as a whole. The The neuronal network of the nematode C. elegans nervous system had been reconstructed using electron micrographs Despite a century of investigation C. elegans based on original data from White et al.In this paper, we advance the experimental phase of the connectomics program We advance the theoretical phase of connectomics C. elegans neuronal network in several ways. A full wiring diagram, especially when conveniently visualized using a method proposed here, helps in designing maximally informative optical ablation Our results should help investigate the function of the Organization of the C. elegans nervous system contains The gram see  of the let al.C. elegans wiring diagram.The new version of the wiring diagram incorporates original data from White The current wiring diagram is considered self-consistent under the following criteria:A record of Neuron A record of gap junction between Neuron N2Y.Although the updated wiring diagram represents a significant advance, it is only about C. elegans nervous system remain as reported previously The basic qualitative properties of the updated The majority of sensory neuron and interneuron categories contain pairs of bilaterally symmetric neurons. Motor neurons along the body are organized in repeating groups whereas motor neurons in the head have four- or six-fold symmetry. A large fraction of neurons send long processes to the nerve ring in the circumpharyngeal region to make synapses with other neurons C. elegans are structurally simple: most neurons have one or two unbranched processes and form en passant synapses along them. Dendrites are recognized by being strictly “postsynaptic” or by containing a specialized sensory apparatus, such as amphid and phasmid sensory neurons. Interneurons lack clear dendritic specialization. It is interesting to note that a given worm neuron has connections with only about The neurons in C. elegans neurons. Gap junctions are channels that provide electrical coupling between neurons, whereas chemical synapses use neurotransmitters to link neurons. The network of gap junctions and the network of chemical synapses are initially treated separately, with each represented by its own adjacency matrix, In the remainder of the paper, we describe and analyze the connectivity of gap junction and chemical synapse networks of C. elegans. Even if directionality existed, such information cannot be extracted from electron micrographs. Thus we treat the gap junction network as an undirected network with a symmetric adjacency matrix, as depicted in Although gap junctions may have directionality, i.e. conduct current in only one direction, this has not been demonstrated in C. elegans. For the linear systems analysis, we do however make a rough guess of the signs of synapses based on neurotransmitter gene expression data.Since chemical synapses possess clear directionality that can be extracted from electron micrographs, we represent the chemical network as a directed network with an asymmetric adjacency matrix, C. elegans have a further limitation that causes some synaptic ambiguity. When a presynaptic terminal makes contact with two adjacent processes of different neurons . As shown in Tables 2A and 2B in In this section, we analyze statistical properties of individual neurons and synaptic connections. To characterize the ability of individual neurons to propagate or collect signals, we compute the degree To visualize the discrete degree distribution, We perform a fitting procedure for the tail of the gap junction degree distribution To characterize the direct impact that one neuron can have on another, we quantify the strength of connections by the multiplicity, Finally, the sum of the multiplicities of all gap junction connections of a given neuron is called the number of terminals, or the nodal strength. The tail of the distribution of the number of synaptic terminals, degree centralityIdentifying neurons that play a central or special role in the transmission or processing of information may also prove useful Having described statistical properties of individual neurons and connections, such as the degree and multiplicity distributions, we now investigate properties that may describe the efficiency of signal transmission across the gap junction network. Traditionally The geodesic distance, A signal originating in one neuron in the giant component must cross gth  see . The disA second measure for signal propagation is the clustering coefficient Small world networks have much higher clustering coefficient relative to random networks without sacrificing the mean path length. For the giant component of the gap junction network, the corresponding ratios are Next we consider how quickly individual neurons reach all other neurons in the network. The normalized closeness of a neuron Restricting to the gap junction giant component, the six most central neurons are AVAL, AVBR, RIGL, AVBL, RIBL, and AVKL. In addition to command interneuron classes AVA and AVB, these include RIBL and RIGL, both ring interneurons, and AVKL, an interneuron in the ventral ganglion of the head. The set of closeness central neurons mostly overlaps with the set of degree central neurons. The correlation between the two centrality measures does not extend to peripheral neurons, as the Spearman rank correlation coefficient Global network properties discussed in the previous section characterize signal transmission while ignoring connection weights. As weights affect the effectiveness of signal transmission and vary among connections, we now analyze the weighted network by using linear systems theory. Although neuronal dynamics can be nonlinear, spectral properties nevertheless provide important insights into function. For example, the initial success of the Google search engine is largely attributed to linear spectral analysis of the World Wide Web We characterize the dynamics of the gap junction network by the following system of linear differential equations, which follow from charge conservation Assuming that gap junction conductance is greater than the membrane conductance, we temporarily neglect the last term and rewrite this equation in matrix form:The system of coupled linear differential equations (6) can be solved by performing a coordinate transformation to the Laplacian eigenmodes. Since the Laplacian eigenmodes are decoupled and evolve independently in time, performing an eigendecomposition of initial conditions leads to a full description of the system dynamics. The survival function of the Laplacian eigenspectrum is shown in What insight can be gained from inspection of the Laplacian eigenmodes? The gap junction network is equivalent to a network of resistors, where each gap junction acts as a resistor. The eigenmodes give intuition about experiments where a charge is distributed among neurons of the network and the spreading charge among the neurons is monitored in time. If the charge is distributed among neurons according to an eigenmode, the relative shape of the distribution does not change in time. The charge magnitude decays with a time constant specified by the eigenvalue. The smallest eigenvalue of the Laplacian is always zero, corresponding to the infinite relaxation time. In the corresponding eigenmode each neuron is charged equally.If the charge is distributed according to eigenmodes corresponding to small eigenvalues, the decay is rather slow. Thus, these eigenmodes correspond to long-lived excitation. The existence of slowly decaying modes often indicates that the network contains weakly coupled subnetworks, in which neurons are strongly coupled among themselves. The corresponding charge distribution usually has negative values on one subnetwork and positive values on the other subnetwork. Because of the relatively slow equilibration of charge between the subnetworks, such an eigenmode decays slowly.As an example of slow equilibration implying a subnetwork that is strongly internally coupled, one might speculate that the eigenmode associated with   on the ‘Another interesting example is the eigenmode associated with  . NeuronsThese two examples demonstrate that spectral analysis can uncover circuits that have been described using experimental studies. The probability of a known functional circuit appearing in an eigenmode by chance is small see . It woulTo prioritize further analysis of eigenmodes for biological significance, it may be advantageous to focus on the slow and sparse modes, where few neurons exhibit significant activity. We can quantify sparseness of normalized eigenmodes by the sum of absolute values of the eigenmode components, also known as the The full set of eigenmodes of the connected component is shown in Figure 2 in C. elegans neurons allows us to estimate the decay times only approximately. Assuming neuron capacitance of To understand timescales, one might wonder what the absolute values of decay constants for various eigenmodes are. Current knowledge of electrical parameters for What is the effect of the dropped term corresponding to the membrane current in (6)? As this term would correspond to adding a scaled identity matrix to the Laplacian, the spectrum should uniformly shift to higher values by the corresponding amount. Thus, even the eigenmode corresponding to the zero eigenvalue would now have a finite decay time. Assuming the membrane conductance of about In addition to highlighting groups of neurons that could be functionally related, spectral analysis allows us to predict, under linear approximation, the outcome of experiments that study the spread of an arbitrarily generated excitation in the neuronal network. Such excitation can be generated in sensory neurons by presenting a sensory stimulus To predict the spread of activity, we may decompose the excitation pattern into the eigenmodes and, by taking advantage of eigenmode independence, express temporal evolution as a superposition of the independently decaying eigenmodes. The initial redistribution of charge would correspond to the fast eigenmodes, whereas the long-term evolution of charge distribution would be described by the slow eigenmodes. Understanding propagation of neuronal activity in response to stimulation (either for the complete network or for ablation studies) may also be carried out directly in the time domain by stepping through the dynamics in (6) or more electrophysiologically realistic nonlinear dynamics. Predictions of experimental results would then be determined by stimulating and measuring exactly as in the experiment itself.Several of the quantitative properties computed thus far measure global network structure or individual neuron properties. Now we analyze the frequency of various connectivity subnetworks among small local groups of neurons. Overrepresentation in the subnetwork distribution often displays building blocks of the network such as computational units Four neurons can be wired into 11 kinds of subnetworks; this distribution is shown in Note that neurons participating in motifs also make connections with neurons outside of the motif, which are traditionally not drawn in putative functional circuits Now we consider the chemical synapse network. Recall that due to structural differences between presynaptic and postsynaptic ends of a chemical synapse, electron micrographs can be used to determine the directionality of connections. Hence the adjacency matrix is not symmetric as it was for the gap junction network.The network that we analyze consists of There are two different definitions of connectivity for directed networks. A weakly connected component is a maximal group of neurons which are mutually reachable by possibly violating the connection directions, whereas a strongly connected component is a maximal group of neurons that are mutually reachable without violating the connection directions. The whole chemical synapse network is weakly connected and can be divided into a giant strongly connected component with The random directed network corresponding to the chemical network is fully weakly connected, even when the degree distribution is taken into account see . A stronSince chemical synapses form a directed network, neuron connectivity is characterized by in-degrees (the number of incoming connections) and out-degrees (the number of outgoing connections) rather than simply degrees. The joint distribution of in-degrees and out-degrees is shown in The survival functions associated with the marginal distributions of in-degrees and out-degrees are shown in Multiplicity of connection, As for the gap junction network, we can also study the distribution of number of synaptic terminals on a neuron. This involves adding the multiplicities of the connections, rather than just counting the number of pre- or post-synaptic partners. The joint histogram (not shown) exhibits similar correlation as for the degree distribution, with Pearson correlation coefficient As for the gap junction network, we can identify central neurons cf.  for the In the strongly connected component, we can define the directed geodesic distance as the shortest path between two neurons that respects the direction of the connections. The distribution of the directed geodesic distance, Figure 1(b) in Although there are several definitions of clustering for directed graphs in the literature For directed networks, measures of in-closeness and out-closeness may be defined using the average directed geodesic distance. In particular, the normalized in-closeness is the average geodesic distance from all other neurons to a given neuron:C. elegans nervous system, signals can propagate efficiently from various sources towards these neurons and that they are in a good position to integrate it.For the giant component of the chemical network, the most in-closeness central neurons include AVAL, AVAR, AVBR, AVEL, AVER, and AVBL. All are command interneurons involved in the locomotory circuit; these neurons are also central in the gap junction network. The in-closeness centrality of command interneurons may indicate that in the The most out-closeness central neurons include DVA, ADEL, ADER, PVPR, AVJL, HSNR, PVCL, and BDUR. Only PVCL is a command interneuron involved in locomotion. The neuron DVA is an interneuron that performs mechanosensory integration; ADEL/R are sensory dopaminergic neurons in the head; and the other central neurons are interneurons in several parts of the worm. The out-closeness centrality of these neurons may indicate that signals can propagate efficiently from these neurons to the rest of the network and that they are in a good position for broadcast.C. elegans, where neurons do not fire classical action potentials Although chemical synapses are likely to introduce more nonlinearities than gap junctions, linear systems analysis can provide interesting insights, especially in the absence of other tools. Such an approach has additional merit in A major source of uncertainty in linear systems analysis of the chemical network is the unknown sign of connections, i.e. excitatory or inhibitory, due to the difficulty in performing electrophysiology experiments. We use a rough approximation that GABAergic neurons make inhibitory synapses, whereas glutamergic and cholinergic neurons form excitatory synapses Similarly to the gap junction network, we write the system of linear differential equations for the chemical synapse network To avoid redundancy we defer analyzing this system of differential equations to the next section, where we consider the combined system including both gap junctions and chemical synapses.We also find subnetwork distributions for the chemical synapse network. Since the network is directed, there are many more possible subnetworks. In particular there are C. elegans network contains similar overrepresented subnetworks as found by analyzing incomplete data Overrepresentation of reciprocal Having considered the gap junction network and the chemical synapse network separately, we also examine the two networks collectively. To study the two networks, one may either look at a single network that takes the union of the connections of the two networks or one may look at the interaction between the two networks.First we look at a combined network, which is produced by simply adding the adjacency matrices of the gap junction and chemical networks together, while ignoring connection weights. Thus we implicitly treat gap junction connections as double-sided directed connections. This new network consists of Naturally, the combined network is more compact than the individual networks. The mean path length Turning to closeness centrality, the most in-close central neurons are AVAL/R, AVBR/L, and AVEL/R, as would be expected from the individual networks. The most out-close central neurons are DVA, ADEL, AVAR, AVBL, and AVAL, which include the top out-close neurons for both individual networks.We can also calculate the degree distribution of this combined network. The Pearson correlation coefficient between the in-degree and out-degree is The neurons with the greatest degree centrality are AVAL and AVAR. As for the chemical synapse network, neuron AVAL has the largest in-degree and AVAR has the second largest in-degree, whereas AVAR has the largest out-degree and AVAL has the second largest out-degree  and 4(b) in As for the chemical synapse network, the tail of the out-number distribution was fit by a power law and the tail of the in-number distribution could not be fit satisfactorily. The tail of the out-number distribution could also be fit by an exponential, albeit with lower likelihood. The multiplicity can be fit satisfactorily by a stretched exponential.In this section we apply linear systems analysis to the combined network of chemical synapses and gap junctions taking into account multiplicities of individual connections. Due to our ignorance about the relative conductance of a single gap junction and of a single chemical synapse, we assume that they are equal. By combining equations (6) and (12) we arrive at:We proceed to find a spectral decomposition for the combined network. To avoid trivial eigenmodes, we restrict our attention to the strongly connected component of the combined network containing What is the meaning of complex eigenvalues? The imaginary part of an eigenvalue is the frequency at which the associated eigenmode oscillates. The real part of an eigenvalue determines the amplitude of the oscillation as it varies with time. Eigenmodes that have an eigenvalue with a negative real part decay with time, whereas eigenmodes that have an eigenvalue with a positive real part grow with time. When examining the temporal evolution of the eigenmodes whose eigenvalues are shown in As for the gap junction network alone, we can look for eigenmodes that may have functional significance. For example, the sixth eigenmode of the combined network, Having the eigenspectrum of the combined network allows one to calculate the response of the network to various perturbations. By decomposing sensory stimulation among the eigenmodes and following the evolution of each eigenmode, one could predict the worm's response to the stimulation. A similar calculation could be done for artificial stimulation of the neuronal network, induced for example, using channelrhodopsin As noted for the gap junction alone, the network may also be studied in the time domain directly by stepping through the dynamics in (13) or more electrophysiologically realistic nonlinear dynamics.We have measured the structural properties of the combined network formed by adding together the adjacency matrices of the gap junction and chemical synapse network, however it is unclear how they interact. The two networks could be independent, or their connections could overlap more or less often than by chance.To investigate how the two networks overlap, we look at local structure. C. elegans. Thus, there may be a functional role for correlation/anticorrelation of the joint presence of gap junction and chemical connections.Durbin had found that chemical and gap junction networks are essentially independent when imposing physical adjacency restrictions Why might the presence of connections in two networks either be correlated or anticorrelated? One possibility is that correlated connections simultaneously perform different functions What are the different functions performed by chemical synapses and gap junctions that could lead to correlation? One possibility is that the two different functions are sign-inverting and non-inverting coupling. Gap junctions are non-inverting: higher potential in a neuron raises the potential in other gap-junction-coupled neurons. Chemical synapses, on the other hand, may be either excitatory (non-inverting) or inhibitory (inverting). When the likelihood computations are repeated considering only neuron pairs where the presynaptic neuron is known to be GABAergic Another measure of the interaction between the two networks is the correlation between the degree sequences. The Pearson correlation coefficient between the gap junction degree and the chemical network in-degree is greater than the Pearson correlation coefficient between the chemical network in-degree and out-degree. The Pearson correlation coefficient between the gap junction degree and the chemical network out-degree is less than the Pearson correlation coefficient between the chemical network in-degree and out-degree. This is shown in The two networks seem to primarily reinforce each other with correlated structure rather than augment each other with anticorrelated connections.Although the reported wiring diagram corrects errors in previous work and is considered self-consistent, one might wonder how remaining ambiguities and errors in the wiring diagram might affect the quantitative results presented. Furthermore there are connectivity pattern differences among individual worms; these individual variations may have similar effects on the analysis as errors and ambiguities.For network properties that are defined locally, such as degree, multiplicity, and subnetwork distributions, clearly small errors in the measured wiring diagram lead to small errors in the calculated properties. For global properties such as characteristic path length and eigenmodes, things are less clear.To study the robustness of global network properties to errors in the wiring diagram, we recalculate these properties in the wiring diagrams with simulated errors. We simulate errors by removing randomly chosen synaptic contacts with a certain probability and assigning them to a randomly chosen pair of neurons. Then, we calculate the global network properties on the ensemble of edited wiring diagrams. The variation of the properties in the ensemble gives us an idea of robustness.First, we explore the robustness of the small world properties and the giant component calculations. We edit wiring diagrams by moving each gap junction contact with Properties for the neuronal network from prior work in Second, we characterize robustness for the linear systems analysis. Because of greater sensitivity of the eigenvalues to errors, we edit wiring diagrams by moving each gap junction contact with  network  are showIn addition to considering the effect of typical random edits, we can characterize the effect of worst-case errors on the eigenvalues using the of norm  . For theElectron micrographs of chemical synapses have a further ambiguity when more than one postsynaptic partner receives input at a release site. We treated such polyadic (send_joint) synapses no differently than other synapses, but one might alternatively determine multiplicity by counting such synapses at Small deviations from equality when weighting gap junctions and chemical synapses to form the combined network yield similar spectral changes as the alternate quantitation of chemical synapses displayed in C. elegans using materials from White et al.We have presented a corrected and more comprehensive version of the neuronal wiring diagram of hermaphrodite We proposed a convenient way to visualize the neuronal wiring diagram. The corrected wiring diagram and its visualization should help in planning experiments, such as neuron ablation.Next, we performed several statistical analyses of the corrected wiring, which should help with inferring function from structure.By using several different centrality indices, we found central neurons, which may play a special role in information processing. In particular, command interneurons responsible for worm locomotion have high degree centrality in both chemical and gap junction networks. Interestingly, command interneurons are also central according to in-closeness, implying that they are in a good position to integrate signals. However, most command interneurons do not have highest out-closeness, meaning that other out-closeness central neurons, such as DVA, ADEL/R, PVPR, etc., are in a good position to deliver signals to the rest of the network.Linear systems analysis yielded a principled methodology to hypothesize functional circuits and to predict the outcome of both sensory and artificial stimulation experiments. We have identified several modes that map onto previously identified behaviors.C. elegans nervous system. To enable comparison of the C. elegans network with other natural and technological networks Networks with similar statistical structural properties may share functional properties thus providing insight into the function of the The tails of the degree and terminal number distributions for the gap, chemical and combined networks (with the exception of the in-numbers) follow a power law consistent with the network being scale-free in the sense of Barabási and Albert C. elegans network are similar to those of the mammalian cortex. In particular, the whole distribution of C. elegans chemical synapse multiplicity is well-fit by a stretched exponential (or Weibull) distribution  We started assembling the wiring diagram by consolidating existing data from both published and unpublished sources. Using J. G. White N2UNext we incorporated R. M. Durbin's data for the anterior portion of the worm, reconstructed from animal Studies based on green fluorescent protein (GFP) reporters mostly confirm the electron micrograph reconstructions described in MOW. A few differences between GFP-stained neurons and White's work have been observed . Notably, the anterior processes of DVB and PVT could have been mistakenly switched in MOW C. elegans where most neuron cell bodies reside. Reconstructions of neurons in the mid-body of the worm, on the other hand, are scant and incomplete. From a combination of published works Most published works have focused on the neck and tail regions of C. elegans archive (Albert Einstein College of Medicine), we uncovered a large number of reconstruction records in White et al.'s laboratory notebooks. These notebooks identified neurons by different color code labels depending on the animal, the location of the neurite , and magnification of the electron micrograph. To ascertain the identity of the neurons, we relied on a combination of color code tables and comparisons of common anatomical structures between electron micrograph prints. In the end, we identified notes for full reconstructions of At the N2U worm prepared by White et al., we produced new high power electron micrographs of this dorsal region. Due to the condition of the sections, only one of every We found that a large section on the dorsal side of the worm, from just anterior to the vulva to the pre-anal ganglion, was never electron micrographed at high power magnification. This dearth of imagery was why so many neurons were missing dorsal side reconstructions. Using original thin sections for the et al. Connections in the posterior region of the animal were also cross-referenced with reconstructions published in From our compilation of wiring data, including new reconstructions of ventral cord motor neurons, we applied self-consistency criteria to isolate neurons with mismatched reciprocal records. The discrepancies were reconciled by checking against electron micrographs and the laboratory notebooks of White For a random network with To apply this method to the weakly connected component of a directed network, we are interested in the undirected network formed by adding a connection between two neurons if there is a connection in either direction. For a random directed network with probability Consider the ensemble of random networks with a given degree distribution Using the computed For the symmetrized chemical network, the generating function corresponding to the measured degree distribution isThe expected fraction of the network taken up by the giant component, Continuing from the previous subsection, we find the derivative of the generating function To find functional forms of the tails of various distributions, we follow the procedure outlined in Let us bound the probability of finding an eigenmode that comprises a random set of neurons. Let Now go through each eigenmode and add to a list all possible unordered Additionally, we can compute the number of all unordered Thus, if a random set of neurons was selected from all possible sets of neurons, the probability Suppose we are interested in putative functional circuits of size Suppose we know The likelihood ratios shown in We used the MATLAB package EigTool http://mit.edu/lrv/www/elegans/. This collection of software may be used not only to reproduce most of the figures in this paper, but also for future connectomics analyses.Note that MATLAB code for computing several network properties is available at The collected data is available from the WormAtlas Text S1Algorithm for directed network drawing.(0.09 MB PDF)Click here for additional data file.Text S2Algebraic form of survival functions.(0.09 MB PDF)Click here for additional data file.Text S3Eigendecomposition.(0.16 MB PDF)Click here for additional data file.Text S4Supporting figures and tables.(0.08 MB PDF)Click here for additional data file.


We report on atomistic simulation of the folding of a natively-knotted protein, MJ0366, based on a realistic force field. To the best of our knowledge this is the first reported effort where a realistic force field is used to investigate the folding pathways of a protein with complex native topology. By using the dominant-reaction pathway scheme we collected about 30 successful folding trajectories for the 82-amino acid long trefoil-knotted protein. Despite the dissimilarity of their initial unfolded configuration, these trajectories reach the natively-knotted state through a remarkably similar succession of steps. In particular it is found that knotting occurs essentially through a threading mechanism, involving the passage of the C-terminal through an open region created by the formation of the native  It has been recently observed that the native structure of proteins can contain knots. These are formed during the folding process and are tightened in a specific (i.e. native) location, along the poly-peptide chain. The existence of knots hence implies a high degree coordination of local and global conformational changes, during the folding reaction. In this work we investigate how the knot is formed and what are the dynamical mechanisms which drive the self-entanglement process. To this end, we report on the first atomistically detailed numerical simulation of the folding of a knotted protein, based on a realistic description of the inter-atomic forces. These simulations show that the knot is formed by following a specific sequence of contacts. The comparison of the findings with those based on simplified folding models suggest that the productive succession of contacts is aided by a concerted interplay of amino acid interactions, arguably including non-native ones. In other protein contexts, this method was shown to yield results consistent with standard extensive MD folding simulations, performed with the same atomistic force field We find that self-knotting of MJ0366 typically occurs at a late folding stage, when about The monomeric unit of the natively-knotted MJ0366 protein consists of 82 amino acids and comprises four We report on the characterization of the folding process of MJ0366 by means of advanced molecular dynamics techniques based on the AMBER99ffSB atomistic force field Specifically, we first generated an ensemble of 100 denatured configurations for protein MJ0366 by unfolding the crystal structure using 100 ps of atomistic molecular dynamics (MD) simulations at high temperature (1600 K) followed by 100 ps of thermalization at 300 K.a posteriori the relative likelihood of each computed folding pathways Next, the folding and knotting dynamics of MJ0366 was studied by carrying out 40–50 folding attempts for each of the 100 denatured configurations, for a total of about 4000 attempted folding trajectories. Simulating so many folding trajectories from an initially unfolded state is presently beyond reach of standard MD simulations even when run on dedicated supercomputers The strength of the rMD scheme is that it allows for efficiently generating an ensemble of trial folding pathways from a given initial denatured state to the known native state, while keeping at a minimum the external work applied to drive the system. In fact, the system dynamics evolves in a completely unbiased way whenever it leads to a higher similarity with the native state, i.e. a larger number of formed native contacts. Conversely, a time-dependent external force is introduced to discourage, though not completely prevent, a decrease of the native similarity. The biased rMD evolution promotes only the overall geometrical similarity with the native state and does not reward specific concerted backbone movements that could lead to knotting. As a matter of fact, knot formation was observed only for a small fraction of the thousands of attempted rMD trajectories, namely 66 of them, covering 31 distinct initially-unfolded states. In all cases, the knotting event corresponded to the formation of the native trefoil knot, thus indicating that incorrect knot formation is not a major source of kinetic trapping for MJ0366.unbiased stochastic dynamics, the DRP selection criterion lessens a posteriori the rMD steering effects.In the DRP approach, only one productive pathway per initial condition was retained, namely the one with the highest statistical weight. This weight, corresponds to the probability that each trial trajectory is generated by an overdamped Langevin dynamics. Notice that, because the weights are calculated with reference to an The selected 31 trajectories were analyzed by monitoring the evolution of several geometrical and topological parameters during the folding process.As a first step we identified the folding stage at which the backbone self-ties into knot. Accordingly, for each trajectory we calculated the percentage of native contacts (overlap) that are formed when the first knotting event occurs. The distribution of these overlaps for the considered trajectories is shown in Next, to characterize the diversity of the folding pathways and the implications for the knotting mechanism, we computed the average “path similarity parameter”, To have a robust indication of the degree of heterogeneity of the selected trajectories, we computed the distribution of We accordingly sought to analyze in detail the folding process to verify that knotting occurs via one dominant mechanism and characterize it.et al.In this regard a valuable term of reference is given by the earlier study of Noel Here, by addressing the same protein folding process with a realistic, non native-centric force-field, it is possible to examine to what extent various aspects of the knotting process are sensitive to the treatment of inter-atomic interactions.As a first step of the analysis, we profiled the folding trajectories along two relevant order parameters: the root mean square distance (RMSD) to the native structure and the RMSD to the native Since in the native MJ0366 structure the C-terminal helix protrudes through the region intervening between the two paired The results shown in the left panel of The detailed inspection of the trajectories indicates that the knotting process almost invariably occurs through the so-called “threading” mechanism, where the In three other cases, the folding was found to occur through the so-called “slipknot” mechanism Finally, in two further cases we observed another knotting mechanism which involves a concerted backbone movement that had not been previously reported for MJ0366. Specifically, in two trajectories when the Videos obtained from the atomistic DRP trajectories which illustrate the three observed knotting mechanisms are included in the on-line SI.It is important to notice that the trajectories associated to the various knotting modes do not present significant quantitative differences regarding the overall solvent accessibility of polar and non-polar residues during the folding process. This point is illustrated in unknotted configuration and nevertheless had a good native similarity .To understand how the interplay of amino acid interaction captured by the realistic force field favours knotting by threading, we have carried out a comparative analysis of the reaction mechanism in successful and unsuccessful folding trajectories. Specifically, the productive, successful set consisted of the 26 trajectories displaying the dominant (threading) knotting mechanism. The non-productive one included an equal number of trajectories that reached an The projection of the unsuccessful trajectories along the two collective coordinates considered before is shown in This conclusion is supported by the detailed inspection of the unsuccessful trajectories, which are exemplified in the sequence of snapshots shown in The relevance of this mechanism for misfolding is highlighted by the fact that all unsuccessful trajectories displayed a late formation of the The fact that the observed dominant knotting mode differs from the one reported previously using pure native-centric force fields suggests that non-native interactions could be relevant for favouring the correct succession of contacts leading to self-knotting (or avoiding unproductive ones). This possibility is particularly interesting in connection with the ongoing discussion about the role that non-native interactions can have in aiding the knotting process even during the early folding To investigate this aspect we generated several folding trajectories for MJ0366 using simplified models where the effect of non-native interactions could be easily turned on or off. Specifically, we considered two different coarse-grained models: one with only native-centric interactions and the other additionally incorporating non-native interactions. The latter included quasi-chemical and screened electrostatic pairwise interactions, as in the recent study of the early folding stages of a trefoil-knotted carbamoyltransferases The folding process presents major differences in the two models. First, they differ significantly in terms of knotting probability. Specifically, for each model we considered an extensive set of 10,000 uncorrelated configurations, equilibrated at the nominal temperature of 300 K. In the native-only case, 12% of the sampled configurations were knotted, while this number had a sixfold increased, to 75%, in presence of non-native interactions. This result aptly complements the atomistic DRP simulations, for highlighting the role of non-native interactions in aiding the formation of the native knotted topology of MJ0366.Secondly, productive trajectories follow different dominant mechanisms in the two models. In fact, when the pure native-centric model is used, 8 out of the 10 trajectories involved the slipknotting mechanism, while the threading one was observed in all trajectories (10 out of 10) with the additional non-native interactions. The latter result, which is in full accord with the atomistic DRP simulations, reinforces the concept that non-native interaction can promote the correct order of contact formation required for self-knotting.This point is further supported by the inspection of the density plots in Based on these results, we can argue that mutations in the In conclusion, the DRP simulations presented here provided the first systematic attempt to characterize the folding process of a natively-knotted protein, MJ0366, using a realistic atomistic force field. MJ0366 knotting is observed to occur via threading at the C-terminal. The comparison of productive and unproductive trajectories (which respectively end up in natively-knotted and unknotted states) further indicates that knotting is aided by the early formation of the native a priori reason to expect that non-native interactions are crucial for guiding the folding process of knotted proteins in general, it is interesting to notice that their important role has been previously suggested for another trefoil-knotted protein carbamoyltransferases While there is no 0 = 7.5 Å is a fixed reference distance.In order to generate an ensemble of trial trajectories connecting a given initial configuration to the native state we used the following variant of the rMD algorithm. At each integration step, we evaluated a collective coordinate (CC) which measures the distance of between the instantaneous contact map and the native contact map:In the rMD algorithm, no bias is applied to the chain when it spontaneously diffuses towards the bottom of the folding funnel, i.e. any time the value of the CC at time In the original formulation of the rMD algorithm The DRP algorithm is used to identify the most probable path in each set of trial rMD trajectories sharing the same boundary conditions. This is done by evaluating the relative probability for each path In both rMD and standard high-temperature MD simulations we used the AMBER ff99SB force field 8 attempted MC moves, corresponding to 1.54 saved frames. MC moves that we have employed were the local crank-shaft and Cartesian moves, whose boldness was chosen such that the acceptance rate was nearly constant and approximately equal to 50The CG folding simulations were based on the model developed in Ref.s α at a sequence distance of 10. If the majority of the 100 stochastic closures return non-trivial Alexander determinants, then the whole conformation can be considered as globally knotted. Because protein knotting can occur through slipknot formation The conformations visited during the MC dynamics were analysed to establish their global and local knotted state. The global topological state was established and assigned by computing the Alexander determinants after suitable closure of the whole protein chain into a ring. For each configuration, this entailed 100 alternative closures where each terminus is prolonged far out of the protein along a stochastically chosen direction, and the end of the prolonged segments are closed by an arc “at infinity” (i.e. not intersecting the protein). As in ref. To quantitatively measure the folding pathways diversity we implemented the analysis described in Camilloni et al.


As largely explained by Lanna et al , the anaAt Beata Vergine Hospital, in Mendrisio, Switzerland, we carry out approximately 1000 shoulder surgeries per year. We are used to practice general anaesthesia with the auxiliary of a laryngeal mask added to ultrasound (US)-guided interscalene brachial plexus block (ISB).The advantage of this combined method is a better miorelaxation of the muscular section where the operation takes part. A minor use of opiate and hypnotics, which are used only during induction with general anaesthesia, lead with low anaesthetic gas dosage. At least, the extension and the action of the local anaesthetic (LA) used for the ISB allows us to set up an adequate analgesic therapy before the pain gets unmanageable.More recently the use of US-guided nerve blockade instead of a nerve stimulation-guided one has brought to an improvement regarding the ISB. The advantages are the visualization of the needle, the nervous structures and the LA injection during the procedure. The use of US-guided ISB reduces the number of attempts and allows the use of ultra-low volumes of LA. This procedure is more effective and successful in most cases and shows a reduction of the complications .The completion of the anaesthetic technique of ISB does not influence surgical schedule as the US-guided nerve blockade concedes an important time saving.Attention has to be focused on postoperative pain control. Even if the ISB ameliorates and lengthens the analgesia, after surgery some patients notice pain not controlled by medical drugs.The application of a continuous infusion of local anaesthetics through an interscalene catheter seems the best available technique to achieve pain relief after shoulder surgery.If the patient is discharged the second postoperative day, it requires a well-organized home health care in order to control the patient.Even though our attention is focused on postoperative pain control, the medical drugs used at present, e.g. Paracetamol (1000 mg each 6 hours), Novalgin (1000 mg each 8 hours) and Morphin (3 mg on demand), are not able to control pain in all patients.To such purpose we are planning an experimental study of US-guided interscalene brachial plexus anaesthesia, using a local anaesthetic (Prilocaine 1 %) with cortisone (Dexamethasone), in order to estimate if the extension of the analgesia given from the combination of these drugs, about 72 hours as we expect, allows us to achieve a better postoperative pain control.Anaesthesiological procedures for shoulder surgery, more than other procedures, have gone through many changes given by the utilization of the US and by the current research of new methods in order to improve postoperative pain control.


Controlling the movement of the arm to achieve a goal, such as reaching for an object, is challenging because it requires coordinating many muscles acting on many joints. The central nervous system (CNS) might simplify the control of reaching by directly mapping initial states and goals into muscle activations through the combination of muscle synergies, coordinated recruitment of groups of muscles with specific activation profiles. Here we review recent results from the analysis of reaching muscle patterns supporting such a control strategy. Muscle patterns for point-to-point movements can be reconstructed by the combination of a small number of time-varying muscle synergies, modulated in amplitude and timing according to movement directions and speeds. Moreover, the modulation and superposition of the synergies identified from point-to-point movements captures the muscle patterns underlying multi-phasic movements, such as reaching through a via-point or to a target whose location changes after movement initiation. Thus, the sequencing of time-varying muscle synergies might implement an intermittent controller which would allow the construction of complex movements from simple building blocks. We perform reaching movements frequently and effortlessly, for example when eating food or using a tool. Reaching is a prototypical goal directed behavior and, as such, has been investigated extensively in human and non-human primates. Kinematic and kinetic analyses of reaching have revealed invariant features suggesting that the central nervous system (CNS) relies on simple rules for movement planning and execution. For point-to-point movements, hand paths are often roughly straight and tangential velocity is “bell-shaped” Morasso, . MoreoveIn contrast, the analysis of the electromyographic (EMG) activity recorded from many muscles acting on the shoulder and elbow joints has revealed complex dependencies of the shape and timing of the EMG waveforms on the movement direction and speed. For reaching in vertical planes, the EMG waveforms are constructed by combining components related to both dynamic and gravitational torques  is a D-dimensional vector that specifies the activation of each muscle at time t and ci(t) is the time-varying combination coefficient for the i-th synergy. Across movement conditions, either the synergies wi or the activation coefficients ci(t), also referred to as temporal components   and Equation 1 can be written as m(t)) Figure . In thiss Figure  m(t)The combination of time-varying synergies can be seen as a special case of anechoic mixture model  and the number of synergies. The optimal number of synergies was selected as the number at which the R2 curve had a change in slope, suggesting that additional synergies only captured small residual amounts of variation attributable to noise.The analysis of the muscle patterns for fast reaching movements in 3D revealed that the complex dependence of the muscle activation waveforms on movement direction results from the combination of 4/5 time-varying synergies  are generated by recruiting the second plus the third synergy, and the fourth synergy with smaller amplitude and later in time. The second and the third synergies are also recruited in a downward movement (fourth column), but with a different balance of activation and a different relative timing. Thus, different muscle patterns underlying reaching movements with different kinematics are captured by selecting a small number of parameters.The reconstruction of the muscle patterns by synergy combination for movements in different directions occurs by recruiting the synergies with different amplitude and at different times Figure . For exaPlotting the dependence of the synergy amplitude coefficients on the movement direction in a polar plot Figure  clearly If movement direction can be controlled by modulating the recruitment of a few time-varying muscle synergies according to a cosine directional tuning, is movement speed also related to synergy recruitment in a simple way? The invariances observed in the arm kinematics and present in the equations of motions for an articulated arm suggest that a simple scaling rule might be used to control speed. Reaching movements between two given locations are executed at different speeds along an invariant path (Soechting and Lacquaniti, The analysis of the muscle patterns for reaching in different directions and with different speeds supports the notion of a simple scaling rule for speed control (d'Avella et al., When reaching a set of different targets in sequence or a target whose location changes after movement initiation, movement kinematics may be complex, with curved paths, and multiple peaks in the tangential velocity. At the kinematic level, such multi-phasic movements can be decomposed as a sequence of superimposed sub-movements, each with the same features of point-to-point movements (Flash and Henis, Sensory feedback is crucial for the control of accurate reaching movements and an internal model of the dynamics of the musculoskeletal system can be exploited to construct an optimal feedback controller (Todorov and Jordan, Reaching muscle patterns are reconstructed by the combinations of a few time-varying muscle synergies. The complex changes of the activation waveforms of individual muscle across movement direction and speed are captured by the modulation in amplitude and timing of these synergies according to simple rules, such as amplitude cosine tuning for direction and time scaling for speed. Multi-phasic reaching movements, such as reaching through a via-point or toward a target whose location changes after movement initiation, appear to be generated by sequencing and superimposing the same small set of muscle synergies identified in point-to-point movements. Thus, the regularities observed in the muscle patterns across movement conditions suggest that muscle synergies are building blocks used by the CNS to control goal directed movement. However, regularities may derive from optimization or task constraints (Todorov and Jordan, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We present a study of an electrical model of electromechanically active carbon-polymer composite (CPC) with carbide-derived carbon (CDC) electrodes. The major focus is on investigation of surface electrode behavior upon external bending of the material. We show that electrical impedance measured from the surface of the CDC-based CPC can be used to determine the curvature of the material and, hence, the tip displacement of a CPC laminate in a cantilever configuration. It is also shown that by measuring surface signals in the process of an actuator’s work-cycle, we obtain a self-sensing collision-detecting CPC actuator that can be considered as a counterpart of biomimetic vibrissae. Ionic EAP (IEAP) is one of two main classes of electroactive materials, where actuation is primarily caused by the displacement of ions inside a polymer matrix . Perhapsetc. by helping navigation in the dark. Moreover, even object recognition is proposed as one of the functions and capability of vibrissae arrays [In Nature, vibrissae  are sensory hairs that respond to mechanical stimuli such as displacement, tension and pressure ,5. VibriIn recent years the range of ionic EAP materials has been extended significantly by means of materials used for electrodes, separator membrane, as well as the electrolyte –12. At fetc.) have become very promising materials for electrodes in several electroactive devices, including IEAP. One of the concepts behind the contribution of porous carbon to the electroactivity of a material is related to the electric double layer emerging at the boundary between carbon and electrolyte, therefore the surface area of this boundary is of great importance. Due to its high specific surface area, nanoporous carbon is one of the most widely used materials for electrodes in electric double-layer capacitors (EDLC) [Lately, carbon and its many allotropes  .i.e., it behaves as an actuator (Carbon-polymer composites (CPCs) are a type of ionic electromechanically active structure, similar to EDLCs in the sense of assembly, as well as the working principle. They consist of two porous carbon electrodes separated by an ion-permeable polymer film. The whole CPC structure—separator membrane and electrodes—contains an ionic liquid as electrolyte. Due to a combination of different charging and ionic effects when relatively low voltage is applied between the surface electrodes, the CPC laminate bends, actuator . A 3-by-Nearly all ionic EAPs also function as sensors: they generate a voltage or electric current when stimulated mechanically . DespiteFurthermore, electromechanically active materials (including CPCs) are considered attractive for biomimetic applications as they are soft, noiseless and can be cut into various sizes and shapes . Additioetc. Using such feedback devices can, due to their size, cost and complexity, remarkably limit the practical application areas of EAP actuators. It is thus desirable to avoid unwieldy off-board sensors for closed-loop control.However, in order to utilize CPC  actuators in real-life application, one must find a suitable technology for closed-loop control of the actuator. To date, the most typically implemented feedback sources for ionic EAPs are laser displacement sensors , video cet al. mounted a strain gage on an IPMC surface electrode to track the movement of the actuator [It is also possible to create a bending actuator system where either some type of material parameter or a sophisticated lightweight sensor is used as the feedback upon tip displacement. For example, Leang actuator , while oactuator ,21. The actuator ,22,23, pConsidering IPMC, the change in resistance of its surface electrodes due to the actuator’s curvature has been of essence when creating a self-sensing device. Thus, investigating the carbon-based electrode of a CPC would be vastly informative in regards to comparing IPMC and CPC. As mentioned above, CPCs are also very similar to EDLCs and therefore the super capacitive effect cannot be neglected . Based oIn this paper, by means of electrical impedance spectroscopy (EIS), a relation between the shape of a CPC actuator/sensor and its electrical parameters  is described. A study into how different components of a CPC actuator affect its behavior and electrical properties is carried out and presented. More specifically, the main focus is placed on investigating these characteristics by measuring signals from the surface electrode of the CPC laminate. Distinctive resistive and capacitive effects emerge on bending of the actuator, thus providing a concept of self-sensing CPC actuator, able to detect obstacles in its path and, hence, act as artificial vibrissae. This paper also extends the concept of self-sensing IPMC to a CPC actuator.When interpreting the gathered EIS data two ideologically different approaches are used. First, CPC is considered a black-box device and therefore, we only describe different curvature dependent electrical effects that allow utilization of CPC as a sensor. The second approach applies the theory of impedance spectroscopy in order to give detailed electrical model of CPC.A CPC actuator is a quite complex system composed of an ionic liquid and special carbon derivative in a somewhat sophisticated structure. Both conductive and isolative components are integrated, while electron conductance is present only in carbon layers; on the other hand, ions can move throughout the CPC laminate. For these considerations, in order to describe an accurate electrical model of a CPC laminate, it is also necessary to investigate the properties of the components that make up the CPC actuator. As the current study focuses on carbon electrode behavior and signals measured from it, the electrode material itself is thoroughly investigated.The remainder of the paper is organized as follows: in Section 2 we briefly describe the CPC material and its fabrication process. In Sections 3 and 4, a series of electrical impedance spectroscopy measurements are presented, resulting in a presumptive equivalent circuit of a CPC actuator and sensor. Section 5 describes a possible application of a CPC actuator-sensor—a groping collision detector.2.i.e., it behaves as an actuator (Carbon-polymer composite (CPC) consists of two porous carbon electrodes separated by an ion-permeable polymer film . The caractuator .The general understanding about the working mechanism of a CPC actuator is not conclusive and is the subject of some dispute. As one plausible explanation, an electro-osmotic effect caused by the voltage applied between the carbon electrodes is proposed. In that scenario, the applied voltage causes a migration of cations towards one surface of the material, and anions move in the opposite direction. Due to the difference in dimensions of anions and cations, the unbalanced distribution of ions causes the side with larger ions to stretch, while the opposite surface is compressed ,17. This2/g, poly(vinylidene fluoride-co-hexafluoropropene)—PVdF(HFP)—as binding polymer and separator, and 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIBF4) as the ionic liquid. A more detailed description of the preparation process of such CPC actuators is given by Torop et al. [The measurements described in the current paper were conducted using a CPC material fabricated in-house. It is composed of boron carbide-derived carbon with a specific surface area of 1,800 mp et al. .All of the experiments described in this paper were conducted on CPC samples with 10 × 30 mm dimensions. The 150 μm thick CDC electrodes are isolated by a separator with a thickness of 30 μm .3.While the structure and chemical composition of the CPC material is defined by the fabrication process, it is not conclusively clear how these components interact with each other in the sense of electrical signals. In order to obtain a thorough understanding of the internal mechanisms of a CPC, electrical impedance spectroscopy (EIS) is utilized. EIS allows characterization of CPC by an equivalent circuit made up of resistors, capacitors, and possibly inductances. This section implements EIS to the surface electrode of the CPC laminate for the purpose of determining the electrical model of a CPC that can explain some of the processes occurring on the surface electrode in the course of bending.et al. [Contrarily to the popular lumped models of the EAP devices –27, we aet al. . It consEZ and MZ, and expect that some of them show definite correspondence between its value and the curvature of the material. The experiments described hereafter study the whole device as well as the components separately using the electrical impedance spectroscopy (EIS) equipment, while different degrees of bending were imposed to the strip of the material, summarizing with a reasonable equivalent circuit.Initially we represent the impedances of the electrodes and the membrane as the general impedances 3.1.κ = 1/r, of bent CPC is directly related to the displacement of one  tip of the actuator, since in the cantilever configuration, the other end of the material always remains fixed. The desired curvature was externally imposed to the CPC actuator by means of a special rig that allowed varying the curvature of the CPC laminate in the range of 15 to 100 m−1  were generated at the sampling rate of 1 kS/s.In these measurements a CPC sheet was placed in a cantilever configuration, as depicted in Δx in In the first series of experiments, the strain of CPC sample was caused by the input driving signal. Continuous movement of the tip occurred for the whole 30 seconds of applied voltage, reaching the displacement of ∼10 mm in the direction of horizontal axis ;the time constant is dynamically changing due to the instantaneous curvature of the actuator.The comparison of voltages along the opposite electrodes of both—unobstructed and fixed actuator—are given in Separating these two processes is a rather complex procedure, however, based on RV) is roughly similar to the previously fixed case. The irregular behavior of the voltage drop along the stretching electrode (LV) indicates that the resistance and apparent capacitance of the corresponding electrode are not constant. Although in Section 4 we showed that no element of capacitance is found in the surface electrode, it can be stated that this outcome complies with fore mentioned results where bending of a CPC actuator causes a significant change in apparent capacitance measured from the surface that is undergoing expansion  an opposing beam forces the actuator back to its initial state. This impediment is unmistakably visible in voltage drops of each surface, hence providing a self-sensing collision detecting CPC actuator. Interestingly this phenomenon appears to have symmetric nature on both voltage drop, thus indicating that this effect is not caused only by the alteration of surface resistance  actuator based on measurements conducted mainly on its surface electrode. It is shown that alteration in resistance of the electrode due to the bending is present for the CPC actuator as well as for the electrode itself. However, a noteworthy effect of capacitance is also measured from the surface electrode of the CPC laminate. When investigating the components of CPC individually, we demonstrate that no detectable capacitance is present on single surface electrode, hence, indicating to a purely resistive nature of CDC-based electrodes. Therefore, this apparent capacitance is caused by the coupling effect of the PVdF membrane separating two electrodes of the CPC actuator. Moreover, whenever measuring signals from any surface of a CPC actuator, one has to consider this capacitive coupling between electrodes that is easily detectable from the surface undergoing expansion. However, the apparent capacitance shows a good correlation to the bending curvature of a CPC sample, thus, providing another means to a position sensing with CPC.As measurements of cross-electrode impedance indicate that no significant alteration of electrical parameters is detected on the separator membrane, most of the electrical phenomena observed during bending must, thus, be contributed to the properties of surface electrode.Empirical resistive and capacitive effects are more prominent when measuring the surface that is being stretched as a result of actuation or external bending. A self-sensitivity of completely blocked or externally manipulated CPC actuator is also presented in this paper. This type of self-sensing system can be utilized as biomimetic vibrissae that allow tactile feedback.Implementation of this type of tactile self-sensing CPC actuator in a robotic application is the subject of planned future work, along with a study into appropriate conditioning circuits to better benefit from the sensorial properties during an actuator’s work-cycle. Also, a study into how mechanical properties (such as elastic modulus) contribute to sensing and actuating properties of CPC is underway.


Intraoperative nerve monitoring (IONM) has many applications in different surgical fields. In head and neck surgery, IONM has been used to perform surgery of the parotid, thyroid and parathyroid glands, preserving the facial and recurrent nerves. However, hypoglossal nerve neuromonitoring has not been addressed with such relevance.A retrospective review of surgeries performed on patients with special tongue and floor of mouth conditions was undertaken to examine the indications that prompted its use. Particular attention was given to the pathology, intraoperative findings and the final outcome of each patient.Four patients, aged between 6 years and 68 years, with complex oral tongue and floor of mouth lesions were reviewed. Three patients were male, aged 22 years and younger, and two of these patients had oral tongue cancers with previous surgery. Oral tongue and neck conditions are challenging since the functions of the hypoglossal nerve are put at risk. The use of IONM technology allowed us to preserve nerve functions, speech and swallowing.Although IONM of the hypoglossal nerve is not a common indication in tongue and floor of mouth lesions, under special conditions its application can be extrapolated to challenging surgical cases, like the ones described. A number of reports have documented that monitoring a specific nerve during an operation can improve patient outcomes -10. IndeThe hypoglossal nerve is the last nerve to arise from the paired hypoglossal nuclei in the caudal medulla. It exits the cranium through the hypoglossal foramen, travels next to the internal carotid artery and vagus nerve, descends toward the carotid bulb and internal jugular vein, and lies next to the posterior belly of the digastric muscle, beneath the submandibular gland to innervate the extrinsic  and intrinsic muscles of the tongue. Therefore, lesions to the nerve not only affect the initial process of swallowing, but also speech, coordinated chewing and breathing ,12. IONMIn the present article, we describe our experience of four patients treated at the Hospital Pablo Tobón Uribe, Medellin, Colombia, on whom IONM of the hypoglossal nerve was used. We were able to achieve extensive resections with good physiological outcomes and no loss of function with a non-neurosurgical indication.A detailed description of Intraoperative neuromonitoring (IONM) of the Hypoglossal nerve is depicted throughout a narrative explanation of four illustrative cases on whom the technique was used . Information about the different steps involved in this novel application of Neuromonitoring is given in order to illustrate the reproducibility of the technology when a surgeon is faced with complex lesions related to the function of the Twelfth cranial nerve.A retrospective review of four patients treated at the Hospital Pablo Tobón Uribe, Medellin, Colombia was undertaken Table . The stuTo monitor the twelfth cranial nerve (XII), ground electrodes were placed on the chest or shoulder, following the procedures of other head and neck surgeries where IONM is used. Two sensing electrodes were placed on each side of the tongue either at the beginning of the surgery or once the hypoglossal nerve was exposed in the neck. The nerve was then stimulated with a manual stimulator provided by the manufacturer. A twitch of the tongue on the ipsilateral side, a dull sound and a graphic spike confirmed the integrity of the nerve. Nasotracheal intubation or a tracheostomy was undertaken; tracheostomy was preferred to allow the surgeon space to perform the procedure without having the orotracheal tube obstruct the view. Intubation was achieved with the use of short-acting, rapid-onset muscle relaxant.Four patients with complex tongue and/or floor of mouth lesions were monitored intraoperatively with IONM. Table The first patient was a 6-year-old boy with a hemangiolymphangioma of the right side of the neck, floor of mouth and tongue, and also on the left side, albeit to a lesser degree. A previous attempt to resect the tumor without considering any other alternative was attempted by another surgical team, but was not successful. Since the symptoms worsened, we performed bilateral nerve dissection (BND) with IONM. The larger, right part of the tumor was approached without being able to obtain a signal/response once the nerve was identified; it was later found that the nerve had been injured in the first surgery. The lesion of the neck and floor of mouth was removed. On approaching the left side of the neck, IONM produced a signal once the left hypoglossal nerve was identified and a careful dissection was performed to avoid injuring this working nerve, leaving a small amount of tumor attached to the most anterior part of the nerve.The second patient was an 8-year-old boy with a lesion on the floor of the mouth and anterior aspect of the tongue. The patient had a history of pulmonary tuberculosis (TB) and was initially given a clinical diagnosis of oral TB. After multiple negative acid-fast bacillus (AFB) smears and cultures, the diagnosis of SCC was established and the patient was referred for surgery. The anterior aspect of the tongue was fixed, suggesting fibrosis or tumor invasion. Therefore, bilateral supraomohyoid neck dissection was performed with IONM.en bloc, regardless of the proximity of a hypoglossal branch; whereas cancer-free areas were preserved. Upon removal of the tumor, stimulation of each hypoglossal nerve branch lead to ipsilateral electromyography (EMG) activity and corresponding contraction of the remaining tongue. The patient was decannulated 1 month after surgery. He was able to swallow a semi-solid diet and was also able to speak. The patient underwent postoperative radiation therapy and was disease-free 14 months after surgery.The electrodes were placed on the most posterior aspect of the oral tongue Figure . Both thThe third patient was a 22-year-old man with a history of SCC of the right tongue. The patient had undergone a right hemiglossectomy without complementary radiation therapy 9 years earlier. He presented with a recurrence in his remaining tongue manifesting as a 3 × 3 cm infiltrative lesion on the left tongue, which was found to be SCC on biopsy. We performed a left and right (revision) supraomohyoid neck dissection, and a left hemiglossectomy. During the operation, the electrodes were placed on the remaining right base of the tongue and the most posterior aspect of the left oral tongue. Remarkably, the right hypoglossal nerve responded to the stimulation by moving the base of the tongue to the ipsilateral side. The left nerve was intact and therefore a left hemiglossectomy was performed, and a radial forearm free flap (RFFF) was used to reconstruct the excised tongue. Surgical margins were cancer-free, and there were no intraoperative or postoperative complications. The patient was decannulated 12 days after surgery, and was able to eat, speak and move the flap with significant improvement in phonation compared to preoperative conditions. He completed his radiation therapy and was disease-free 9 months after surgery.The fourth patient was a 68-year-old woman. The patient was unable to swallow and had severe sleep apnea with failed nocturnal continuous positive airway pressure (CPAP). She was operated on owing to a large obstructing macroglossia resulting from amyloidosis. A partial glossectomy under IONM was performed by placing the electrodes on the most posterior part of the patient’s oral tongue. Forty percent of the patient’s oral tongue together with the most anterior aspect of the base of tongue was excised in a rhomboid fashion. Pathological examination of the removed tissues confirmed amyloidosis but also showed multiple myeloma. Following surgery, the patient remained unable to swallow and was was not able to be decannulated as her base of tongue remained swollen. In preparation for her first chemotherapy cycle, a surgical gastrostomy with placement of a G-tube was performed (the endoscope could not be advanced to the esophagus). Unfortunately the patient died owing to complications treating the multiple myeloma.IONM is a technique that allows surgeons to preserve critical nerves during surgical procedures. Critical anatomical areas can be securely preserved by testing a particular structure before operating, which can avoid complications and provide the surgical team with the final status of the nerve ,7,9,13.IONM indications are varied since its early use in acoustic neuroma has spread to other surgical specialties, such as neurosurgery, skull base, and head and neck surgery -4. High Regarding the hypoglossal nerve, its anatomical relations and involvement by lesions in the neck, an experienced head and neck surgeon should be able to identify the main trunk in the neck; Walshe stated that ‘there is no substitution for a good anatomical knowledge when identifying the hypoglossal nerve in the neck’. The hypoglossal nerve is identified in the neck owing to its size and usual anatomical location, although anatomical variations of the nerve have been described ,13.Hypoglossal nerve monitoring can be achieved by head and neck surgeons familiar with neuromonitoring of the facial and recurrent nerves, by following the procedures of other head and neck surgeries where IONM is used . This isDifferent from various neurosurgical indications, it could be questioned whether head and neck surgeons should be required to schedule surgery with IONM for every patient with a tongue lesion. In thyroid surgery, there is a debate regarding the need to use the technology for every case regardless of different conditions  -7. In paAs described, two of the young male patients had SCC and, in these difficult cases of oral tongue and floor of mouth lesions, the surgical team did not want to compromise positive margins in order to preserve function. For the avoidance of doubt, we do not use the IONM technology on regular cases. Prior to sectioning the tissue, multiple frozen sections were taken from the normal-appearing tongue or floor of mouth mucosa tissue, to identify the minor hypoglossal nerve branches and direct the resections of the lesions. If they were identified near the tumor they were sacrificed, regardless of proximity to a nerve branch. It was rewarding to know that at the end of each case, the young patients had good amplitude wave EMG responses in both nerves and a positive functional outcome of mobility of the tongue itself. These positive results allowed us to remove the tracheostomy in two out of three patients; only the fourth patient remained with the tracheostomy cannula in place, even though the patient’s tongue remained mobile with functional hypoglossal nerve.We choose to place the electrodes directly onto the posterior aspect of the anterior tongue instead of the extrinsic muscles in order to maintain the intrinsic musculature and obtain an optimum result. Energy to stimulate the nerves varied from 0.5 mA to 1.0 mA, with an average of 0.8 mA .To the best of our knowledge, Walshe’s study on hypoglossal nerve stimulation is the only published article with true head and neck indications , howeverFinally, the system helped us to identify a previously injured nerve on a young patient with a large volume lesion on the oral tongue and floor of mouth. This was not detected prior to surgery since the patient was barely moving his tongue displaced by the hemangiolymphangioma. The surgical team was able to remove the lesion and preserve the left working hypoglossal nerve.Head and neck surgery applications of IONM have been mainly restricted to thyroid, parathyroid and parotid surgery. Although monitoring the hypoglossal nerve is not a regular indication of this technique, surgeons should be aware of the potential ‘nonconventional’ applications that the technology can offer to challenging surgical cases like the ones described.Written informed consent was obtained from the patients for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.AFB: Acid-fast bacillus; BND: Bilateral nerve dissection; CPAP: Continuous positive airway pressure; EMG: Electromyography; IONM: Intraoperative nerve monitoring; RFFF: Radial forearm free flap; SCC: Squamous-cell carcinoma; TB: Tuberculosis.CSD and JPD gave instructional courses on neuromonitoring to surgeons in Colombia and Latin American in 2012, sponsored by Medtronic. However, this study was not supported by any company.CSD, AL, AP performed the operations. DU assisted the surgeries. JPD gave input in neuromonitoring. All authors read and approved the final manuscript.


SRY (sex determining region on Y chromosome) located on the short arm of chromosome Y. The differentiation of male external genitalia  occurs between the 9th and 13th weeks of pregnancy and requires adequate concentration of testosterone and the conversion of this to another more potent androgen, dihydrotestosterone, through the action of 5α-reductase in target tissues.Androgen is a generic term usually applied to describe a group of sex steroid hormones. Androgens are responsible for male sex differentiation during embryogenesis at the sixth or seventh week of gestation, triggering the development of the testes and penis in male fetuses, and are directed by the testicular determining factor: the gene This report describes the case of a teenage girl presenting with a male karyotype, and aims to determine the extension of the mutation that affected the AR gene. A Caucasian girl aged 15 was referred to our laboratory for genetic testing due to primary amenorrhea. Physical examination, karyotype testing and molecular analysis of the androgen receptor were critical in making the correct diagnosis of complete androgen insensitivity syndrome.AR gene may cause either complete or partial androgen insensitivity syndrome. The case reported here is consistent with complete androgen insensitivity syndrome, misdiagnosed at birth, and consequently our patient was raised both socially and educationally as a female. It is critical that health care providers understand the importance of properly diagnosing a newborn manifesting ambiguous genitalia. Furthermore, a child with a pseudohermaphrodite phenotype should always undergo adequate endocrine and genetic testing to reach a conclusive diagnosis before gender is assigned and surgical interventions are carried out. Our results show that extreme care must be taken in selecting the genetic tools that are utilized for the diagnosis for androgen insensitivity syndrome.Sex determination and differentiation depend on a cascade of events that begins with the establishment of chromosomal sex at fertilization and ends with sexual maturation at puberty, subsequently leading to fertility. Mutations affecting the  SRY (sex determining region on Y chromosome) located on the short arm of chromosome Y. The differentiation of male external genitalia in the penis, scrotum and penile urethra occurs between the 9th and 13th weeks of pregnancy and requires adequate concentration of testosterone and conversion of this to another more potent androgen, dihydrotestosterone (DHT), through the action of 5α-reductase in target tissues [Androgen is a generic term usually applied to describe a group of sex steroid hormones. Androgens are produced primarily by a male's testes. However, some small amounts are also produced by the ovaries in females and by the adrenal gland in both sexes. Androgens are responsible for male sex differentiation during embryogenesis at the sixth or seventh week of gestation, triggering the development of the testes and penis in male fetuses, and are directed by the testicular determining factor: the gene  tissues .AR gene is a protein-coding gene located at Xq11.2-q12. It spans over 90 kb and codes for a protein that functions as a steroid-hormone-activated transcription factor  is relatively rare. In childhood, the most common clinical presentation is the presence of bilateral inguinal hernias. Individuals not diagnosed during childhood are detected after puberty because of primary amenorrhea. Patients with the complete form of AIS have female external genitalia, an absence or thinning of pubic hair and the absence of a uterus .The AR NTD is relatively long and displays the greatest sequence variability among nuclear receptors. It is very flexible and displays a high degree of intrinsic disorder. In addition, the NTD has a variable number of homopolymeric repeats, the most important of which is a polyglutamine repeat that ranges from eight to 31 repeats in normal individuals, with an average length of 20 base pairs .The DBD is centrally located in the AR and it is the most conserved region within the nuclear receptor family. This region has nine cysteine residues, eight of which are involved in forming two zinc fingers, and a C-terminal extension. The first zinc finger, most proximal to the NTD, determines the specificity of DNA recognition, whereas residues in the second zinc finger are involved in AR dimerization. Two AR monomers in a head-to-head conformation bind as a homodimer to androgen response elements which are direct or indirect repeats of the core consensus 5'-TGTTCT-3' or more complex response elements with diverse arrangements of AREs. The C-terminal extension is important for the three-dimensional structure of the DBD and it plays a role in mediating the AR selectivity of DNA interaction .AR gene [The hinge region has long been considered to be a flexible linker between the DBD and LBD in the AR. More recently, however, this region was shown to be involved in DNA binding as well as AR dimerization. It was suggested that the hinge region also acts to attenuate transcriptional activity of the AR gene .AR gene cause X-linked androgen insensitivity syndrome (AIS) characterized by androgen unresponsiveness, which affects proper male sexual development both at embryogenesis and at puberty. As a genetic disorder, AIS presents problems to affected people and their families, and is a major medical challenge for health providers. This impaired response to androgen results from an incapacity or reduced capacity of the AR to transactivate androgen-responsive genes in target cells, and leads to abnormal differentiation and development of male internal and external genitalia, leading to male pseudo-hermaphroditism [Mutations in the roditism .AR gene result in AIS (OMIN #300068). Depending on the extent of the AR defect, the phenotype can vary. Three forms are classified, complete (CAIS), partial (PAIS), and mild (MAIS). All forms of AIS are X-linked traits inherited as a recessive disorder. Despite a normal male karyotype , individuals affected with CAIS  have female external genitalia, blind vaginas, an absent uterus and female adnexa, female breast development, and abdominal or inguinal testes. Partial androgen insensitivity results in hypospadias and micropenis with gynecomastia [Most mutations on the comastia .in situ hybridization (FISH) and PCR in order to determine the nature and the extent of the mutation that affected the AR gene.The aim of the investigation reported here was to provide a genetic diagnosis of a teenage girl with normal male karyotype using fluorescence A 15-year-old Caucasian girl was referred to a laboratory in Goiânia, Goiás, Brazil, for genetic testing due to primary amenorrhea. Her medical history included removal of an abdominal mass as a newborn. The tissue removed was referred to as an umbilical hernia. Her G-band karyotype revealed a diploid set of chromosomes, including 22 pairs of homologous autosomes and one pair of sex chromosomes, compatible with a 46, XY male chromosome complement.The geneticists at our laboratory concluded that the mass withdrawal from the abdomen of our patient was, in fact, testes, and that our patient had a condition known as cryptorchidism; a reproductive change characterized by a failure of the movement of one or both testes from the abdominal cavity to the scrotum.AR gene and to detect the AR gene, respectively. We prepared a culture of T lymphocytes in RPMI 1640 medium, supplemented with 20% fetal calf serum and 2% of phytohemagglutinin. Metaphasic preparations were made by conventional methodology. The slides for FISH were prepared with a micropipette, with about 15 μL of the material set. Only slides of good quality (in terms of metaphase) were selected by phase contrast microscope, and were subjected to FISH using the LSI Androgen Receptor SpectrumOrange (Xq12) probe . For PCR, primers were used for exons 1, 4, 6, 7 and 8 of AR [We performed PCR and FISH to verify mutations of the exons 1, 4, 6, 7 and 8 of the  8 of AR .In situ hybridization with the LSI AR probe indicated the presence of the gene in all analyzed cells. However, genomic DNA extracted from peripheral blood leukocytes assessed by PCR revealed coding sequence abnormalities for the AR gene, which lacked exons 1 to 7 indicating large deletion spanning the proximal region of the gene. Figure The results reported in this paper underline the fact that great care must be taken when selecting genetic testing tools to be utilized to reach the proper diagnosis for AIS. If a child reaches his or her teenage years undiagnosed due to clinical challenges presented by ambiguous genitalia it further complicates matters, as there could be several genetic events subjacent to that outcome, ranging from total or partial deletion of the gene to point mutation, that efficiently silences the gene and therefore leads to AIS. Here, we report that FISH alone was not able to properly diagnose our patient, despite the proximal deletion within the AR observed on PCR Figure . This reAR gene may cause either complete or partial AIS. The case of our patient reported here is consistent with CAIS, misdiagnosed at birth, and she was consequently raised socially and educationally as a female. It is critical that health care providers understand the importance of properly diagnosing a newborn with ambiguous genitalia. Prompt evaluation of both clinical and genetic findings is crucial to determine proper gender assignment and the detection of life-threatening conditions [Sex determination and differentiation depend on a cascade of events that begins with the establishment of chromosomal sex at fertilization and ends with sexual maturation at puberty subsequently leading to fertility. Mutations affecting the nditions . Furthernditions .The presentation of a patient, and specifically a neonate, with abnormal genital development represents a difficult diagnostic and therapeutic challenge. Referral to a center with experience in the diagnosis and management of disorders of sexual development is advised, where an emphasis should be placed on psychological and genetic counseling ,15.Written informed consent was obtained from the patient's next-of-kin for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.COAM was involved in collating the information, reviewing the literature, and preparation of the manuscript. DMS was involved in collating information regarding the case and getting informed consent from our patient. ADC was involved in the review of literature and revising the manuscript critically. All authors read and approved the final manuscript.