In recent years there have been tremendous advances in our ability to rapidly and cost-effectively sequence DNA. This has revolutionized the fields of genetics and biology, leading to a deeper understanding of the molecular events in life processes. The rapid technological advances have enormously expanded sequencing opportunities and applications, but also imposed strains and challenges on steps prior to sequencing and in the downstream process of handling and analysis of these massive amounts of sequence data. Traditionally, sequencing has been limited to small DNA fragments of approximately one thousand bases (derived from the organism's genome) due to issues in maintaining a high sequence quality and accuracy for longer read lengths. Although many technological breakthroughs have been made, currently the commercially available massively parallel sequencing methods have not been able to resolve this issue. However, recent announcements in nanopore sequencing hold the promise of removing this read-length limitation, enabling sequencing of larger intact DNA fragments. The ability to sequence longer intact DNA with high accuracy is a major stepping stone towards greatly simplifying the downstream analysis and increasing the power of sequencing compared to today. This review covers some of the technical advances in sequencing that have opened up new frontiers in genomics. Fully understanding the language of DNA requires the complete determination of the order of bases in the genome of humans (or other organisms of interest). Acquiring that knowledge promises to yield more complete insights into biological variations and etiology of diseases. In the dawn of sequencing, reading the order of the four bases of DNA was a cumbersome process. Sequencing was made possible, although still difficult, by the introduction of the chemical degradation method of Maxam and Gilbert  and SangThe most commonly used DNA sequencing technologies, and their challenges and limitations, are described below. A summary table of the features of each technology is provided in The ingenuity of the chain-termination technique lies in the use of chain-terminating nucleotides, dideoxynucleotides that lack a 3'-hydroxyl group, restricting further extension of the copied DNA chain. Early Sanger sequencing required the sequencing process to be split into four separate reactions. Each reaction involves a single-stranded DNA template, a DNA primer and a DNA polymerase in the presence of a mixture of the four unmodified nucleotides, one of which is labeled, and a type of modified chain-terminating nucleotide. Fragments of varying length are synthesized after primer hybridization and polymerase extension, all having the same 5' end but terminated by a chain-terminating nucleotide at the 3' end. Adding only a fraction of the terminating nucleotide ensures the random incorporation of dideoxynucleotides in only a small subset of molecules. Since different chain-terminating nucleotides are used in the four sequencing reactions, all combinations of termination can be produced. The generated 3'-terminated DNA templates are then heat-denatured and fractionated by gel-electrophoresis, running products of all four sequencing reactions in parallel . Using rSeveral enhancements have been made to the original method developed by Sanger. These include: labeling the chain-terminating nucleotides with spectrally distinct fluorescent dyes enabling: a single tube and lane to be used in the fragment generation and fractionation steps, respectively , 4; elimThe most widely used sequencing platforms in genetic research today are the massively parallel sequencing platforms, which have inherited many features from Sanger sequencing, such as the use of polymerases for synthesis, modified nucleotides and fluorescent detection. Another feature is that they require the DNA to be clonally amplified forming a consensus template prior to sequencing. These sequencing methods have been called next generation sequencing, high-throughput sequencing methods, or second-generation sequencing. However, as the sequencing technologies continue to develop, it is hard to keep referring to old and novel emerging sequencing technologies as belonging to a certain generation. Therefore, it is preferable to categorize them by their most prominent common feature, such as being massively parallel or single-molecule sequencing methods. Currently, there are five competing massively parallel sequencing technologies, each with specific strengths and weaknesses. These are described and discussed below.Melamede originally outlined the concept of sequencing-by-synthesis (SBS) in 1985, in a report of efforts to detect nucleotide incorporation events by measuring nucleotide absorbance . PyroseqIn SBS, the event that is detected is the incorporation of nucleotides into growing DNA strands. As the nucleotides are incorporated by the polymerase, pyrophosphate (PPi) and protons are generated. In pyrosequencing, the PPi is used in an enzymatic cascade to generate a light burst. The PPi is converted by ATP sulfurylase to ATP, which is then used by the firefly enzyme luciferase to generate photons, providing a light signal that is proportional to the number of nucleotides being incorporated. The challenge is to detect the flashes of light from each unique DNA template while sequencing numerous templates in parallel. Spatially separating each sequencing reaction on beads deposited in small wells on a picotiter plate elegantly solved this problem.Massively parallel pyrosequencing begins with fragmentation of the DNA and adaptor ligation. Single-stranded DNA templates are then bound on beads and emulsion PCR is performed, clonally amplifying each DNA template in aqueous microreactors isolated by oil. The emulsion is then broken and the beads carrying DNA are separated from empty beads in a process called enrichment. The enriched beads are deposited in the small wells on the picotiter plate together with primer and DNA polymerase. Ideally, only one of these beads will fit into each well. Smaller beads are also added, carrying the enzymes responsible for the light generation using PPi. Nucleotides are then passed over the substrate in a laminar flow of solutions applied in a predetermined order, and the flashes of light in each well representing incorporation events are recorded. Efficient removal of reaction by-products, which could otherwise perturb the sequencing reaction, is facilitated by the laminar flow. Hence, the sequence of the DNA templates is determined from the knowledge of their location, the order of the flow of nucleotides and records of each flash of light from each well –12. The The comparable read lengths and increased sequence capacity compared to traditional Sanger sequencing made the massively parallel pyrosequencing ideal for de novo sequencing, re-sequencing of genomes and metagenomic studies . James WA fundamental gain in sequencing throughput was achieved when the second massively parallel sequencing system to be commercialized was launched in 2006. The technology is based on SBS using four colored reversible dye termination nucleotides and was first outlined in 2001 by a small company, Solexa , 17, whiSBS using reversible dye termination begins with fragmentation of the DNA and ligation of adaptors. Instead of using emulsion PCR to spatially separate each DNA template, as in the massively parallel pyrosequencing approach, another technique is employed. The DNA templates are hybridized via the adaptors to a planar surface, where each DNA template is clonally amplified by solid-phase PCR, also known as bridge amplification. This creates a surface with a high density of spatially distinct clusters, each cluster of which contains a unique DNA template. These are primed and sequenced by passing the four spectrally distinct reversible dye terminators in a flow of solution over the surface in the presence of a DNA polymerase. Only single base extensions are possible due to the 3' modification of the chain-termination nucleotides, and each cluster incorporates only one type of nucleotide, as dictated by the DNA template forming the cluster. The incorporated base in all clusters is detected by fluorescence imaging of the surface before chemical removal of the dye and terminator, generating an extendable base that is ready for a new round of sequencing. The most common sequencing errors produced in reversible dye termination SBS are substitutions , 19.One of the major drawbacks of reversible dye termination chemistry is the limitation in read length, as it is difficult to reach 100% efficiency of base incorporation and cleavage in each cycle. When the system was launched, it featured a high- quality read length of only 30–36 bases , 20, draIn nature, DNA ligases are essential enzymes for repairing nicks and breaks in DNA and are involved in high fidelity reactions in both DNA replication and repair . In 2005Initially, the SOLiD platform yielded many more bases than the Illumina platform, but the recent upgrades to the Hiseq2000 chemistry make this instrument's yield significantly higher than that for SOLiD. However, in the library preparation steps the SOLiD platform resembles massively parallel pyrosequencing. Clonal amplification is achieved by emulsion PCR after creating an adaptor-ligated fragment library. The enriched beads are then randomly deposited on a glass slide and attached to its surface by a 3' modification introduced on the targets covering the beads. In subsequent sequencing by chained ligation, oligomers of eight nucleotides, octamers, are used as detection probes instead of single nucleotides. Each octamer contains two known bases at the 3' end followed by three degenerate nucleotides and three nucleotides that can hybridize to any other nucleotide, called universal nucleotides. Four spectrally distinct dyes are employed, each of which is carried by four octamers, creating a total probe set of 16 octamers, covering all possible combinations of the two known bases. The dye is attached to the 5' end of each octamer. To initiate the first sequencing cycle, the DNA-covered beads are incubated with a sequencing primer, the 16 octamer probes and ligase. Only a perfectly hybridized probe will be joined to the sequencing primer by the ligase. The probes are imaged to decode the first two bases, and then chemically cleaved to remove the last three 5' bases. This cycle of probe hybridization and ligation, fluorescent imaging and chemical cleavage is repeated ten times, interrogating every fifth base. The created fragment is then stripped from the beads and a second ligation round is performed using a sequencing primer annealing one base upstream of the previous one. Hence, each base is associated with two color calls. Progressively shifting the position of the sequencing primer and repeating the ligation round using ten cycles each time yields color calls, which can be decoded to obtain a linear sequence in color space. Translating from color space to an ordinary nucleotide sequence is most accurately performed by aligning the color space reads to a color space reference genome. As a consequence of the color space alignment, errors can be efficiently corrected since only certain color combinations are allowed, facilitating distinction between nucleotide substitutions, represented by a two-color change in adjacent nucleotides, and sequence errors –28. The Life Technologies has recently announced a replacement of the cumbersome emulsion library preparation step with a technique called wildfire, which resembles the bridge amplification performed in the Illumina platform. This promises a reduction of the library preparation time as well as a higher density on the chip surface, enabling a higher throughput. The SOLiD system is best suited for re-sequencing projects demanding low error rates , transcrIn 2010, Complete Genomics Inc published a proof-of-principle paper demonstrating its complete human genome sequencing capabilities, using a sequencing method sharing several features with polony sequencing , 30. RatComplete Genomics performs sequencing by unchained ligation using its combinatorial probe-anchor ligation (cPAL) technology. The DNA to be sequenced is fragmented to 500 bases and four adaptors are inserted into each genomic fragment by repeated cutting with restriction enzymes and intramolecular ligation. The resulting circles are then amplified in solution into coils of single-stranded DNA , called The reasons that Complete Genomics are keeping their sequencing platform in-house are, at least partly, related to the considerable challenge of mapping and assembling the highly repetitive human genome using short, 35-base-paired-end reads and the rather demanding cPAL library preparation. Illumina has responded to Complete Genomics' initiative by also offering complete human genome sequencing as a service, using reversible dye termination chemistry. It remains to be seen if most sequencing will be performed in-house at hospitals and sequencing centers, or outsourced to sequencing companies.All massively parallel sequencing platforms described so far use optic detection to decipher a DNA sequence. Exchanging optic detection for electric detection would potentially have several advantages since it would avoid the need for modified nucleotides, expensive optics and the time-consuming acquisition and processing of large amounts of fluorescent image data. Hence, it promises cheaper, space-efficient and more rapid sequencing. Pourmand and colleagues  outlinedThe process is very similar to that of the massively parallel pyrosequencing, developed earlier by Rothberg and co-workers. It involves use of the same library preparation steps, with an adapter-ligated fragment library, which is clonally amplified on beads using emulsion PCR. A DNA polymerase is used to incorporate nucleotides on a primed DNA template with a predetermined flow of nucleotides. However, the focus is shifted from the PPi to the other by-product of nucleotide incorporation, the hydrogen ion. The enriched beads from the emulsion PCR are deposited in small wells (each equipped with a sensor capable of detecting free protons) on an ion chip. As the DNA polymerase adds nucleotides to the DNA template hydrogen ions are released, and the resulting free proton shift is detected by the sensor and transformed to an electric signal. The ion chip is based on the most widely used technology to construct integrated circuits, facilitating low-cost, large-scale production and should confer excellent scalability. However, as in massively parallel pyrosequencing, homopolymeric regions give rise to insertions and deletions, indels, due to the nonlinear electric response upon incorporation of more than five nucleotides .Escherichia coli provided an excellent example of the value of a sequence platform with a modest output but fast sequencing run time when monitoring outbreaks of pathogens [Sequencing of Gordon Moore's genome  demonstrathogens , 36. TheThe massively parallel sequence methods are quite diverse in terms of sequencing biochemistry, but they share many common features. Their library preparation steps begin with random fragmentation of DNA followed by ligation of platform-specific adaptors at the end of each fragment. These adaptors are then used in amplification of the fragment on a solid surface by a polymerase, except in cPAL amplification, which is performed in solution. The amplification products are spatially clustered on an array before sequencing. The sequencing process itself is performed by an orchestrated automated series of enzyme-driven biochemical and fluorescent imaging data acquisition steps. Only the newest system, the Ion Torrent, based on free proton shifts, is capable of electric detection. All of these platforms also have the capability to read both ends of a DNA fragment, called paired-end sequencing. This feature is instrumental in resolving repetitive regions in genomes and quantifying transcript isoforms in RNA-Seq.The rate-limiting step in the sequencing process has traditionally been the sequencing reaction. However, that started to change towards the end of the HGP, when the capacity of sequencing instruments began to exceed the rate at which new samples could be prepared for sequencing. Current sequencing instruments produce several orders of magnitude more data than conventional Sanger sequencing, shifting the rate-limiting steps to library preparation and data analysis. The challenge is to keep the processes of sample preparation, sequencing reaction and data analysis balanced. Hence, automation of library preparation and quality control steps has played a vital role in keeping up to speed with the increase in sequencing power. The generally short read lengths coupled to the enormous amount of data to be analyzed and reduced raw accuracy, compared to Sanger sequencing, has introduced many challenges in the downstream data analysis . HoweverMassively parallel consensus sequencing has become the dominant sequencing technology, but other approaches have emerged that avoid amplification of the DNA template prior to sequencing. The aim of these technologies is to sequence single DNA molecules, preferably in real time. Potential benefits of using single-molecule sequencing are: the minimal quantities of input DNA required; elimination of amplification bias; asynchronous synthesis; fast turnaround times; and the capacity to investigate the characteristics of individual DNA molecules. A comparison of consensus and single-molecule sequencing, as well as the most common errors for each sequencing technology is shown in Helicos Biosciences introduced the first single-molecule DNA sequencer, the Heliscope sequencer system in 2008 . The sysScience in 2009. The platform introduced several interesting novel features, including immobilization of the DNA polymerase instead of the DNA, real-time monitoring of the synthesis process and potential read lengths of several kilobases of DNA [Pacific Biosciences presented the first single-molecule, real-time SBS sequencing platform in a proof-of-principle paper in s of DNA .–21 L), enabling minimization of background noise, parallelization, and monitoring of single-molecule DNA polymerization. The DNA template is allowed to diffuse into the ZMV in the presence of primer and nucleotides with fluorescent labels attached to the phosphate chain. The DNA polymerase latches onto the DNA template and begins incorporating the distinctly labeled nucleotides. The DNA sequence is determined by recording each nucleotide incorporation event dictated by the DNA template. Nucleotides that are being incorporated remain for a longer time in the optical detection volume than freely diffusing nucleotides, facilitating the separation of background noise generated by a high concentration of labeled nucleotides from true nucleotide incorporation events. The label attached to the phosphate chain is cleaved off during the elongation process. The polymerases are randomly immobilized among the ZMWs, leading to only a third of them being occupied by a single polymerase. The rest of the ZMWs are either empty or contain two or more polymerases, which dramatically reduces the potential capacity of the ZMW array. SMRT sequencing has high error rates, mainly due to failure to detect all incorporations leading to indels [The single-molecule real-time (SMRT) sequencing relies on a processive DNA polymerase immobilized via a biotin-streptavidin linkage at the bottom of a nanostructure called a zero-mode waveguide (ZMW) . This sto indels , 43.SMRT sequencing has several interesting features that can be exploited. A circular template can be used, enabling a high-quality consensus sequence to be obtained by allowing the polymerase to make multiple passes along the same DNA template . The phyIn 1996, Deamer and colleagues demonstrated that single-stranded DNA  or RNA Several problems must be resolved before nanopore sequencing can be fully exploited. The nanopores are too long to provide single-base resolution, as several nucleotides in the pore contribute to each change in electric current. In addition, the high speed of DNA translocation through the nanopores complicates the electrical measurements, making the nucleotide identification signals difficult to interpret . HoweverRecently, Oxford Nanopore Technologies announced its ability to sequence the entire 48-kilobase Lambda genome on both sense and antisense strands using their strand sequencing method. Commercialization of the nanopore-based sensing chemistry on an electronic-based platform, the GridION, and a disposable portable device for electronic single molecule sensing, the MinION, is scheduled for 2012. Oxford Nanopore Technologies claims competitive accuracy and ultra-long read-length single-molecule data, which will deliver a complete human genome in 15 min using multiple GridIONs. It is too early to tell whether Oxford Nanopore Technologies can fully deliver on these promises, and the acid test for the sequencing technique will be the assessment of the early tester. However, if the promise of competitive accuracy and ultra-long reads holds true it will cause another paradigm shift in the sequencing field.Sequencing technologies have advanced at a rapid pace in recent years and the advance shows no signs of slowing down. This has created an imbalance between the sequencing per se and other procedures involved, both upstream and downstream, i.e. sample preparation and sequence data analysis and storage. Bar coding of samples coupled to automation of steps prior to sequencing has the potential to handle most upstream process issues. The true challenges lie in the downstream steps of sequence data analysis, handling and storage. The assembly and mapping algorithms need to become more efficient and accurate to handle the massive amounts of sequence data and extract as much pertinent information from them as possible. Long-term storage of sequence data and subsequent analysis is cumbersome and expensive, thus, in the future, it might be cheaper and easier to simply re-sequence samples as the information is needed. There are, of course, several challenges remaining in the actual sequencing reactions. Two of the most urgent are increasing the read lengths and the quality of the sequence reads, enabling resolution of common repeats and duplicated regions. Currently, the massively parallel sequencing and single-molecule platforms are complementary, and no single technology is best suited for all applications. Thus, it remains to be seen which sequencing technology will cause the next sequencing paradigm shift.Sequencing has traditionally been reserved for large and very well-funded research centers, but the cost per base sequenced started to decrease during the end of the HGP. The pace of cost reduction accelerated even further with the launch and subsequent evolution of the massively parallel sequencing instruments seen in recent years. What was once restricted to a privileged few can now be accessed by many, leading to a revolution in the availability of sequencing power.The democratization of sequencing is evident from the constantly increasing number of applications and analyses that involve sequence data. Sequencing is today applied in diverse areas, such as forensics, agriculture, biofuel production, paleontology, domestication, and medicine to name but a few. This ongoing revolution promises to bring sequencing to nearly every aspect of life.


The Arabian Peninsula is a key region for understanding climate change and human occupation history in a marginal environment. The Mundafan palaeolake is situated in southern Saudi Arabia, in the Rub’ al-Khali (the ‘Empty Quarter’), the world’s largest sand desert. Here we report the first discoveries of Middle Palaeolithic and Neolithic archaeological sites in association with the palaeolake. We associate the human occupations with new geochronological data, and suggest the archaeological sites date to the wet periods of Marine Isotope Stage 5 and the Early Holocene. The archaeological sites indicate that humans repeatedly penetrated the ameliorated environments of the Rub’ al-Khali. The sites probably represent short-term occupations, with the Neolithic sites focused on hunting, as indicated by points and weaponry. Middle Palaeolithic assemblages at Mundafan support a lacustrine adaptive focus in Arabia. Provenancing of obsidian artifacts indicates that Neolithic groups at Mundafan had a wide wandering range, with transport of artifacts from distant sources. The Arabian Peninsula is fast becoming a key region for understanding palaeoenvironmental change and its relationship to human occupation history. Major recent improvements have been made in our understanding of the Palaeolithic record of the region see: . Recent Palaeoenvironmental studies of cave speleothems e.g.,  and palaHomo sapiens presence in the Arabian interior, which further supports the notion of a dispersal out of Africa Here we report the first reliable discoveries of Middle Palaeolithic and Neolithic sites in association with the former lakeshores of the Mundafan palaeolake, in the Rub’ al-Khali of Saudi Arabia. We associate our new archaeological finds with previous environmental research Given the importance of the environmental research carried out in the Mundafan palaeolake region, pilot archaeological research was undertaken. The present article reports on the discovery of both Middle Palaeolithic and Neolithic sites along the south-eastern shores of the palaeolake, suggesting that climatic cycles have most probably influenced the history of human occupations in Arabia. All necessary permits were obtained for the Najran-Mundafan fieldwork and analyses, which complied with all relevant regulations from the Saudi Commission for Tourism and Antiquities, Kingdom of Saudi Arabia.2). The lithic scatters are exclusively characterized by the presence of flakes, cores and tools, and no other kinds of archaeological remains were identified. The temporal delimitation of the sites is sometimes difficult to precisely determine, as their status of proper individual ‘site’ can be biased by repeated occupations  were discovered, labeled MDF-01 to MDF-21 . All of upations . Lithic upations , though The distribution of archaeological sites in 2, 100km2 and 50km2 contributing area at the equator) to allow clear examination of the palaeohydrological characteristics of the region at a range of scales , located in the southern Saudi Arabian province of Najran, represents one of the largest lacustrine deposits on the Arabian Peninsula, formerly radiocarbon dated by Mcclure to MIS 3 2 . This di2 . Examinit 100 ka . These aThe lake is found at the juncture between the Asir mountains, the Tuwayq mountains and the Rub’ al-Khali desert . The valThe palaeohydrological reconstruction of the region shows that the principal source of water for the lake is via channels draining the Asir Mountains . The neiThis ancient drainage course is visible as a valley partially exposed beneath obscuring dunes in satellite imagery and DEMs, and is corroborated by the flow analyses, which plot a drainage course following this system, seen as the large scale rivers presented in Different views have been published regarding the activity of these drainage channels within the Rub’ al-Khali, with the most recent activity being ascribed to pluvial phases during the early Pleistocene McClure Capra sp./Ovis sp.), oryx (Oryx sp.), gazelle (Gazella sp.), horse and ass (Equus sp.), camel (Camelus sp.), wild cattle , Hippopotamus amphibious, and birds such as ostrich Struthio sp.  yielding Middle Palaeolithic lithic assemblages  and longer term fluctuations of lake depth and area need to be conducted.A total of 8 surface sites are attributed to the aceramic Neolithic of the Rub’ al-Khali tradition  (see for methods: th century AD Ostrich egg shells were also identified at MDF-13 and MDF-14. Ostrich fossils have not been recovered at Mundafan , p.182. Homo sapiens into this marginal environment.Survey in the Mundafan palaeolake basin revealed, for the first time, Middle Palaeolithic occupations. Recovery of Middle Palaeolithic assemblages corresponds with recent environmental and geoarchaeological studies that indicate at least three lacustrine wet phases in MIS 5 The Mundafan Neolithic arrowheads are pertinent typological indicators, and although well known from surface sites The Mundafan Neolithic sites do not appear to be sedentary locations on the basis of the absence of architectural features, grindstones, domesticated faunal remains, and relatively low artifact densities. The prevalence of projectiles and other weaponry is probable evidence of hunting activities. Mundafan would have been a favorable setting for short-term hunting along the lakeshore. The presence of rare obsidian artifacts demonstrates Mundafan’s participation in long-distance mobility systems that included relations with the obsidian-rich mountainous zones of Yemen, some 400–500 km away from the site.The archaeology associated with Lake Mundafan is not an isolated occurrence . NeolithNew environmental studies, remote sensing research and archaeological reconnaissance survey at Mundafan is beginning to shed light on the relationship between climate change and human presence. Currently, there is no clear evidence for the presence of Upper Palaeolithic or Late Palaeolithic industries at Mundafan, seemingly confirming an Arabia wide pattern


Few existing interactive rehabilitation systems can effectively communicate multiple aspects of movement performance simultaneously, in a manner that appropriately adapts across various training scenarios. In order to address the need for such systems within stroke rehabilitation training, a unified approach for designing interactive systems for upper limb rehabilitation of stroke survivors has been developed and applied for the implementation of an Adaptive Mixed Reality Rehabilitation (AMRR) System.The AMRR system provides computational evaluation and multimedia feedback for the upper limb rehabilitation of stroke survivors. A participant's movements are tracked by motion capture technology and evaluated by computational means. The resulting data are used to generate interactive media-based feedback that communicates to the participant detailed, intuitive evaluations of his performance. This article describes how the AMRR system's interactive feedback is designed to address specific movement challenges faced by stroke survivors. Multimedia examples are provided to illustrate each feedback component. Supportive data are provided for three participants of varying impairment levels to demonstrate the system's ability to train both targeted and integrated aspects of movement.The AMRR system supports training of multiple movement aspects together or in isolation, within adaptable sequences, through cohesive feedback that is based on formalized compositional design principles. From preliminary analysis of the data, we infer that the system's ability to train multiple foci together or in isolation in adaptable sequences, utilizing appropriately designed feedback, can lead to functional improvement. The evaluation and feedback frameworks established within the AMRR system will be applied to the development of a novel home-based system to provide an engaging yet low-cost extension of training for longer periods of time. Sensorimotor rehabilitation can be effective in reducing motor impairment when engaging the user in repetitive task training . VirtualFeedback used for rehabilitation training must also be adaptable in design, allowing for changes in training intensity and focus. Yet few existing augmented reality rehabilitation environments effectively communicate multiple aspects of movement performance simultaneously, or furthermore, do so in a manner that is adaptable and generalizes across multiple training scenarios.In our companion paper, Lehrer et al present a methodology for developing interactive systems for stroke rehabilitation that allow for adaptive, integrated training of multiple movement aspects . While tWe now provide an overview of the AMRR system and participant experience, followed by a more detailed discussion of the applied design methodology within the system's implementation. An action representation for reach and grasp training is presented with accompanying methods for quantifying the representation's kinematic features, which allow for measurable evaluation of performance and generation of media-based feedback. Descriptions of how the AMRR feedback addresses specific movement challenges are then provided, with corresponding multimedia examples. An overview of the system's adaptation of the feedback and training environments demonstrates how AMRR training can be customized for each stroke survivor. Finally supportive data from three participant cases are presented to demonstrate the system's ability to promote integrated improvement of several movement features. Correlations between performance improvements in trials following the presence of observable feedback are also presented in support of the feedback design's efficacy in promoting self-assessment by the participant. A full results paper evaluating the use of AMRR therapy in comparison to traditional therapy will be provided in a forthcoming paper after the conclusion of a clinical study currently underway. The main intent of this paper is to provide a detailed description of the implemented methodology for interactive feedback within the AMRR system based on principles established in .The Adaptive Mixed Reality Rehabilitation (AMRR) system provides detailed evaluation information and interactive audiovisual feedback on the performance of a reach and grasp task for the upper extremity rehabilitation of stroke survivors. See additional file Figure The system is used by stroke survivors presenting clinical symptoms consistent with left-sided motor area lesions resulting in right-sided hemiparesis, who were right hand dominant prior to stroke. Each participant must demonstrate active range of motion in the right arm, with the following minimum movement thresholds to ensure they can complete the reaching task: shoulder flexion of at least 45°, elbow ROM of at least 30°-90°, forearm pronation or supination of at least 20°, wrist extension of at least 20°, and at least 10° active extension of the thumb and any two fingers. Each participant must earn a score greater than 24 on the Mini Mental State Exam and demonstrate acceptable levels of audio and visual perception. Our sensory perception test assesses color blindness, the ability to detect basic properties of musical sounds, such as pitch, timbre, loudness, and the ability to perceive structural characteristics of the feedback such as movement of images and rhythm acceleration .A participant receives 1 hour of AMRR therapy, 3 times a week for 1 month, for a total of 12 therapy training sessions. An average of 8-12 sets of 10 reaches are practiced per session depending upon the participant's ability and fatigue. Between sets the participant is able to rest, while also interact with the clinician to discuss the last set. During a therapy training session, the participant is seated at a table that is lowered or raised to provide various levels of support for the affected arm. The table also allows various target objects to be mounted and adjusted in location. Visual and audio feedback is presented on a large screen display with stereo speakers in front of the participant. While seated at the table, the participant performs a reaching task to a physical target, a cone to grasp or a large button to press, or virtual target, which requires the completion of a reach to a specified location with the assistance of audiovisual feedback. Physical and virtual target locations are presented either on the table to train supported reaches, or raised to variable heights above the table to train unsupported (against-gravity) reaches. At each height, targets can be placed at three different locations to engage different joint spaces in training.In virtual training , each reach begins with a digital image appearing on the screen, which breaks apart into several minute segments of the image, referred to as particles. As the participant moves his hand towards a target location, the hand's forward movement pushes the particles back to reassemble the image and simultaneously generates a musical phrase. Any aspect of the digital feedback, however, may be turned on or off for reaching tasks to physical targets, depending on the needs of the participant, to provide mixed reality tasks and associated training. See additional file The same abstract representation is applied across different reaching tasks  and various target locations in three-dimensional space, as viewed in additional file The AMRR system utilizes an action representation, which is necessary for simplifying the reach and grasp task into a manageable number of measurable kinematic features. Kinematic parameters are grouped into two organizing levels: activity level and body function level categories, and seven constituting sub-categories: four within activity and three within body function, presented in Figure From the three-dimensional positions of the markers worn by the participant, pertinent motion features are derived and used to compute all kinematic attributes. The quantified evaluation of these kinematic attributes is based upon four types of profile references: (a) trajectory reference, (b) velocity reference, (c) joint angle reference and (d) torso/shoulder movement reference. Each type of reference profile is derived from reaching tasks performed to the target locations trained within the AMRR system by multiple unimpaired subjects. These reference values, which include upper and lower bounds to account for variation characteristic of unimpaired movement, are scaled to each stroke participant undergoing training by performing a calibration at the initial resting position and at the final reaching position at the target. Calibrations are performed with assistance from the clinician to ensure that optimal initial and final reaching postures are recorded, from which the end-point position and joint angles are extracted and stored for reference. Real-time comparisons are made between the participant's observed movement and these scaled, unimpaired reference values. Therefore, in the context of the AMRR system, feedback communicating "inefficient movement" is provided when the participant deviates from these scaled unimpaired references, beyond a bandwidth determined by the clinician. Figure Activity level kinematic features . To reduce the distortion of the image, the participant must adjust his end-point in the direction opposite of the image stretch. See additional file Formation of the image, as the most prominent and explicit stream among the feedback mappings, not only provides a continuous frame of reference for trajectory distance and shape but also communicates progress towards achieving the goal of the completed image. Furthermore, by using visual information on the screen to complete the action, and thus not simultaneously focusing visually on his hand, the participant reduces reliance on visual monitoring of his end-point.Principles Applied: Visual feedback is best suited for communicating three-dimensional spatial information. Particle movement is directly linked to end-point movement in order to explicitly describe the end-point's spatial deviation from or progress towards achieving an efficient trajectory to the target. The feedback is delivered concurrent to action and continuously to allow the participant to observe movement of his end-point by monitoring formation of the image, and when needed, apply this information for online control of his movement to adjust for vertical or horizontal deviations.Movement Challenge: Sometimes stroke survivors are unable to utilize online information during task execution to develop a movement strategy, and require feedforward mechanisms to assist with planning proceeding movements.Feedback Components: A static visual summary communicates overall maximum trajectory deviation after each reach is completed to facilitate memory of real-time trajectory error. The summary presents a series of red bars. Their location on the screen  represents where error occurred in terms of vertical and horizontal coordinates . Visual perspective is used to communicate the distance at which error occurred  through spatial depth. A deviation occurring in the beginning of the movement appears closer to the viewer in perspective space, while deviations that occur later appear further away. The number of red bars conveys the magnitude of trajectory error. See the inefficient reach presented in additional file Principles Applied: Visual perspective is used to communicate the reaching distance as spatial depth. The summary provides an abbreviated history of the continuous particle movement by explicitly illustrating the magnitude (number of bars) and direction (location on screen) of trajectory errors. Presenting an offline terminal visual summary allows the participant to make an overall comparison of timing, location and magnitude of his trajectory deviations within the context of the entire reach. This display may also facilitate the implicit processing of the connection to memory of performance on other aspects of movement . Connecting real-time movement to offline contemplation can inform feedforward planning of successive movements.Movement Challenge: From the volitional initiation of movement until the completion of the reaching task, stroke survivors often have difficulty planning and controlling acceleration, trajectory speed, and deceleration of their movement across a defined space. This challenge makes relearning efficient movement plans difficult.Feedback Components: The musical phrase generated by the participant's movement is designed to help monitor and plan the timing of movement, as well as encourage completion of the action goal. The end-point's distance to the target controls the sequence of chords of the musical phrase. The reach is divided into four sections with different musical chords played for each. The sequence of chords follows a traditional musical pattern (with some randomized variation to avoid repetitiveness) that underlies many popular songs and is thus more likely to be familiar to the participant. The participant may intuitively associate each part of the reach  with a corresponding part of a musical sequence and be motivated to finish the reaching task to complete a familiar audio composition. If the end-point deviates from an efficient trajectory towards the target, the musical chords detune for the duration of deviation to place in time the occurrence of the deviation . See additional file End-point speed is mapped to the rhythm of the musical phrase. The participant's movement speed results in a "rhythmic shape" (change of rhythm over time) that most strongly encodes the end-point's acceleration during reach initiation, the deceleration when approaching the target, and the overall range of speed. In additional file Principles Applied: Audio feedback is best suited for communicating temporal movement aspects. Musical feedback is controlled by the end-point's speed and distance, and communicates the end-point's concurrent progress towards the target in a continuous manner. In accompaniment to explicit visual monitoring of the image formation, the audio feedback communicates changes within the end-point's temporal activity and encourages implicit information processing of the rhythm as a singular, remembered form . Memory of the musical phrase supports feedforward mechanisms for planning future movements and facilitates comparison across multiple reaches . The detuning of the harmonic progression adds a timestamp to the visual stretching of the image to assist feed-forward planning.Movement Challenge: Many stroke survivors do not exhibit a bell-shaped velocity profile characteristic of unimpaired reaching movements as a result of difficulties with timing and executing an efficient trajectory.Feedback Components: Simultaneous feedback streams describing the participant's end-point behavior can help the participant in relating the temporal and spatial aspects of his reach. The acceleration/deceleration pattern communicated by the rhythmic shape of music assists the participant in understanding speed modulation. The shrinking size of the image and harmonic progression communicate his distance and overall timing to reach the target. Coupling these simultaneous mappings allows for changes in speed to be connected to distance and facilitate the development of an integrated space-time plan. Figure Principles Applied: As a complex aspect of movement, the velocity profile cannot be effectively expressed as a singular feedback component, for a singular mapping would not allow the participant to determine which aspect of movement  requires adjustment that generalizes to multiple types of reaching tasks. Therefore, feedback on the velocity profile is observed through extracted information processing, in which the participant integrates information from both visual and audio streams reflecting directed distance and speed. Form integration of relevant audio and visual mappings into a unified velocity profile is encouraged by feedback feature selection: all involved components are concurrent and continuous mappings of end-point movement [Extracted information processing of the velocity profile may facilitate feedforward planning of acceleration and deceleration patterns along a given reaching distance to a target.movement . ExtractBody function level feedback must assist with challenges impeding the relearning of premorbid movement patterns of specific body structures relevant to reach and grasp performance. Feedback on body function parameters is coarse and discrete, thereby temporarily directing attention to specific body structures without distracting from completing the action goal. When a clinician must focus training on regaining functional independence primarily through teaching compensatory mechanisms, training for improved body function may not be appropriate ,21. AccoMovement Challenge: Many stroke survivors cannot achieve the appropriate timing or range of forearm pronation to complete a reach and grasp task.Feedback Components: Feedback on forearm rotation is related to end-point activity by controlling the angle of image orientation while particles come together to form the image. Image rotation is controlled by the difference between reference forearm orientation angles at a given distance to the target and the observed forearm orientation. Excessive supination causes clockwise rotation, while excessive pronation causes counter-clockwise image rotation. The size of the rotated image communicates where forearm rotation error occurred relative to the distance from the target.Principles Applied: Visual feedback communicates forearm spatial orientation in an explicit manner, which provides concurrent, continuous information throughout the reach to assist the participant's online control.Movement Challenge: Many stroke survivors have difficulty with sufficiently extending the elbow and appropriately timing elbow extension.Feedback Components: Percentage of elbow extension is mapped to a musical sequence performed by a digitized orchestra. In additional file Principles Applied: Communication of elbow extension is provided as audio feedback to assist with timing of movement. Percentage elbow extension is mapped in a continuous manner, which communicates changes in magnitude of extension within a reach. Elbow extension feedback is provided concurrently to movement and may be used for online control and/or memory of its temporal relationship to other mappings can be used for feedforward planning. Underscoring achievement with an increasingly rich orchestral sound is a standard technique of film scoring [implicit information processing, memory of successful reaches may be compared with the orchestral quality of other reaches within a set. scoring . ThroughMovement Challenge: To compensate for lack of extension during a reach, many stroke survivors use excessive shoulder movement (elevation and/or protraction) and excessive torso movement (flexion and/or rotation).Feedback Components: Compensatory movements are signaled by distinctive sounds that can interrupt the musical phrase (foreground) generated by end-point movement. Excessive shoulder compensation during a reach causes a cymbal sound while excessive torso compensation causes a crackling sound. These compensation indicators may be enabled individually or simultaneously, and are activated when the participant moves beyond an acceptable range of movement. Because these sounds do not combine well with the musical foreground in terms of harmony, timbre and rhythm, they draw the participant's attention to body structures exhibiting inefficient movement strategies.Principles Applied: Unique audio feedback indicators provide explicit indication of either shoulder or torso compensation concurrent to action for online control and correction of error. These auditory cues allow for intermittent monitoring of specific body functions amidst continuous end-point monitoring. As audio indicators, implicit information processing of these sounds caused by compensatory movements may be integrated into memory of the overall musical phrase and overall performance to assist with feedforward planning.Movement Challenge: Lack of intersegmental joint coordination while reaching for an object in three-dimensional space often results in inappropriate timing of movement at each joint.Feedback Components: The relationship among different audio mappings connects individual joint function to end-point behavior. During a successful reach, as the target is approached, the rhythmic progression driven by the end-point decreases in speed, and the volume and richness of orchestral strings mapped to elbow extension increases. The peak of orchestral richness is reached at the completion of the grasp and is synchronous to the image completion, the music progression resolution, and the sounding of the success cue (triangle sound), as demonstrated in the first reach in additional file extracted information process by the user.Principles Applied: Music (audio) is a very effective communicator of temporal relationships between separate but parallel events [concurrently to their movement, and remembered for extracted processing after the event [feedforward planning [Continuous feedback streams that should be correlated are given different individual characteristics  but follow a similar harmonic progression. Intermittent feedback indicators are used that can be related to continuous detailed feedback yet can still be identified as separate events.l events . Multiplhe event ,25. The planning ,27. ContThe connection between feedback components and corresponding movement components must be intuitive and easily perceived. Simplifying training by only offering feedback on one element at a time is not an optimal solution. Some stroke survivors cannot connect individually learned aspects of performance into a complete, generalizable, sustainable strategy . The higAs shown in the previous section, the AMRR system facilitates integration of multiple media streams through (a) appropriate selection of feature choices for feedback mappings , (b) compositional strategies that integrate closely related streams , and (c) parallel action and feedback narratives . These three strategies establish formal coherence between action and media.The AMRR system is highly adaptable to maintain an appropriate level of challenge and engagement based on a participant's impairment and progress. Numerous combinations of mediated feedback and task types allow for a wide variety of training scenarios for each participant.The primary components of the AMRR system that may be adapted include target type and location, selection and sensitivity of media components, and training environment. Three task types are trained within the AMRR system: reach, reach to push a button, and reach to grasp a cone. Different task types may be applied for focused training of specific aspects of movement, such as encouraging increased speed by using a button instead of a cone as the target. Various target locations engage different joint spaces in training. The four primary target locations used within the AMRR system include supported (on the table) ipsilateral (SI), supported lateral (SL), against-gravity (elevated from table) ipsilateral (AGI), and against-gravity lateral (AGL), listed in order of complexity of joint space utilized.Training is conducted in sets of ten reaches. Task type and feedback may be adjusted after each set. The clinician may guide the training focus for each set by selecting which media components to use. The clinician's choices also control the level of feedback complexity provided to the participant. Furthermore the sensitivity of each enabled feedback component may be independently adjusted, which allows the clinician to set the appropriate level of difficulty for each movement attribute. A hull is the adjustable amount of error in a participant's movement that the system will tolerate before giving feedback showing error Figure . AdjustaDifferent training environments may be used within the AMRR system by controlling the amount of media-based feedback  and task  components applied. The AMRR system provides for gradual transitions between virtual and physical training by using three types of training environments: physical , hybrid  and virtual . Multiple available variants (gradations) of these environments enable the clinician to shift training on a continuum towards a more virtual  or moreBefore training with the AMRR system begins, a participant is evaluated by the evaluating clinician and an attending physician to determine and rank the movement aspects requiring focused training. This collection of movement aspects from the preliminary assessment is called the impairment profile and may be used to guide the adaptive training. For each set of ten reaches, the treatment clinician typically chooses one or two limitations from the limitation profile on which to focus. Assisted by the media specialist, the clinician may then select the appropriate task components (target type and location), media-based feedback components (which feedback components to enable and respective sensitivities) and type of training environment  to utilize for training. Sequential modifications of these training components and movement aspects form the adaptive training. After each set, the system's quantitative evaluation, coupled with the clinician's direct observation, inform the adaptation decisions for the proceeding set. The AMRR system's ability to adapt along several dimensions throughout training allows for a customized training experience for each participant. For more description on the dynamics of training in application please refer to Chen et al, in which two example patient case studies are presented .The study protocol has been reviewed and approved by the Institutional Review Board at Arizona State University. All participants signed a consent statement and authorization forms. Copies of these signed forms are available for review by the Editor-in-Chief of this journal upon request. No participant's facial identity is revealed within the media associated with this manuscript.Training using the AMRR system is evaluated using changes in kinematic performance and standard clinical assessments as outcomes, each of which are measured prior to and after four weeks of training. Kinematic performance is measured from 10 reaches, unassisted by feedback, to each of the 4 trained target locations for a total of 40 reaches. Full clinical assessments include the Motor Activity Log (MAL), the Stroke Impact scale (SIS), the upper extremity portion of the Fugl-Meyer Assessment (FMA), and the Wolf Motor Function Test (WMFT) -32.Pre and post values are presented here from the WMFT and the upper extremity motor function portion of the Fugl-Meyer Assessment. The WMFT consists of a series of functional tasks relevant to activities of daily living that are timed and rated for quality by the evaluating clinician . The FASSupportive kinematic data are provided within this paper to demonstrate successful application of the system's feedback components to achieve focused and integrated improvements in three representative participants with different movement impairments. The raw pre and post training measurements of 12 kinematic features related to activity and body function movement aspects trained using the AMRR feedback are presented with respect to the three participants' training needs. Pre and post kinematic performance were analyzed using the Wilcoxon rank-sum Test due to the small sample number of reaches . Statisttrials). Individual trials are compared in sequential pairs to evaluate the correlation between observable feedback in one trial and improved performance of kinematic variables in the trial immediately following. Percentage improvement in performance of a specific kinematic variable after the presence of feedback was calculated as follows:In addition to kinematic measures and standard clinical assessments for evaluation, methods are being developed to better gauge the added value of AMRR feedback to rehabilitation training on the level of individual reaches . If the error feedback was not triggered (due to low feedback sensitivity) or the feedback component was turned off for that trial, the trial k was classified as no feedback (NFB).2. Within these selected trials, if error feedback was triggered, the trial k (pk mean) was compared to the mean performance of all immediately following trials k+1 (pk+1 mean) by calculating the percentage change:3. For the two resulting sets of FB and NFB trials, the mean performance of all initial trials 4. The difference in percentage improvement between the NFB and FB sets of trials was calculated:Results are presented from the pre and post evaluations of three stroke survivors who have each trained with the AMRR system for a period of 12 weeks. Table Figure Figure Among the kinematic parameters listed in Figure different kinematic variables . For example, for all 3 participants, torso forward compensation improved more after trials that triggered feedback on horizontal or vertical trajectory error  than after trials that had significant torso compensation error but did not trigger image stretching.Table Table The generalizable value of AMRR training is supported by improvements in both the WMFT FAS scores Figure  and kineTable An example of training using feedback on correlated aspects of movement can be seen in Participants 2 and 3, who each began training with insufficient elbow extension as a top-ranked movement impairment and experienced most of his focused training in torso compensation. This example illustrates how a more direct mapping encouraging explicit information processing on torso compensation was also utilized to promote elbow extension. Multiple types of feedback (e.g. feedback encouraging explicit versus implicit processes) catered in design to communicate performance of specific aspects of movement (trajectory versus joint relationships) can allow for both targeted and integrated training of multiple movement aspects. For example, in Participant 2's training, feedback on trajectory and speed were utilized to train individual performance aspects with resulting improvements, or were utilized in parallel to focus on training velocity bellness, which also improved. Improvement in joint correlation also utilized the integration of individual intermittent mappings in the context of continuous streams of activity level feedback in all 3 participants, for example, to improve performance of both individual joint function and integrated joint correlation.Training also accommodates focus on activity level versus body function level movement aspects. Participant 1, rated with mild impairment, was able to maintain the majority of her activity level performance while improving all of her body level aspects that related to focused training. While over half of Participant 2's training focused in torso compensation and elbow extension, he experienced significant improvements across most features presented in both body function and activity level training. Participant 2's significant worsening in shoulder compensation could potentially be addressed with longer treatment periods allowing for focused training on this parameter.different kinematic parameters may suggest that AMRR feedback promotes integrated training; it can allow the user to derive relationships between different aspects of movement through an extraction process (i.e. gain understanding after longer term experience with multiple feedback components), such as the effect of proximal body movements on distal end-point activity. Differences among the participants' performance improvements in relation to the triggered feedback demonstrates that the effectiveness of each feedback mapping can vary by user, providing support that an adaptable feedback paradigm catering to the needs of each participant is necessary.Table Future work will include analysis of relationships between other feedback mappings that encourage more implicit processes  and correlated performance improvements. These aspects of movement, which require longer experience with the system, must in turn be assessed across multiple trials or sessions.The AMRR system has been used and accepted by both stroke survivors and clinicians through a series of studies that demonstrate its reliability and validity ,16,17. WCurrently a home-based adaptive mixed reality rehabilitation (HAMRR) system is under development that uses the design principles of the AMRR system to allow stroke survivors to continue their treatment over multiple months of therapy at home. Multimodal sensing methods and audiovisual feedback provide concurrent and summary information on task performance to assist the stroke survivor in self-assessing his movement. Within each week of training, a computational adaptation component automatically customizes weeklong therapy sessions based on a clinician's goals for the stroke survivor. The adaptation is structured as a semi-supervised framework, using a probabilistic graphical network representation based on . The sysThe methodology for designing a mixed reality system for stroke rehabilitation discussed in our companion paper has beenThe authors declare that they have no competing interests.NL, TR, YC and SLW contributed to the concepts and of the paper. NL and YC prepared the manuscript. TR, SLW and MD provided editing and consultation. All authors read and approved the final manuscript.QuickTime movie. Depicts a participant interacting with the system in the presence of a physical therapist.Click here for fileQuickTime movie. Depicts motion capture tracking in parallel to a participant interacting with the system.Click here for fileQuickTime movie. Depicts a participant interacting with different targets and target locations within the system.Click here for fileQuickTime movie. Depicts the visual feedback generated by (1) an efficient reach, followed by a reach exhibiting (2) trajectory error, communicated as image stretching. The box and diamond appearing before and after each reach is a visual representation of the location of the participant’s right hand, provided to assist the participant in returning his or her hand to a consistent rest zone on the table.  Click here for fileQuickTime movie. Depicts the image formation and plays the musical phrase generated when a participant performs (1) an efficient reach, followed by (2) a reach with a large horizontal trajectory deviation to the right that generates image stretching and a detuning musical phrase, followed by (3) a slow reach without a prominent rhythmic shape.Click here for fileQuickTime movie. (1) Depicts image rotation when a participant incorrectly times forearm rotation, (2) the orchestral accompaniment generated when a participant extends his elbow while performing an efficient reach, (3) the shoulder compensation sound generated when a participant uses excessive shoulder compensation during the reach, and (4) the torso compensation sound generated when a participant uses excessive torso compensation during the reach.Click here for fileQuickTime movie. (1) Depicts image formation and plays the musical phrase with orchestral accompaniment generated when a participant extends his elbow while performing an efficient reach, and (2) depicts image formation, stretching, and summary of error, and plays the shoulder compensation sound amidst the musical phrase, generated when a participant uses excessive shoulder compensation during the reach, and as a result performs related trajectory deviations.Click here for file


Inves[in vivo  or code [in vivo . In the [in vivo , a phenoIn this work we investigate the functional role of PFC microcircuits in the expression of persistent activity, focusing on the contribution of nonlinear dendritic properties to the induction, termination, and coding of upcoming state transitions.prior to the induction and/or termination of persistent firing contain predictive information of the upcoming state-transition(s), which is readily available to downstream neurons. These findings suggest that PFC microcircuits may serve as tunable and predictive modules of persistent activity and subsequently working memory.Towards this goal we developed a layer V PFC microcircuit consisting of 7 pyramidal neurons and 2 interneurons implemented in the NEURON simulation environment. Modelling equations for the biophysical mechanisms used have been reported in ,7. All n


In Western music, the major mode is typically used to convey excited, happy, bright or martial emotions, whereas the minor mode typically conveys subdued, sad or dark emotions. Recent studies indicate that the differences between these modes parallel differences between the prosodic and spectral characteristics of voiced speech sounds uttered in corresponding emotional states. Here we ask whether tonality and emotion are similarly linked in an Eastern musical tradition. The results show that the tonal relationships used to express positive/excited and negative/subdued emotions in classical South Indian music are much the same as those used in Western music. Moreover, tonal variations in the prosody of English and Tamil speech uttered in different emotional states are parallel to the tonal trends in music. These results are consistent with the hypothesis that the association between musical tonality and emotion is based on universal vocal characteristics of different affective states. Carnatic music) is especially useful for such comparison because, as in Western music, the emotions associated with various tone collections  are well documented ragas associated with positive/excited and negative/subdued emotions compare with the use of tones in Western melodies composed in the major and minor modes. In particular, we wished to know whether there is a common denominator in the way musical tonality is used to convey emotion across cultures, and, if so, its probable basis.Different sets of tones and tone-relationships are used to convey particular emotions in a variety of musical cultures raga in which it is composed. Ragas are analogous to modes in Western music in that they specify a particular collection of tones and tone-relationships. Ragas, however, are more complex because they also prescribe specific patterns of ascending and descending movement and gamakam  ragas are associated with specific emotional themes called rasasrasas, two of which parallel the emotions associated with the major and minor modes and are thus suited to a cross-cultural comparison. These are Haasya, the rasa of joy, happiness, and mirth; and Karunaa, the rasa of sadness, grief, and pity ragas selected for comparison are associated with these rasas.The tonality of a Carnatic melody is organized by the ragas are derived is the 12-tone octave division shown in gamakams (see above) The basic set of tones and tone-relationships in Carnatic music from which Haasya rasa, the selected ragas were Bilahari and Mohanam. For the Karunaa rasa, the selected ragas were Naadanaamakriya, Punnaagavaraali, and Varaali . All melodies were traditionally notated using the intervals listed in For the  Varaali  [8], [9]raga . In both cases interval size was calculated as the difference in cents between the fundamental frequencies (F0s) of the two notes in question. Melodic intervals thus describe variations in F0 over time, whereas tonic intervals describe the place of each F0 within the tonal context of the raga. Interval size data was tallied in 100-cent bins from −1200 to 1200 for melodic intervals, and from 0 to 1200 for tonic intervals. Bin counts were normalized as percentages of the total number of melodic or tonic intervals in a melody and averaged across melodies in one of the emotional conditions examined to obtain average distributions of interval size. Differences between emotional conditions were evaluated for statistical significance using Mann-Whitney U-tests. For melodic intervals comparisons were between the proportion of intervals smaller and larger than a major second  and the tonic note of the Kodu” and “Nijam” in Tamil. In both cases the speakers were instructed to use the quality of their voice to convey either positive/excited or negative/subdued affect. The recordings were made in a sound-attenuating chamber with an omni-directional capacitor microphone coupled to a solid-state digital recorder. Data were saved on flash memory cards in .wav format .Speech was recorded from 20 native speakers of Indian Tamil  and 20 native speakers of American English ; the speakers ranged in age from 19–72 and had no significant speech or hearing pathology. To mitigate the possible influence of foreign intonation patterns, speakers were required to have lived in the country of their native language for at least 10 years. All subjects gave written informed consent as required by the Institutional Review Boards of Duke University and the National University of Singapore. Speakers read monologues and bi-syllabic expressions in their native language. The monologues consisted of 10 paragraphs, 5 with emotionally positive/excited content and 5 with negative/subdued content . Given the large number of recordings (2000), a subset comprising 240 recordings  was randomly selected for evaluation. Raters listened to each recording, categorizing it as expressing joy or sadness and scoring the quality on a five point scale . The accuracy of emotion recognition and average quality of expression were assessed in terms of speaker language and rater language. The intended emotions were accurately recognized and comparable in terms of quality of expression in both languages .For each recording, the fundamental frequency and relative intensity of each 10 ms time-step was calculated using Praat's U-tests comparing the proportion of intervals smaller and larger than a major second (see above).For each recording, prosodic intervals were calculated as the frequency difference in cents between adjacent voiced intensity maxima. Like melodic intervals in music, prosodic intervals thus describe changes in frequency over time. Interval size data was tallied in 25-cent bins from −1200 to 1200. Bin counts were normalized as percentages of the total number of prosodic intervals in a recording and averaged across recordings in an emotional condition to obtain representative distributions of interval size. In keeping with the analysis of melodic intervals in music, differences between emotional conditions were evaluated for statistical significance using Mann-Whitney U-tests .For each recording, normalized spectra were calculated for each voiced speech segment. Each segment's spectrum was calculated by applying a Hamming window and a fast Fourier transform , whereas negative/subdued raga melodies comprise more intervals smaller than a major second .The average distributions of melodic interval sizes in Western melodies composed in the major and minor modes are shown in The average distributions of prosodic interval sizes in positive/excited and negative/subdued Tamil and English speech are shown in The average normalized spectra of voiced segments in positive/excited and negative/subdued Tamil and English speech are shown in This analysis of Carnatic and Western music shows that melodic intervals in both traditions are generally larger in melodies associated with positive/excited emotion, and smaller in melodies associated with negative/subdued emotion. Similarities are also apparent with respect to major and minor tonic intervals, being more prevalent in melodies associated positive/excited and negative/subdued emotions respectively in both traditions. There are however, differences in the particular tonic intervals emphasized most in making these emotional distinctions see . The difIn contrast to at least one other report Spectral features differentiate excited and subdued speech less well than prosodic differences. In English, speech spectra show a greater proportion of energy in the higher harmonics of negative/subdued relative to positive/excited speech, thus emphasizing smaller intervals. Tamil speech, however, does not exhibit this pattern so clearly see . Given tIn previous work on the affective impact of the major and minor modes we focused on spectra, reporting parallel differences in music and American English speech One way of explaining the common cross-cultural pattern in the use of melodic intervals to convey emotion is mimicry of the universal tonal characteristics of the human voice in different emotional states This similarity, however, does not demand a causal relationship between these two domains. It remains possible, for example, that the association between melodic interval size and emotion in music is entirely the product of exposure to arbitrary cultural conventions that just happen to accord with speech. This possibility notwithstanding, such an interpretation is at odds with considerable evidence. In addition to the present results examining tonality, correlations between vocal and musical emotional expression have been documented with respect to tempo, intensity, timbre, and F0 in a variety of languages and musical traditions . Second, infants spontaneously produce vocalizations with acoustic properties related to their affective status long before the development of musical abilities The use of musical tones to convey emotion is similar in Western and Carnatic Indian melodies. Melodic intervals are smaller in melodies composed in tonalities associated with negative/subdued emotion and larger in melodies composed in tonalities associated with positive/excited emotions. Melodic interval size as a function of emotion in music parallels the variation of interval size with emotion in speech prosody. Although musical interval size is also related to speech spectra, this association is less robust across cultures. These observations, together with physiological, developmental, and phylogenetic evidence, suggest that the affective impact of musical tonality is generated by association with the characteristics of voiced speech in corresponding emotional states.Figure S1Assessment of emotion expression in speech recordings. (A) Accuracy of emotion recognition. Native speakers of Tamil (N = 3) and English (N = 3) rated a subset of the Tamil and English speech recordings Click here for additional data file.Figure S2Complete statistics for melodic interval comparisons. (A) Overlays of the distributions underlying the mean percentages shown in the insets of U-tests used to assess differences between the distributions in A for statistical significance.(TIF)Click here for additional data file.Figure S3Complete statistics for tonic interval comparisons. (A) Overlays of the distributions underlying the mean percentages shown in U-tests used to assess differences between the distributions in A for statistical significance. (C and D) Data presented in the same format for the mean percentages shown in (TIF)Click here for additional data file.Figure S4Complete statistics for prosodic interval comparisons. (A) Overlays of the distributions underlying the mean percentages shown in the insets of the monologue panels of U-tests used to assess differences between the distributions in A for statistical significance. (C and D) Data presented in the same format for bi-syllabic-expressions.(TIF)Click here for additional data file.Figure S5Complete statistics for average normalized spectra comparisons. (A) Overlays of the distributions underlying the mean percentages shown in the insets of U-tests used to assess differences between the distributions in A for statistical significance.(TIF)Click here for additional data file.Table S1Comparison of fundamental and formant frequencies in Tamil and English speech. (A) Comparison of mean fundamental frequencies and mean peak frequencies for the first and second formants in Tamil and English speech (from the monologue recordings). The results of two-tailed independent samples t-tests used to assess language differences for statistical significance are also shown. (B) Same as (A), but for the bi-syllabic expression recordings. Praat's (DOC)Click here for additional data file.Text S1Discussion of fundamental and formant frequencies in English and Tamil speech. This file discusses the results of the comparison of fundamental and formant frequencies in Tamil and English shown in (DOC)Click here for additional data file.Text S2Further discussion of spectral similarities This file compares past (DOC)Click here for additional data file.


Current meta-omics developments provide a portal into the functional potential and activity of the intestinal microbiota. The comparative and functional meta-omics approaches have made it possible to get a molecular snap shot of microbial function at a certain time and place. To this end, metagenomics is a DNA-based approach, metatranscriptomics studies the total transcribed RNA, metaproteomics focuses on protein levels and metabolomics describes metabolic profiles. Notably, the metagenomic toolbox is rapidly expanding and has been instrumental in the generation of draft genome sequences of over 1000 human associated microorganisms as well as an astonishing 3.3 million unique microbial genes derived from the intestinal tract of over 100 European adults. Remarkably, it appeared that there are at least 3 clusters of co-occurring microbial species, termed enterotypes, that characterize the intestinal microbiota throughout various continents. The human intestinal microbial metagenome further revealed unique functions carried out in the intestinal environment and provided the basis for newly discovered mechanisms for signaling, vitamin production and glycan, amino-acid and xenobiotic metabolism. The activity and composition of the microbiota is affected by genetic background, age, diet, and health status of the host. In its turn the microbiota composition and activity influence host metabolism and disease development. Exemplified by the differences in microbiota composition and activity between breast- as compared to formula-fed babies, healthy and malnourished infants, elderly and centenarians as compared to youngsters, humans that are either lean or obese and healthy or suffering of inflammatory bowel diseases (IBD). In this review we will focus on our current understanding of the functionality of the human intestinal microbiota based on all available metagenome, metatranscriptome, and metaproteome results Bacteroidetes and Firmicutes  are determined by the microbiota composition and have a direct effect on the host epithelial cell energy availability. A microbiota stimulated with probiotic microbes can even decrease the incidence of infant diarrhea and atopic eczema due to host immune stimulation , other environmental bacteria form the basis for the microbiota instead of vaginal and faecal bacteria from the mother, reportedly resulting in a substantial reduction of bifidobacteria  and elderly individuals (0.6) . The centenarian group also showed a dominant portion of Firmicutes and Bacteroidetes . The Firmicutes/Bacteroidetes ratios obtained for the centenarians, elderly and young adults were 3.6, 5.1, and 3.9, respectively. However, there was a significant decrease in the Firmicutes subgroup Clostridium cluster XIV and an increase in Bacilli in the centenarian group. Furthermore, there was a significant increase in several facultative anaerobes, members of the Proteobacteria phylum, many of which constitute opportunistic pathogens. This rearrangement of the microbiota does not seem to be in favor of the aging subjects that showed an increased level of circulating inflammatory cytokines. These were inversely associated with bacteria belonging to Clostridium cluster XIV and Clostridium cluster IV that include the main butyrate-producers in the gut. Butyrate has been associated with a range of health effects from anti-inflammatory properties to enhancement of intestinal barrier function , elderly (60–80 years old) and an additional group of centenarian citizens (~100 years old), clearly demonstrated that the process of ageing coincides with decreasing microbiota diversity  compared with Firmicutes (40%). However, the proportions of the major phyla showed extraordinary variation between individuals, with the proportion of Bacteroidetes ranging from 3 to 92% and Firmicutes from 7 to 94%. In addition to the general composition, also the core microbiota of the elderly differed substantially with that of young adults, characterized by a shift to a more Clostridium cluster IV-dominated community in the elderly. The microbiota of the elderly showed temporal stability for the majority of subjects as revealed by analysis of 3-month follow-up samples.Recently, pyrosequencing of tagged PCR-amplified 16S rRNA genes was applied to characterize the fecal microbiota of 161 seniors aged 65 years and older in the ELDERMET consortium  lead to rapid changes in the microbiome composition but was not sufficient to shift individuals between the two enterotypes described in the same study  containing several probiotics on the gut microbiome of gnotobiotic mice colonized with a model human gut microbiota and monozygotic twins  and Firmicutes gene clusters encoding ABC-type sugar transport systems.Metagenomic approaches combined with studies using gnotobiotic animals colonized with only a few known microorganisms or even the entire human fecal microbiota provide a powerful tool for examining the relationship between the host and the functionality of the microbial community under controlled conditions. A study of humanized gnotobiotic mice transplanted with either fresh or frozen adult human fecal microbial communities into germ-free C57BL/6J mice revealed a stable and heritable colonization which enabled a diet intervention, where the mice were switched from a low-fat, plant polysaccharide to a high-fat, high-sugar diet  representing the most studied disorders to date. Most research about potential differences of microbiota related to different disease states has so far focused on describing the composition and diversity of the microbiome in patients compared to healthy subjects, and consequently revealing interesting associations between them. In order to get a better understanding of the underlying mechanisms of the relationship between the microbial communities and specific disorders, functional microbiomic approaches need to be employed.Despite exhaustive research efforts, the etiology and pathogenesis of IBD, including Crohn's disease (CD) and ulcerative colitis (UC) have stayed unclear. The causes of these intestinal diseases are most likely linked with both human gene- and microbiome-associated factors  compared to healthy subjects. In addition to F. prausnitzii, also other core members of the microbiota, such as Roseburia, were less abundant in ICD. Interestingly, a separate study analyzing the same samples showed clear shifts in metabolic profiles corresponding to the same bacterial groups  mice to germ-free wild-type mice causes an increase in fat mass in the recipients, indicating that the obese microbiota has an increased capacity to harvest energy from the diet  5, which is a transmembrane protein recognizing bacterial flagellin, in a mouse model results in a phenotype resembling human metabolic syndrome (Vijay-Kumar et al., The complexity of the microbiota–host interactions has been the prime obstacle in defining microbial functionality at a post-genomic level. The recent technical advances in analyzing genomes, transcriptomes and proteomes of complex bacterial consortia and intra- and interspecies metabolic networks help to tackle this problem and will enable systems-level analyses of the crosstalk between the microbiota and the host.There are multiple reports providing circumstantial evidence to support the concept that microbiota composition and activity influence host metabolism and disease development. These examples include the differences in microbiota composition and microbiota expressed proteins of breastfeeding as compared to formula-fed babies (Schwartz et al., 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.


A single olivocerebellar (OC) axon gives rise to about seven branches that terminate as climbing fibers (CFs). Branching patterns of an OC axon, which are classified into local, transverse, and longitudinal types, are highly organized, in relation to the longitudinal molecular  compartmentalization and the transverse lobulation of the cerebellum. Local branching is involved in forming a narrow band-shaped functional subarea within a molecular compartment. On the other hand, transverse and longitudinal branchings appear to be involved in linking mediolaterally separated molecular compartments and rostrocaudally separated lobular areas, respectively. Longitudinal branching occurs frequently between equivalent molecular compartments of specific combinations of lobules. These combinations include lobule V-simple lobule and crus II-paramedian lobule in the pars intermedia and hemisphere, and lobules I–V and lobule VIII in the vermis. The longitudinal branching pattern not only fits with mirror-imaged somatosensory double representation of the body in the pars intermedia, but it also suggests a general rostrocaudal link exists for the whole cerebellum across the putative rostrocaudal boundary in lobule VIc-crus I. Molecular compartments of the cerebellar cortex originate from the Purkinje cell (PC) clusters that appear in the late embryonic stage, when the immature OC projection is formed. Some clusters split rostrocaudally across crus I during the development of cortical compartments, which would result in longitudinal branching of OC projection across crus I. Supposing that the branching pattern of OC axons represents an essential organization of the cerebellum, longitudinal branching suggests a functional and developmental links between the rostral and caudal cerebellum across lobule VIc-crus I throughout the cerebellar cortex. The cerebellar cortex is subdivided two ways: transversely by its lobular folding and longitudinally by PC subset compartments that are defined by the expression patterns of certain molecules, such as aldolase C or zebrin II  afferents, which originate exclusively from the inferior olive (IO) as the OC projection, are particularly well-organized in a topographic sense. CFs that arise from a specific IO subdivision form synapses onto PCs in a particular aldolase C compartment that are about 0.1–0.5 mm in mediolateral width but elongated in the longitudinal direction , are characterized by their divergent branching patterns  compartments in the medial and lateral pars intermedia, respectively, in lobule V in cats  simple lobule and caudal lobule V vs. crus II and paramedian lobule in the pars intermedia and hemisphere, (2) lobules II–V vs. copula pyramidis in the pars intermedia, and (3) lobules II–V and lobule VIII in the vermis  generally project and converge to a distinct neuronal group in the lateral vestibular nucleus  with their local branches . Thus, the transverse branching does not seem to represent the general projection pattern of OC axons. Transverse branching of OC axons makes multiple longitudinal narrow areas that are separated mediolaterally, receive the same OC input, and project to the similar subarea in the DCN. Functional significance of this organization has not been much clarified. Further studies, including comparative ones, may be required to better characterize the transverse branching.The most frequently observed category of longitudinal branching is the one across crus I in the pars intermedia and hemisphere. Branching between lobule V and paramedian lobule in C1 zone in cats (above) belongs to this category. Branching between lobules II–V and the copula pyramidis and branching between the simple lobule and the crus II-paramedian lobule, which are the most common pattern in the rat  in the mouse , our analysis of PC cluster development has shown some clusters are “split” into rostral and caudal clusters underneath other clusters in crus I (Fujita et al., Branching patterns of OC axons can be classified into local, transverse, and longitudinal types. These branching patterns of OC axons are tightly related with the functional organization of the cerebellar cortex. The local branching is in accordance with the microzonal organization of the cerebellar cortex. The transverse and longitudinal branching patterns are in accordance with the compartmental organization of the cerebellar cortex and related to the lobular organization. The longitudinal branching, in particular, is a basis of the mirror-imaged double somatotopic representation of the body in the cerebellum. Attempts to relate the OC branching patterns to development of cerebellar compartmentalization and lobulation may be helpful in untangling the complicated compartmental and lobular organization of the cerebellar cortex.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.


Sensory hair cells in auditory and vestibular organs rely on active mechanisms to achieve high sensitivity and frequency selectivity with respect to weak stimuli. Self-sustained oscillations in hair cells occur on two very different levels. First, the mechano-sensory hair bundle itself can undergo spontaneous mechanical oscillations. Second, self-sustained electric voltage oscillations across the membrane of the hair cell have been documented in the inner ear of lower vertebrates. The functional significance of these self-sustained voltage oscillations is currently unknown.We used a Hodgkin-Huxley type model of the baso-lateral ionic currents of bullfrog sacculus to study genesis of spontaneous voltage oscillation patterns and how the spontaneous oscillations shape the response of the hair cell to external mechanical stimuli. To examine the influence of inevitable fluctuations on the dynamical regimes, we included a stochastic transduction current originating in the Brownian motion of the hair bundle and channel noise arising due to the finite number of mechano-electrical transduction channels .+-activated (BK) potassium currents. We found that for large values of BK conductance the system is either at equilibrium or exhibit tonic oscillations. For small values of BK and large values of K1 conductances the dynamics of the model shows diverse patterns of activity including quasi-periodic oscillations, large-amplitude periodic spikes, and bursts of spikes. In particular we found a peculiar transition to bursting through quasiperiodic oscillations with two independent frequencies corresponding to a 2D torus in the phase space of the system. Within small patches of parameter space at the transition from spiking to bursting and at the spike adding transition, voltage dynamics are chaotic. Furthermore, we showed that thermal fluctuations of mechano-electrical transduction current can lead to chaos in a wide area of parameter space.We determined the bifurcation structure of the model in terms of two important ionic conductances, associated with the inwardly rectifier (K1) and Ca2f > 5 Hz. Hence, an oscillatory voltage compartment might constitute a biophysical implementation of a high-gain amplifier. Cells poised in the chaotic regime of low BK and high K1 conductances showed poor tuning, but provided a high sensitivity to low-frequency variations of external stimuli. In summary, this study shows that the electrical oscillator found in saccular hair cells contributes significantly to nonlinear amplification of external mechanical stimuli. This further supports the idea of nonlinear oscillators playing a crucial role in the operation of the inner ear.We found a high sensitivity and frequency selectivity for the regime of regular spontaneous oscillations in response to sinusoidal stimuli with frequencies