The way long-term synaptic plasticity regulates neuronal spike patterns is not completely understood. This issue is especially relevant for the cerebellum, which is endowed with several forms of long-term synaptic plasticity and has been predicted to operate as a timing and a learning machine. Here we have used a computational model to simulate the impact of multiple distributed synaptic weights in the cerebellar granular-layer network. In response to mossy fiber (MF) bursts, synaptic weights at multiple connections played a crucial role to regulate spike number and positioning in granule cells. The weight at MF to granule cell synapses regulated the delay of the first spike and the weight at MF and parallel fiber to Golgi cell synapses regulated the duration of the time-window during which the first-spike could be emitted. Moreover, the weights of synapses controlling Golgi cell activation regulated the intensity of granule cell inhibition and therefore the number of spikes that could be emitted. First-spike timing was regulated with millisecond precision and the number of spikes ranged from zero to three. Interestingly, different combinations of synaptic weights optimized either first-spike timing precision or spike number, efficiently controlling transmission and filtering properties. These results predict that distributed synaptic plasticity regulates the emission of quasi-digital spike patterns on the millisecond time-scale and allows the cerebellar granular layer to flexibly control burst transmission along the MF pathway. By operating in a continuously changing environment, neuronal networks have evolved precise processes regulating the number and positioning of spikes  .A granular-layer model was built using the EDLUT simulator (Centro de Investigación en Tecnologías de la Información y las Comunicaciones (CITIC) in the University of Granada.The differential equations were solved using the fourth order Runge–Kutta method with a fixed time-step of 100 μs. Simulations of 4-s activity trials were carried out on a 152-node cluster located at the Figure The granular-layer model which has been simulated in this work is composed of the following elements:Mossy fibers (MFs) (350 fibers): MFs convey the input stimuli and activate GrCs and GoCs.Granule cells (GrC) (4500 neurons): The population of GrCs has a number of connections per cell (from MFs) that follows a Gaussian distribution with a mean of four connections and a standard deviation of one connection  (27 neurons): Each GoC receives excitatory connections from 100 GrCs  (300 neurons): Each SC receives excitation from 100 GrCs and inhibits the activity of 4.5 GoCs (on average). The SCs have only been included in the model when explicitly stated. Otherwise, the connections between SC and GoC have been disabled.Since this work is focused on temporal properties, while neglecting specific topologies like the center-surround organization imposed by lateral inhibition  and resting conductance (Grest),Neurons were modeled using modified versions of the LIF model . Finally, Eq. 1B determines g∞,NMDA(Vm), the gating function of the NMDA channels accounting for voltage-dependent magnesium block  and NMDA (N-methyl d-aspartate) receptor-mediated conductance respectively, which provide excitation, and gGABA represents the GABA receptor-mediated conductance, which provides inhibition. τAMPA, τNMDA, and τGABA are the decaying time constants of each receptor type. The synaptic conductances were modeled as decaying exponential functions, which provide a reasonable accuracy as well as computational efficiency  and synaptic receptor (Eqs 2A–C) have been chosen to model granule cell, stellate cell, and Golgi-cell dynamics  in response to the first spike of the MF burst and the number of spikes  elicited in response to the whole burst. In order to determine the activity state of neurons under combined excitatory and inhibitory synaptic drive, we have defined the GrC excitatory/inhibitory conductance balance using the following equation.E/I g balance (t) represents the average excitatory/inhibitory balance at time t, P represents the set of cells under study. iE and iI represent all the sources of excitation and inhibition. g,i,jAMPA(t) and g,i,jNMDA(t) represent the AMPA-receptor and NMDA-receptor-mediated conductances at the j-th excitatory synapse reaching the cell i, as previously defined in Eq. 3. Similarly, gi,jGABA,(t) represents the GABA-receptor conductance at the j-th inhibitory synapses reaching the cell i. In Eq. 4, the excitatory conductances (AMPA and NMDA) have been considered with negative sign, and the inhibitory conductance (GABA) has been considered positive. Indeed, a negative value at the conductance balance indicates the predominance of excitation over inhibition in the cell.In order to investigate the consequences of distributed synaptic plasticity on spike timing, we have analyzed the impact of weights at the different synapses of the granular layer in a spiking network model Figure . Backgrotime-window effect, which predicts that the feed-forward inhibitory loop passing through the MF-GoC-GrC connections can curtail the GrC response . Following a MF burst, the E/Ig balance showed an initial negativity (net excitatory conductance) followed by a large positivity (net inhibitory conductance), which was eliminated by switching-off GoC-GrC transmission  function of the circuit was analyzed by evaluating the relationship between input and output bursts in GrCs, which represent the output element of the circuit. I/O plots for the first GrC spike showed the GrC response as a function of the time-offset with respect to MF discharge Figure B. By couplateau effect is that, as the GrC response increases due to higher MF-GrC weight, the inhibitory feed-back loop is more intensely activated preventing a further increase in GrC firing . Finally, MF-GrC synaptic weights had an impact on the spike pattern emitted by GrCs, which changed from a small number of singlets in the LTD state toward a higher amount of singlets and some doublets in the LTP state  PF responses. The synaptic weights at GoC-GrC connections influenced the strength and duration of the inhibitory window. In the feed-back inhibitory loop, regulation of weights at the GrC-GoC synapses transformed the increasing activity at the PFs in shorter, and more precise excitatory windows. The inhibitory connections impinging onto GoCs effectively controlled generation of late spikes. The control of synaptic weights in the feed-forward and feed-back inhibitory loops and in the interneuron inhibitory network indeed allowed generating a large variety of patterns in GrCs determining the number and timing of emitted spikes. While long-term synaptic plasticity has been demonstrated at the MF-GrC synapse LTD may indeed occur at the PF – GoC synapse  The nature of GrC synaptic receptors is such that they can precisely control both first-spike timing on the sub-millisecond scale through AMPA receptors . The composition of the output burst is critical for cerebellar network computation, as shown by the powerful regulation exerted by NMDA receptors (D’Angelo et al., As long as spike bursts are regulated to achieve specific computational effects, the granular-layer circuit needs to maintain a homeostatic balance in order to prevent saturation of PF activity Marr, . The gratime-window mechanism (D’Angelo and De Zeeuw, in vivo, sensory stimulation generates short spike bursts and whole-cell recordings from GrCs show generation of a new burst (Chadderton et al., in vivo in response to MF bursts show on average five EPSCs and one to three spikes in GrCs (e.g., see Duguid et al., in vivo, an apparent reduction of response duration was observed when the inhibitory circuit was blocked and a clear anticipation of the response emerged when LTP was induced at the MF-GrC synapse (Roggeri et al., in vivo in a way consistent with that predicted here.By fully implementing the The importance of this quasi-digital GrC spike pattern becomes evident when considering that PC responses are differentially sensitive to the number of spikes transmitted by GrCs along the PFs. First of all, the PF-PC synapse shows a pronounced short-term facilitation, so that single PF spikes are not transmitted but transmission becomes effective with two or more spikes (Casado et al., time-window mechanism (D’Angelo and De Zeeuw, These simulations show that distributed synaptic plasticity fully implements the These simulations also address one major issue in cerebellar modeling. Theoretical models of the cerebellum assume that learning is driven by some optimization factors related to gain, signal-to-noise ratio, and mutual information transfer. Although some models consider only learning at the PF-PC synapse (e.g., Schweighofer 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.


Arabidopsis thaliana Col-0 ecotype (Col) as our focal plant co-cultured in vitro with different neighbors [A. thaliana Ler ecotype (Ler) or Capsella rubella (Cap)]. Principal component and cluster analyses revealed that both root secreted secondary metabolites and proteins clustered separately between the plants grown individually  and the plants co-cultured with two homozygous individuals  or with different individuals (Col-Ler and Col-Cap). In particularly, we observed that a greater number of defense- and stress- related proteins were secreted when our control plant, Col, was grown alone as compared to when it was co-cultured with another homozygous individual (Col-Col) or with a different individual (Col-Ler and Col-Cap). However, the total amount of defense proteins in the exudates of the co-cultures was higher than in the plant alone. The opposite pattern of expression was identified for stress-related proteins. These data suggest that plants can sense and respond to the presence of different plant neighbors and that the level of relatedness is perceived upon initial interaction. Furthermore, the role of secondary metabolites and defense- and stress-related proteins widely involved in plant-microbe associations and abiotic responses warrants reassessment for plant-plant interactions.The mechanism whereby organisms interact and differentiate between others has been at the forefront of scientific inquiry, particularly in humans and certain animals. It is widely accepted that plants also interact, but the degree of this interaction has been constricted to competition for space, nutrients, water and light. Here, we analyzed the root secreted metabolites and proteins involved in early plant neighbor recognition by using  How organisms recognize each other has been at the forefront of inquiry ever since people began to wonder when babies begin to perceive themselves as unique individuals separate from their mothers. In the 70 s, studies were conducted in which chimpanzees were found to recognize themselves in mirror reflections but monkeys failed to do likewise Proteus mirabilis that allows colonies from the bacteria to differentiate between self and non-self by the development of boundaries between the colonies Ambrosia dumosa when it encounters a clonal individual as compared to a different individual from the same species. Subsequent studies demonstrated self/non-self recognition of roots in different plant species, such as strawberry The ability of organisms to differentiate each other has been studied at various levels in the context of self- and non-self recognition. One study found a molecular determinant, the ids (identification of self) locus, on the bacterium Most biochemical and molecular studies related to plant recognition have focused on the aerial parts of plants, describing how volatiles from leaves and flowers can establish communication with other plants Drosophila melanogasterIt was reported that in ants A. thaliana respond to different plant neighbors. We choose to look at root exudates to determine whether neighbor identity can influence the quantity and/or quality of signals secreted by the roots. For this purpose, we selected A. thaliana ecotypes Col-0 and Landsberg erecta (Ler), and Capsella rubella (Cap) due to their similarities/differences in their genome composition. A. thalaiana ecotypes Col and Ler have close genome relationship, but are not identical, based on genetic diversity and genome sequence comparison analyses A. thalianaA. thaliana ecotype Col-0 would be higher when grown with Cap than when grown with ecotype Ler or with the same individual (Col-0). For this purpose, A. thaliana Col-0 ecotype was co-cultured with different neighbors exhibiting different degrees of relatedness and assumed levels of competition. A. thaliana Col-0 ecotype was cultured with a similar homozygous Col-0 individual, with Ler ecotype or with Cap. The interaction time between the plant individuals was kept to a minimum (co-cultured three days) to identify early biochemical markers in the root exudates that are involved in neighbor differentiation.Previous studies have shown that plants exhibit competition behavior towards other plants by producing more number of roots. When a given plant encounters an individual from a different species the degree of competition (by means of measuring root production) increases compared to when the plant encounters an individual from the same species Arabidopsis thaliana Col-0 ecotype (Col), A. thaliana Lansberg erecta ecotype (Ler) and Capsella rubella (Cap) seeds were purchased from Lehle Seeds  and ABRC . The seeds were surface sterilized with 3% (v/v) sodium hypochlorite for two minutes followed by three washes with sterile distilled water. The seeds were germinated on solidified MS Plantlets were grown in Magenta boxes as described above. Three days post transfer, the exudates of 40 plants or 40 co-cultures were collected, pooled and centrifuged at 8,000 g for 15 min at 4°C to remove the root sheathing. The supernatants of Col, Col-Col, Col-Ler, Col-Cap, Ler, Ler-Ler, Cap and Cap-Cap were filtered through a 0.2 µm syringe filter and the filtrate was concentrated to 20 ml by lyophilization. The concentrated root exudates were desalted and further concentrated to 500 µl by passing through Amicon Ultra Centrifugal Filter Devices  and used for proteomic studies. Root-exuded proteins were stored at −80°C until needed. For each combination of plants, three biological replicates  were used. The protein concentration of the samples were determined as described by Bradford After separating the proteins from the root exudates by using Amicon ultra centrifugal devices, the fraction containing phytochemicals was freeze dried , dissolved in 10 ml of distilled water and the pH was adjusted to 3.0 with 1N HCl. The liquid was partitioned three times with an equal volume of ethyl acetate . All three EtOAc fractions were pooled and the remaining water residues were removed using sodium sulfate as a drying agent. The dried concentrate was dissolved in 100 µl of methanol (MeOH) for subsequent HPLC-MS analysis.The phytochemicals extracted from liquid media were chromatographed by gradient elution on a 150 mm×4.6 mm reverse phase, C18 column (Dionex). The chromatographic system  consisted of two P680 pumps connected to an AS1–100 automated sample injector and detected at 280 nm with a UV-visible detector. Mass determination of the peaks was analyzed by an MSQ-MS detector system . A gradient was applied for all separations with a flow rate of 0.7 ml min-1. The gradient was as follows: 0–3 min, 90.0% water and 10% methanol; 3–43 min, 10.0 to 90% (v/v) methanol, 90 to 10% (v/v) water; 43–51 min, 90.0% (v/v) methanol and 10% (v/v) water.https://xcmsonline.scripps.edu/) Alignment of the chromatographic datasets by retention time and mass was performed using the XCMS software  equipped with a 12-bit camera by using first the Phoretix 2D Expression software  from three different biological replicate gels for each treatment. A spot volume was calculated and each spot was assigned a normalized spot volume as a relative portion of the total value prior to analysis by ANOVA. Each individual protein spot was then matched with the identical protein spot from each replicate gel. For the 2DE gels analysis, we also used the software PDQuest Advanced (Bio-Rad Laboratories) version 8.0.1. Three biological replicate gels of each treatment were normalized using local regression model Loess. Quantitative analysis set for each sample type was created using triplicate gels. Proteins that consistently displayed 3-fold or greater increase or decreased expression when compared to the control (Col) were validated and reported. Proteins that remained unchanged, up to 1.5-fold were also validated and reported. Boolean analysis set was created to evaluate intersections of two or more sets of spots. Cumulative spot intensities were calculated for each of the functional categories  found in the protein profiles (128 identified proteins). Interaction-specific differences between protein profiles were tested using a multivariate ANOVA  and the exudate profiles comprised all 128 identified proteins. Alternatively, a separate ANOVA was used to test interaction-specific differences for the cumulative spot intensities of each categorized proteins.Differentially accumulated 2-DE protein spots observed during the interactions between plants of the same individual and plants of different individuals were excised and separately digested with trypsin prior to mass spectrometry analyses as part of the protein identification process. Silver-visualized protein spots were manually excised from the gels. These gel plugs were transferred to polypropylene 96-well plates and destained according to Sumner et al., The protein digests were analyzed using a nanoscale HPLC system  consisting of an autosampler (Famos), a precolumn switching device (Switchos), and a nano HPLC pump system (Ultimate). Samples (5 µl) were loaded onto a C18 precolumn  for desalting and concentrating at a flow rate of 50 µl/min using mobile phase A (5% ACN and 95% water containing 0.1% formic acid). The desalted peptides were then eluted from the precolumn and separated on a nano analytical C18 column  at a flow rate of 200 nL/min. Peptides were eluted with a linear gradient of 5–40% mobile phase B (95% ACN and 5% water containing 0.08% formic acid) over 40 min. The separated peptides were directly analyzed with an ABI QSTAR Pulsar I hybrid quadrupole time-of-flight mass spectrometer  equipped with a nanoelectrospray ionization source (Protana). QTOF-MS and tandem mass spectral data were acquired using information-dependent acquisition (IDA) with the following settings: charge state selection from 2 to 5, an intensity threshold of 10 counts/s for tandem experiments, and a collision energy setting automatically determined by the IDA based on the m/z values of each precursor ion.For protein identification, the acquired mass spectral data were queried against the NCBI non-redundant protein database (NCBInr), downloaded on 4-22-2003, using the MASCOT  search engine We first analyzed the secondary metabolites present in the root exudates of the different treatments. Principal component analysis (PCA) and cluster analysis by the Ward method were performed on the qualitative and quantitative data of 191 mass features. Based on the PCA analyses, we observed clear groupings between the plants grown individually  and the plants co-cultured with two homozygous individuals  or with different individuals (Col-Ler and Col-Cap)  & 2. FurWe also analyzed the proteins secreted in the root exudates of the different treatments were detected using 2-DE-gel electrophoresis  and idenA total of 128 protein spots were excised for identification based on the following criteria: 1) protein spots observed in all three replicates of each treatment, 2) protein spots that showed changes both quantitatively and qualitatively between the treatments, and 3) a spot was present at a sufficient concentration for nano LC-QTOFMS identification. PCA and cluster analysis by the Ward method were performed on the qualitative and quantitative data of the 128 proteins with the help of the web server tool Metaboanalyst Qualitative differences in the protein secretion profiles were observed when Col, Ler and Cap were grown individually . For exaTwenty-five proteins were secreted by Col when grown alone, but were absent when co-cultured with the same homozygous individual. These proteins were: glycosyl hydrolases (spots # 14 & 16), peroxidases , defense-related proteins , secretory protein-related (spot # 74), myrosinase binding protein-related (spots # 92 & 97), xylosidase (spot # 128) and proteins  of unknown function . SimilarUnlike the Col-Col treatment, new proteins were observed in the co-culture treatments of Ler-Ler compared with Ler grown alone  and Cap-Qualitative differences in protein secretions were observed when Col was co-cultured with different individuals (Col-Ler and Col-Cap) and compared with their partners  grown alone. For instance, Col secretes 11 unique proteins  present only in Col grown alone and not in Ler or in the co-culture of Col-Ler . BesidesSimilar trends were also observed when comparing Col, Cap and the co-culture of Col-Cap. For instance, Col secreted 18 unique proteins  were present only in Col grown alone and not in Cap or in the co-culture of Col-Cap . BesidesIn addition, we found that specific proteins were secreted at higher levels depending upon their neighbor identity. For example, the secretion levels of subtilisin-like proteases (spots # 4 & 5) are higher in the co-culture Col-Col compared with other co-culture treatments and the plants grown alone. Similarly, the secretion level of chitinase (spot # 84) was higher in the co-culture of Col-Ler compared with other treatments. Finally, the secretion level of another chitinase (spot # 66) was higher in the co-culture of Col-Cap compared with other co-cultures and the plants grown alone . BesidesIt is noteworthy to mention that the protein exudates collected from the co-cultures are from both plants and that we can’t distinguish from what plant these came from. However, we can distinguish if the co-culture induced the secretion of specific proteins as compared to the plants cultured alone. For instance, one protein [PR5 (spot # 126)] was specifically secreted in the co-culture Col-Ler but not secreted in the co-culture treatments Col-Col and Col-Cap or when the plants  were grown alone . SimilarThe dataset was analyzed based on the number of proteins  related to each functional category  and the The pattern and quantity of overall protein secretion under each functional category varied depending upon the neighbor’s identity with some apparent trends. For example, the total secretion levels of defense proteins increased with the presence of a specific neighbor genotype as compared to the control plant Col grown alone (Col < Col-Col < Col-Ler < Col-Cap) . On the Competition between plants is a major component of the ecology field and for the most part these studies measure the outcome of the interaction  In the scientific literature, many plant biochemical studies are conducted using single plants in an effort to simplify the experimental conditions. However, our analyses show that the root secreted metabolites and proteins of the single plants are significantly different from that of plants growing with a neighbor. When plants were grown alone, it was observed that they released a higher number of different proteins related to defense, but the cumulative secretion level of these proteins was rather low compared to the co-cultures. Furthermore, the cumulative secretion levels of specific defense related proteins increased depending upon the neighbor identity . Thus, iThe present studies suggest that the biochemistry of a single plant might not be the same in field studies involving monocultures of the same plant or polycultures of different plants. A recent study reported that the production of glucosinolates in Arabidopsis leaves was higher in high density of plants grown in pots as compared to low densities It is unknown if the different root exudate protein profiles contribute to the sensing and recognition of plant neighbors or whether these are responses to the recognition event. The actual sensing of the neighbor might be based on touch among roots Secondary metabolites have been largely credited to be involved in plant-plant interactions  with the assumption that these compounds tend to be phytotoxic and persistent in the soil. However, our study showed that the root secreted secondary metabolites could be potentially involved in plant neighbor recognition as it has been demonstrated previously in the context of legume-rhizobia interactions In this study, we have presented a first glance of root secreted proteins and metabolites involved in the early biochemical events of plant neighbor recognition. The data set presented here suggest that the early events of plant competition involve more than nutrient sensing and these events also include a biochemical realization that a given plant is interacting with a similar or different individual. The realization of differences among interacting plant neighbors is complex and, in part, involves a differentiation in both the quantity and identity of proteins in the root exudates, which appears to be influenced by the level of relatedness to its neighbor. The implications of this study are multipronged but it seems that the understanding of the ways that crops behave in monocultures vs. polycultures will allow us to device strategies by which the plants could better defend themselves.Figure S1A representative two-dimensional proteomic map of the total secreted proteins of the plants grown alone, co-cultured with the homozygous individual and co-cultured with the different individuals. The molecular masses (kDa) of protein standards are indicated to the left side of the gel and the isoelectric point (pI) is indicated at the top of the gel. The arrows with numbers represent the proteins identified which are listed in (TIF)Click here for additional data file.Figure S2Diagram depicting the total number of proteins identified in the 2-D gels of each treatment analyzed by PDQuest 8.0.1. Different treatment names are shown inside the boxes and the numbers represented on top of each box account for the total number of protein spots identified. Numbers represented next to the lines are the number of proteins commonly shared between the two treatments.(TIF)Click here for additional data file.Table S1List of all root secreted metabolites mass features and their abundance values from all the experimental conditions.(PDF)Click here for additional data file.Table S2List of all root secreted proteins and their abundance values (spot intensity) from all the experimental conditions.(PDF)Click here for additional data file.Table S3Univariate ANOVA comparing each individual protein across treatments.(PDF)Click here for additional data file.Table S4Total secreted proteins by category of individually grown or plants co-cultured with homologous or different individuals.(PDF)Click here for additional data file.Table S5Univariate ANOVA comparing each protein category across treatments.(PDF)Click here for additional data file.Table S6Multivariate ANOVA comparing protein profiles by category across treatments.(PDF)Click here for additional data file.


Ataxia-telangiectasia is known for cerebellar degeneration, but clinical descriptions of abnormal tone, posture, and movements suggest involvement of the network between cerebellum and basal ganglia. We quantitatively assessed the nature of upper-limb movement disorders in ataxia-telangiectasia. We used a three-axis accelerometer to assess the natural history and severity of abnormal upper-limb movements in 80 ataxia-telangiectasia and 19 healthy subjects. Recordings were made during goal-directed movements of upper limb (kinetic task), while arms were outstretched , and at rest. Almost all ataxia-telangiectasia subjects (79/80) had abnormal involuntary movements, such as rhythmic oscillations (tremor), slow drifts (dystonia or athetosis), and isolated rapid movements (dystonic jerks or myoclonus). All patients with involuntary movements had both kinetic and postural tremor, while 48 (61%) also had resting tremor. The tremor was present in transient episodes lasting several seconds during two-minute recording sessions of all three conditions. Percent time during which episodic tremor was present was greater for postural and kinetic tasks compared to rest. Resting tremor had higher frequency but smaller amplitude than postural and kinetic tremor. Rapid non-rhythmic movements were minimal during rest, but were triggered during sustained arm postures and goal directed arm movements suggesting they are best considered a form of dystonic jerks or action myoclonus. Advancing age did not correlate with the severity of involuntary limb movements. Abnormal upper-limb movements in ataxia-telangiectasia feature classic cerebellar impairment, but also suggest involvement of the network between the cerebellum and basal ganglia. Ataxia-telangiectasia (A–T) is a recessively inherited multi-system disorder with prominent neurodegeneration, immunodeficiency, radiosensitivity, and enhanced risk for lymphoreticular malignancy. Pathological studies in A–T have focused predominantly upon abnormalities of the cerebellar cortex, leading early investigators to identify the disorder as a form of cerebellar ataxia, hence its iconic name We evaluated abnormal limb movements in A–T with three intents: (1) to quantify the severity of tremor and non-rhythmic adventitious movements, (2) by a cross-sectional approach to determine whether, and to what extent, these movement disorders correlate with increasing age, and (3) to determine whether these movement disorders have specific features that support either a pure cerebellar etiology or are instead better explained by mixed cerebellar and extra-cerebellar correlates.The study was approved by The Johns Hopkins Institutional Review Board (NA-00014314). The written informed consent was obtained for the experiment protocol. In instances when participants were minors or children, the written informed consent was obtained from their guardians. The consent form was also approved by the Johns Hopkins Institutional review board. The investigation was conducted according to the principles expressed in the Declaration of Helsinki. The guardian of the subject of the video recording (as the subject was a minor) had given written informed consent, as outlined in the PLOS consent form, to publication of this video. Subjects with A–T were recruited from The Johns Hopkins A–T Clinical Center (ATCC). All subjects met the previously defined criteria for the diagnosis of A–T A light-weight three-axis accelerometer, mounted on to the dorsum of the middle phalange of the index finger, was used to record movements. Subjects held their arms outstretched against gravity , comfortably rested their forearms and hands in their lap (rest), and performed slow back-and-forth finger-to-nose movements (kinetic task). Limb movement during each task was recorded for 120 seconds. The signal was recorded at 100 Hz sampling rate.Each axis of a three-axis accelerometer will record inertial acceleration, and if it changes in orientation an associated change in acceleration attributable to gravity Accelerometry is inherently associated with high-frequency sampling noise. A moving average algorithm was used to filter the sampling noise. Cycle-by-cycle analysis was performed on smoothed three-dimensional composite vectors. The following steps were followed to define an individual cycle: (1) Data was normalized and de-trended data with the mean intensity . This permits data to be realigned along the abscissa such that the peaks of the cycles remained positive and the troughs negative. (2) The ‘x’ co-ordinate of the intersection of the data trace with the abscissa  was recorded. The x-coordinate of the first data point that crosses the abscissa marked the beginning and the subsequent data point marked the end of the given cycle. 3) With this definition of cycle, its width was computed. The inverse of the cycle width yields the cycle frequency and the difference between the peak and trough measures the cycle intensity. In this technique, the “intensity” of movement was measured in the form of acceleration. (4) We were then able to assess cycle-by-cycle variation of tremor frequency (i.e. standard deviation (SD) of the frequency) within the defined tremor intervals. rhythmic movement with a relatively fixed frequency over the determined interval. In this study intervals of tremor were defined as those times when the frequency of three or more consecutive cycles varied by 2 Hz or less.The transient episodes of tremor were determined according to the criteria defining tremor as a Accelerometer recordings were performed on 80 A–T subjects  and 19 age-matched control subjects. Rhythmic movements that fulfilled the definition of tremor were observed when the A–T subjects were at rest (resting tremor), with outstretched upper limbs holding them in a sustained position against the constant force of gravity , and while reaching between the examiner’s finger and subject’s own nose or chin (kinetic tremor). Postural and kinetic tremor was present in 79 of 80 A–T subjects, while a resting tremor was also present in 48 (61%). The intensity of postural and kinetic tremor was higher compared to resting tremor in all A–T subjects. None of the control subjects had recordable postural, resting, or kinetic tremor.2, respectively . The median values of postural, resting and kinetic percent tremor time were 16%, 6%, and 19.5% respectively . PercentThese quantitative differences between postural, kinetic, and resting tremor suggest that postural (or kinetic) and resting tremor all have central etiology, but possibly discrete correlates.Rapid non-rhythmic adventitious movements were present in all A–T subjects, but were not identified in any of the controls. These movements were of two types – slow movements appearing like athetosis or dystonic drifts, and fast movements resembling myoclonus or dystonic jerks.2  and further to 4.5 mm/s2 in kinetic condition .Rapid myoclonic or dystonic movements were also present during rest but increased with activity. Accelerometry allowed quantitative measurement of the rapid, non-rhythmic adventitious movements. In order to compare the severity of these movements amongst different subjects and conditions, we normalized the intensity of these movements per unit time. During resting condition, non-rhythmic movements had normalized median intensity of 1.1 mm/sThe quantitative characteristics of non-tremor movements suggest possible role of limb muscle activation to increase their incidence. The results point to the impairment of neural networks between the cerebellum and basal ganglia.In our cross-sectional study there was no relationship between the tremor frequency, tremor intensity, or non-tremor movement intensity and age in any of the three recording conditions. This lack of a relationship extended across a wide range of ages, including the pre-teen years of declining neurologic function and later years of relative stability . Weak vaProgressive degeneration of the cerebellar Purkinje neurons is the hallmark neuropathology of A–T The abnormal movements characterized here include postural, kinetic, and resting tremors mixed with other non-rhythmic involuntary movements. There were two types of non-rhythmic movements: slow irregular movements appearing like athetosis or dystonic drifts, and relatively rapid non-rhythmic movements resembling dystonic jerks or myoclonus. Analysis of three dimensional accelerometer recordings permitted the separation of tremor from non-rhythmic rapid adventitious movements.Resting tremor was generally of lower intensity and higher frequency compared to postural and kinetic tremor. The combination of features identified in subjects with A–T are similar to those described with disturbances of the cerebello-thalamic pathway Cases of genetically confirmed A–T have recently been described in which the dominant feature is dystonia The neurodegeneration of A–T is complex, with many dimensions that manifest independence with respect to one another Video S1Movie clip depicts disorders of upper-limb movement in one subject with A-T.(WMV)Click here for additional data file.


PT Askes (Persero) uses a combination of fee-for-service (FFS) and package tariff as a provider payment mechanism. Based Wouters, Annemarie et al (1999), FFS system has a high financial risk for payor. The objectives of this paper are to give a preliminary comparison between INA-CBGs (which is Indoesian DRG) and Askes’ tariff (MoH Act number: 416/Menkes/Per/II/2011), to estimate financial risks associated with the adequacy of premium if using INA-CBGs and to gain recommendation for Askes in a preparation as an Organizing Bodies of Social Security in 2014. These three objectives are answered by calculating 10 most expensive inpatient diagnoses of Askes 2011 and comparing the cost and Length Of Stay (LOS) of both tariff. Askes’ tariff doesn’t have severity level, whereas INA-CBGs has three severity levels with five different accommodation classes . The study uses variety of the lowest cost , the moderate cost  and the high cost .The results showed that (1) by using III accommodation with a severity level i: Askes’ tariff higher than INA-CBGs with financial risk decrease to 73%, (2) by using II accommodation with a severity level ii: Askes’ tariff still higher than INA-CBGs with financial risk about 143%, (3) by using I accommodation with a severity level iii: Askes’ tariff lower than INA-CBGs with higher financial risk, approximately 234%. Overall, Askes’ LOS is controllable compare to the ALOS of INA-CBGs. This study recommends the alternative provider payment mechanism for Askes. If Askes uses INA-CBGs with III accommodation it will minimize the financial risk. Whereas if Askes uses II and I accommodation, then the adequacy of the existing premium must be taken into account, i.e. by the estimation of financial risk. As a leading social health insurance company in Indonesia, which will have a big number of member in 2014, PT Askes has to train the staffs about DRG . On considering the complexity of the DRG scheme, rather to make the new one, it is a good beginning to start with the existing DRG which is INA-CBGs from MoH, and then gradually re-calculate the cost weight and base rate and update it yearly. Nevertheless, the adequacy of the existing premium must be taken into account. Hopefully, the health care costs for the whole population of Indonesia will be more controllable in the future.


How specific visual perceptions transform into corresponding long-term memories remains unknown. Here, I will review recent Blood Oxygenation Level-Dependent functional Magnetic Resonance Imaging (BOLD fMRI) in humans together with molecular biology studies  aiming to understand how the retinal image gets transformed into so-called visual (retinotropic) maps. The broken object paradigm has been chosen in order to illustrate the complexity of multisensory perception of simple objects subject to visual —rather than semantic— type of memory encoding. The author explores how amygdala projections to the visual cortex affect the memory formation and proposes the choice of experimental techniques needed to explain our massive visual memory capacity. Maintenance of the visual long-term memories is suggested to require recycling of GluR2-containing  The McMillan Thesaurus dictionary  defines  A majority of the outside-world stimuli reach our brain via visual and auditory channels. A photoreceptor layer of each retina captures an instant snap shot of the scene, which is then transmitted, filtered, and reproduced by means of the electrical activity of neurons in the visual cortex , 19 Fig. The actInterestingly, the written words FACE or HOUSE, giving an indication about an image category to be presented, enhanced selectively BOLD response in the left FFA, or parahippocampal area . The antThe present paper focuses on the molecular biology of visual perception in human as compared to that of rodents Sections  and 3. Iβ [d-aspartate receptors (NMDAR) and AMPAR, which causes ion influx into the cell. The overall spiking profile of a given L2/3 pyramidal neuron depends on the extent of its targeting by other neurons expressing GABAergic, cholinergic, adrenergic, and other receptor subtypes [Visual object perception starts with the formation of the retinal image and its transfer from the retina as parallel electrical signals  by the optical tract to the lateral geniculate nucleus (LGN) Figures  and 2. Iβ  and pyraβ   [A drop in the neuron firing is constantly readjusted by insertion/removal of AMPAR –56 and Nia NMDAR . This giia NMDAR , 61. In he NMDAR . On the on (LTD) , 63. LTPon (LTD) , 65. Theon (LTD) .The Rho GTPase family kinases  communicate with the calcium-dependent kinase CAMKII and are able to spread laterally tens of microns away from the original LTP site –69. ImpoThe information transfer across the visual cortex takes place mainly at synaptic contacts between the axon terminals and spines equipped with glutamate receptors. Kwon and Sabatini  have recIn contrast to that, the spine enlargement in older animals (>20 days) required the electric activity-dependent LTP, the TrkB receptor activation by BDNF and downstream MAPK- and CAMKII signalization pathways.1R receptor (L2/3 of V1), and other factors [vice versa [Zilberter and colleagues  studied  factors . The exc factors ), and vice versa .Formation of the retinotropic map in early visual brain areas requires both excitatory and inhibitory circuits. The excitatory signal propagates preferentially along certain dendrites of direction-sensitive L2/3 pyramidal neurons, while other dendritic branches are kept under the inhibitory control of somatostatin-(SOM-), parvalbumin-(PV-), and calretinin-(CR-) positive GABAergic interneurons , 83. In Perception of the broken coffee cup by humans has been chosen here since such irregular object shapes appear to be good substrate for the visual rather than the lingual-type of LTM encoding. Humans are simply missing words to describe the exact shape of the missing fragment. From an fMRI standpoint, the coffee cup belongs to the category of neutral, nonliving, and motionless objects of round shapes. In practice, our visual perception of a white coffee cup does not elicit emotion. However, the same does not apply for a broken coffee cup. The accidental drop of the ceramic object on the floor usually results in its damage and practically instantaneous expression of emotions.In infants, the initial moment of surprise shifts to the sudden feeling of joy and irresistible desire to break another object. In older children, the feeling guilt and fear mix together and may lead to the diverse strategies and attempts to hide the accident. In adults, the coffee cup breaking is often accompanied by swearing, which tends to calm down our initial excitement and anger. While the human brain is able to efficiently erase visual memories encoding the exact shape of a broken sugar cube we drop in our coffee, we seem to remember the shape of the broken coffee cup even though the accident happened some time ago. We are not likely to forget in what shape our car was following a traffic accident, even though the accident happened many years ago. Thus, it seems the more personal the story gets and the more value we assign to the object, the more efficient are the processes of long-term memory formation, maintenance, and retrieval. It has been noticed only very recently that amygdala activation takes place not only during intense emotional events, but also when evaluating an object's value . InteresAt first glance, our visual perception of the coffee cup and its broken counterpart should be rather similar. The only difference resides in the exact shape of the missing fragment. Our brain must capture such contour differences quite early during the retinotropic map formation in early visual areas. Once the fragment contours captured by V1–V4, other visual brain areas responsible for the stereoscopic depth processing and analysis of complex 3D shapes need to be recruited . The lefβ-adrenergic receptors. Norepinephrine binding to β2-adrenoreceptor causes release of the Gs subunit from a GPCR. This activates adenylate cyclase, which generates locally high levels of cAMP and recruits β-arrestin. The latter protein binds also to the β2-adrenoreceptor and activates B-Raf and ERK signalling pathways [ζ expression levels [Let us take a look at how the memories of the broken coffee cup could have been formed and shaped from the first seconds to several hours following the accident. Initially, the visual cortex keeps the sensory information about the broken object contours in its short-term visual memory system . Multiplpathways . In animpathways , 108. Empathways , 108–111pathways  and nighpathways –115 of tn levels . This faIndependently from the exact location of the LTM storage site, neuron clusters and individual neurons sensitive to our broken coffee cup shape are unique in the sense that they are not linked to any other event, place, or time in our life. The fact that the human brain is able to locate such specific LTM-keeping clusters among millions of others, retrieve object-place associations, activate the amygdala and instantly express emotions is quite remarkable. Emotions are expressed instantly following the visual perception of the broken object and memories retrieved in a fraction of the second even years later. These observations reflect the fact that related neural networks were formed at the same time, are wired together and, therefore, reactivated together.Let us consider what separates neuron populations encoding the broken coffee cup shape compared to those keeping, for example, our credit card number. The five- or six-digit numeric code is usually learned within a day or two and related LTM reactivated almost daily during four year credit card period. On the other hand, remembering the shape of the broken coffee cup seems to be effortless, and related LTM may be retrieved sometimes years later without daily memory training. It is likely that the six-digit code-encoding neuron cluster would be either lost or inaccessible for LTM retrieval following the same time period. A simple explanation of the phenomenon could be that our brain allocates very little resources to storage of numeric representations in the intraparietal cortex . EventuaBOX1: Forms of visual memory. The vision science makes the distinction between the iconic memory (<500 ms), visual short-term memory (vSTM), and visual long-term memory (vLTM). Slighte and colleagues  have shoζ (myr-zip peptide (myristoylated PKMζ inhibitor).Where do we store visual long-term memories marked by emotions? Sacco and Sacchetti  have recζ  and are ζ and the injection of alkaloid chelerythrine . Joels and Lamprecht [+ ATPase Thorase is able to disassemble the AMPAR-GRIP1 complex and induce AMPAR endocytosis and LTD [ζ working together with the NSF ATPase ? In the opposite scenario, the mushroom-like spine could experience a more serious damage and apoptotic cell death. The spine growth and shrinkage in rat visual cortex has recently been investigated in vivo by two-photon imaging microscopy [ζ inhibitor application in any brain area. Since PKMζ presence in LTM-expressing spines leads to doubling of GluR2/3 receptors at the postsynaptic membrane [Zip-peptide, the lysine 281 mutation to tryptophan (K281W) in PKMrythrine  into LTMse PIN-1 . A seconse PIN-1 . Axon guse PIN-1 . The BDNteasomes . On the synapses . Emotionsynapses , 142. Elsynapses . Such syablished , its impablished , 144 werlleagues  have sugamprecht  have recamprecht . GluR2/3 and LTD . One of  and LTD . This seF ATPase . The exa control  and was  control . The rel control . GluR2/3 control ). Co-loc control . Would lcroscopy . Howevermembrane , the immOne of the goals of the 21st century neuroscience will be to understand our vision on a molecular level. Recently available novel technologies and experimental approaches allow studying brain structure-function relationship in animal models. Classical loss-of-function and gain-of-function studies have been extended for the light-controlled neuron activation, the incorporation of non-canonical amino acids into newly synthesized brain proteins , or 3D-rζ, GluR2/3 and postsynaptic NSF using high-resolution neuron array tomography in post-mortem human brain tissues would be of great interest. Equally, it will be important to identify AMPA receptor subtypes involved in the LTM maintenance across the human mesolimbic reward circuit. In this regard, it is interesting that the injection of two PKMζ inhibitors  within the nucleus accumbens core [In spite of the exciting developments in neuroscience, as briefly outlined above, a general consensus regarding how the human brain handles memory formation, storage and retrieval is yet to be reached. BOLD fMRI is commonly used to track which brain areas get activated during sound hearing or picture viewing. However, it remains to be understood where resulting LTMs are stored. Even though the GluR2/3 receptors are less abundant (ca. 10–15%) than GluR1/2 and GluR1 homomers in the rat hippocampal CA1 neurons , it is lens core  and VTA ens core  blocked Very little is known about the distribution of the ionotropic and metabotropic glutamate receptors in the human fusiform gyri. This concerns the AMPAR subunit composition, postsynaptic-density proteins and enzymes involved in the LTM formation and maintenance. The surgical dissection of the left FG resulted only in difficulties with orthographic processing such as retrieval of word spelling from its meaning in writing  . On the 2+-dependent signalization network downstream of NMDA, AMPA and mGluR receptors.Changes in the composition of membrane receptors and membrane proteins affecting communication between the excitatory or inhibitory neurons.Many of plasticity-related phenomena rely on calcium-permeable glutamate receptors and CaAt the single neuron level, changes in the spine volume and protein composition due to frequency- or chemical-dependent forms of learning such as LTP or LTD. Long-term memory formation usually requires alternation of LTP and LTD in order to switch between the transcription and translation machinery steps needed for the local protein synthesis within the spine. A small protrusion on a dendrite surface equipped with membrane receptors and hundreds of proteins and enzymes. Spine volume and shape often indicate whether it is implicated in neuron-to-neuron communication , eventually, the memory storage (mushroom-like spines). In order to maintain neuron firing profile imposed by the neural network, the number of glutamate receptors, their subunit composition, and phosphorylation pattern change depending on the spine location on the dendrite in respect to the action-potential generating somatic cell compartment.In spite of important degree of compartmentalization and specialization, the spine is far from being a closed system. It communicates very rapidly by means of diffusing kinases with other spines residing on the same dendritic branch. This way the spine reports about the progress of its synaptic learning. Similarly, spines communicate with the nucleus even though on somewhat longer time scale (hours). This usually takes place when delivery of specialized cargo proteins is required and such proteins are not available locally or might not be possible to synthesize within the spine.Blood Oxygenation Level-Dependent functional Magnetic Resonance imagingfollows local changes in the blood flow and local concentration gradient of the paramagnetic deoxy-hemoglobin molecule. Point-to-point topography of the retinal image reconstructed by electrical activity of neurons in anatomically distinct visual cortex areas.Visual maps may be conveniently measured by high-resolution BOLD fMRI.Genetic manipulations in animal models resulting in deletion of the gene of interest or, on the other hand, introduce novel gene belonging to another organism. Optogenetic gain-of-function studies rely on introduction of genes encoding ion pump proteins from the opsin family sensitive to certain wavelengths in the visible range.


Mammalian olfactory receptors (ORs) are subject to a remarkable but poorly understood regime of transcriptional regulation, whereby individual olfactory neurons each express only one allele of a single member of the large OR gene family.ab initio motif-finding algorithms. We found strong enrichment of binding sites for the O/E family of transcription factors and for homeodomain factors, both already known to be involved in the transcriptional control of ORs, but did not identify any novel enriched sequences. We also found that TATA-boxes are present in at least a subset of OR promoters.We performed a rigorous search for enriched sequence motifs in the largest dataset of OR promoter regions analyzed to date. We combined measures of cross-species conservation with databases of known transcription factor binding sites and Our rigorous approach provides a template for the analysis of the regulation of large gene families and demonstrates some of the difficulties and pitfalls of such analyses. Although currently available bioinformatics methods cannot detect all transcriptional regulatory elements, our thorough analysis of OR promoters shows that in the case of this gene family, experimental approaches have probably already identified all the binding factors common to large fractions of OR promoters. Mammals can detect and discriminate many thousands of odorous molecules in our environments using millions of neurons in the olfactory epithelium. These neurons express members of one of the largest gene families in the mammalian genome, the olfactory receptors (ORs) . In mousEach neuron in the olfactory epithelium expresses only one member of the OR gene family; furthermore, only one of the two parental alleles of the selected OR is expressed in each cell -8. IndivTransgene studies have shown that a small region of a few hundred base pairs upstream of the OR transcription start site (TSS) can act as a minimal promoter to drive transcription of a reporter construct in a pattern that seems mostly faithful to that of the endogenous gene, although occasional differences are observed in zonal restriction of transgenes -16. It tO/E-1 , O/E-2, O/E-3 and O/E-4 . We excluded some cDNAs that showed unusual splice patterns [et al. [DR065530 - DR065963].We previously reported end-sequences of 1264 OR cDNAs from adult and embryonic olfactory epithelial cDNA libraries  among a patterns  and a sm [et al.  [GenbankIn order to assign each EST or RACE sequence to the corresponding OR gene, we first defined a genomic "domain" for each OR, whereby any cDNA mapping to that domain on the correct strand would be assigned as being transcribed from the corresponding gene. We defined the upstream edge of the domain as being 1 kb closer than the nearest edge of the next OR ORF upstream (or 100 kb upstream of the ORF's start if no other OR was found within that distance); we defined the downstream edge of the domain as being 1 kb downstream of the end of the gene's ORF.We used cDNA or RACE product sequence accession numbers as queries in UCSC's Table Browser to look up their map positions in the mouse genome assembly (July 2007). We then used Bioconductor's IRanges package to compare those cDNA positions to the set of OR gene domains as defined above. cDNAs whose position overlapped more than one OR domain were excluded from further analysis. For each gene domain, we assigned base-pair positions present in any cDNA/RACE sequence and/or in the ORF of the gene domain as part of the transcript. Because UCSC's cDNA-genome mapping algorithm reports separate alignment blocks whenever an indel occurs, we merged any transcribed segments that were separated by < 10 bp, as these gaps are very unlikely to represent true introns. After this merge, we removed from further analysis 49 OR genes for which only a single transcribed exon appeared to be present, as these might represent cDNAs that are only partial transcripts or genomic contaminants of the cDNA library. We also removed 11 apparent transcribed pseudogenes . We removed 2 additional ORs for which the cDNA/RACE sequences implied an unusual gene structure (an intron downstream of the ORF), leaving 432 intact ORs for which we assigned the furthest upstream base of the transcript as the TSS.In order to ensure that our final dataset did not contain any close duplicates, we grouped the 1151 intact ORs by sequence similarity. We determined pairwise synonymous nucleotide divergence of the 1151 intact OR coding regions using an in-frame nucleotide alignment as input to PAML's codeml algorithm (version 4.3) , run in In order to search for candidate binding sites for transcription factors with known sequence specificity, we purchased a license to use the MatBase database and MatInspector search algorithm  , and again using a threshold 0.1 units lower than the optimized threshold ("opt-0.1"), which results in about ten times as many matches as does "opt". We used the OUTFILES = 2 option to obtain tab-delimited text output, which we then parsed and analyzed in R. Many transcription factor binding sites are at least partially palindromic, so that MatInspector often predicts overlapping sites for the same matrix on opposite strands.Transcription factor binding sites are represented by short and degenerate position weight matrices (PWMs). This degeneracy means that when genomic sequence is searched with a PWM, there is often a non-trivial level of false-positive site predictions. We wanted to determine whether the number of predicted sites in the 200-bp regions nearest our set of predicted TSSs was more than would be expected in "background" non-promoter sequences. We also performed all the tests described below on 500-bp regions upstream of the TSS: for simplicity, below we describe analysis of only the 200-bp regions. As described in the main text, we performed two statistical tests to address this question, using two different sets of background sequences. In the first test, we compared the number of predicted sites in the 200 bp nearest to the predicted TSS with the number predicted in the preceding 200-bp region. It is possible that some sites in that previous 200-bp region do indeed represent real TF binding sites, reducing the ability of our test to detect truly functional sequence motifs in the TSS-proximal region, but given the unusual GC-content of our promoters, we decided that it was best to use neighboring regions as controls rather than anything yet farther from the predicted TSSs. We used a one-tailed binomial test to assess whether the number of sites in our regions of interest was statistically significantly greater than that in the background region, with a null hypothesis that transcription factor binding sites are equally likely to be found in each of the 200-bp regions. Because we performed each test on 177 PWM families, we conservatively required a p-value of < 0.05/177 (< 0.000282) in order to declare statistical significance (the Bonferroni correction).In our second test, we randomized the DNA sequence of each 200-bp promoter region, using the shuffle program from the SQUID package  , supplyiWe investigated the use of an alternative type of negative control sequence: GC-matched promoters from non-OR genes, as one would not expect many of them to share the same enriched motifs as OR promoters. However, it proved impossible to assemble a truly appropriate non-OR promoter control set. First, OR promoters have such extremely low GC content that we struggled to obtain a matched set; only ~280 promoters in the mouse genome had similarly low GC content. Those GC-matched non-OR promoters still had much higher conservation scores than our OR-promoter set (data not shown) - it was thus not possible to match promoters on GC content and conservation levels. This difference in conservation levels is difficult to interpret given the fact that the OR family has experienced many recent duplications and deletions disrupting orthology, and given that OR coding regions appear to be evolving under much lower selective constraints in some species (e.g. primates) than others (e.g. rodents), so that it seems likely their promoter regions are also under lower selective constraint. The sequences we analyzed that immediately precede the OR promoter sequences are only very slightly different in GC content and conservation level to the promoter dataset Figure , and areWe used UCSC's Table Browser to obtain multiple-species alignments of the 200-bp promoter regions. These are portions of whole-genome alignments that were generated by a sophisticated computational pipeline based on the multiz and TBA programs . One cavSeveral algorithms exist that estimate the evolutionary selective pressure acting on each nucleotide of a set of aligned sequences, given a phylogenetic tree defining the evolutionary relationships between the sequences. We chose to use the SCONE algorithm , subtracIn order to examine OR promoters in other species, we used UCSC's liftOver utility to obtain orthologous TSS coordinates in the rat, human, dog and cow genomes. For a given base-pair range in a reference genome assembly, liftOver provides the coordinates of the aligned region from another species, using the same whole-genome alignments we discuss above. We considered an alternative approach of using the orthologous sequences present in the multiple sequence alignments discussed above, but in many cases the alignments contained < 1 kb of orthologous sequence concentrated around the TSS, and in some cases aligned sequences comprised several non-contiguous pieces of the orthologous genome. We wished to examine a more extended, continuous stretch of sequence centered around the orthologous promoter, rather than only those regions that were found to align using whole-genome alignment procedures. As input to liftOver, we used 21-bp regions centered around the predicted TSSs . After obtaining orthologous coordinates for those 21-bp regions, we took the center position as the predicted orthologous TSS, and extended 2 kb each side of that TSS to create 4-kb regions. The resulting dataset of 4-kb regions contained some redundancy . We removed such redundancy by merging overlapping 4-kb regions, and taking the most upstream 4 kb of each merged region . Almost all of the predicted orthologous promoter regions are near OR genes in the corresponding genomes.In order to determine the proportion of O/E sites predicted using MatInspector's less stringent parameters that appear evolutionarily conserved, we performed the following analysis, which utilizes the mammalian species tree and multiple sequence alignments of OR promoter regions described above. We used ungapped versions of each sequence in the multiple sequence alignments as input to MatInspector (again using the less stringent parameters). We used a custom Bioconductor script to convert the position of all predicted O/E sites in ungapped sequences to positions relative to the multiple sequence alignment, and therefore relative to the reference mouse sequence. For each mouse O/E site, we could then determine the list of species that had overlapping O/E site predictions. We used that species list to extract a subtree from the full species tree and determined the length of that subtree: subtree length thus provides a measure of how evolutionarily conserved each site is. We then filtered mouse O/E sites according to various minimum tree length thresholds . We also masked all predicted TATA boxes ("opt") between position -61 and -10 relative to the TSS. We recognize that this approach is very conservative (masks more sequence than necessary) because there is a relatively high false-positive prediction rate for TATA boxes; we preferred to mask too much sequence than to identify enrichment of transcription factors with TATA-box-like matrices.As well as masking those regions from the real promoter set, we recorded the positions of the masked sites and also masked the equivalent regions from (a) each of the 10,000 shuffled datasets, (b) the dataset of preceding 200-bp regions, and (c) the lists of conservation scores. We then repeated MatInspector runs and the three statistical analyses using masked sequences and scores. This strategy ensured that an equivalent amount of sequence was being analyzed in the real and control datasets.We used three programs  to identify novel sequence motifs using various sets of RepeatMasked promoter region sequences as input.th, 2010) from the Genome Bioinformatics site [First, we used MEME  to searcics site .We ran Weeder  locally For MotifSampler , we firsCAGE: cap analysis of gene expression; EST: expressed sequence tag; LCR: locus control region; OR: olfactory receptor; ORF: open reading frame; PWM: position weight matrix; RACE: rapid amplification of cDNA ends; RNA-seq: massively parallel cDNA sequencing; TF: transcription factor; TSS: transcription start site; UTR: untranslated region;The authors declare that they have no competing interests.JY designed the study, analyzed the data and drafted the manuscript; RL and BT participated in study design and edited the manuscript; RL performed initial data analyses. All authors read and approved the final manuscript.Table listing olfactory receptor genes and pseudogenes in the July 2007 mouse genome assembly. See Methods for details of how gene names were determined.Click here for fileTable listing TSS locations and data sources. This table shows all TSS locations we collected from a high-throughput RACE study [et al.).CE study , our hybCE study  and variCE study ,52,85-87Click here for fileFigure showing that most O/E sites predicted using MatInspector's less stringent parameters are evolutionarily conserved. The solid gray line shows all mouse O/E sites predicted using MatInspector's less stringent parameters. For reference we include a dotted gray line showing all mouse O/E sites predicted using MatInspector's default parameters. As in Figure 1, coverage is calculated as the proportion of promoter sequences containing a predicted O/E binding site at each base-pair, averaged over 20-bp windows, sliding along promoters 1 bp at a time. In panel A, the colored lines show predicted mouse O/E sites that remain after we apply an evolutionary "filter" to MatInspector predictions on a multiple sequence alignment (see Methods). For example, the red line shows mouse O/E sites that have overlapping O/E sites in a set of mammals with tree length of at least 1.5. Note that the conservation filter removes many of the O/E sites far away from the TSS and selects for likely functional sites near the TSS. In panel B, we explore the effect of allowing various amounts of "slide" (see Methods), where sites in other species are no longer required to overlap but could be various distances away, accounting for evolutionary "turnover" [urnover" .Click here for fileFigure showing enrichment of transcription factor binding sites 200 bp before TSS using less stringent MatInspector parameters. These plots follow the same layout as those in Figure 2 but use transcription factor site predictions made using MatInspector with less stringent parameters (see Methods).Click here for fileTable summarizing statistical tests on matrix families for 200-bp promoter region (default MatInspector parameters). Results of our statistical tests for enrichment and conservation for all matrix families after masking O/E sites and TATA boxes using default MatInspector parameters. See legend to Table 1.Click here for fileTable summarizing statistical tests on matrix families for 200-bp promoter region (less stringent MatInspector parameters). Results of our statistical tests for enrichment and conservation for all matrix families after masking O/E sites and TATA boxes using less stringent MatInspector parameters. See legend to Table 1.Click here for fileSummary of statistical tests for 500-bp promoter region. See legend to Table 1. This table gives equivalent statistics for a larger putative promoter region comprising 500bp before the TSS. Binomial tests here show comparison with the preceding 500-bp region.Click here for fileTable summarizing statistical tests on matrix families for 500-bp promoter region (default MatInspector parameters). See legend to Additional File Click here for fileTable summarizing statistical tests on matrix families for 500-bp promoter region (less stringent MatInspector parameters). See legend to Additional File Click here for fileTable summarizing statistical tests on individual matrices for 200-bp promoter region (default MatInspector parameters). Results of our statistical tests for enrichment and conservation for individual matrices after masking O/E sites and TATA boxes using less stringent MatInspector parameters. See legend to Table 1. In addition, for selected matrices, we obtained gene symbols of the corresponding transcription factor(s) (using MatBase and/or the Mouse Genome Database: [atabase: ), and usatabase: . We provClick here for fileTable summarizing statistical tests on individual matrices for 200-bp promoter region (less stringent MatInspector parameters). Results of our statistical tests for enrichment and conservation for individual matrices after masking O/E sites and TATA boxes using less stringent MatInspector parameters. See legend to Table 1 and Additional File Click here for fileTable summarizing statistical tests on individual matrices for 500-bp promoter region (default MatInspector parameters). See legend to Additional File Click here for fileTable summarizing statistical tests on individual matrices for 500-bp promoter region (less stringent MatInspector parameters). See legend to Additional File Click here for file


The classic model of eukaryotic gene expression requires spatial contact between a distal enhancer and a proximal promoter. To control gene expression, various models of enhancer-blocking insulators have been proposed, including a decoy model and a topological model . The decHere we use the polymer model of chromatinized DNA and simulations of Brownian polymer dynamics to study the topological model of enhancer-blocking insulators. We consider one- and two-loop topological elements and assess spatial contacts between various regions of DNA induced or suppressed by an topological elements.We find that a loop formed in the region between an enhancer and a promoter in fact facilitates enhancer-promoter contacts and does not act as an enhancer-blocking insulator. However, we find that sequestration of an enhancer (or promoter) within a loop is a plausible mechanism for topological insulators. Both the facilitating and insulating effects of topological elements are more dramatic with two-loop elements.Our polymer simulations demonstrate that the loop-forming topological elements are capable of both facilitating and insulating enhancer-promoter interactions. We note that the biological model of insulator activity around H19 and Igf2 genes in mouse liver cells presented by Kurukuti et al. [


A protein at equilibrium is commonly thought of as a fully relaxed structure, with the intra-molecular interactions showing fluctuations around their energy minimum. In contrast, here we find direct evidence for a protein as a molecular tensegrity structure, comprising a balance of tensed and compressed interactions, a concept that has been put forward for macroscopic structures. We quantified the distribution of inter-residue prestress in ubiquitin and immunoglobulin from all-atom molecular dynamics simulations. The network of highly fluctuating yet significant inter-residue forces in proteins is a consequence of the intrinsic frustration of a protein when sampling its rugged energy landscape. In beta sheets, this balance of forces is found to compress the intra-strand hydrogen bonds. We estimate that the observed magnitude of this pre-compression is enough to induce significant changes in the hydrogen bond lifetimes; thus, prestress, which can be as high as a few 100 pN, can be considered a key factor in determining the unfolding kinetics and pathway of proteins under force. Strong pre-tension in certain salt bridges on the other hand is connected to the thermodynamic stability of ubiquitin. Effective force profiles between some side-chains reveal the signature of multiple, distinct conformational states, and such static disorder could be one factor explaining the growing body of experiments revealing non-exponential unfolding kinetics of proteins. The design of prestress distributions in engineering proteins promises to be a new tool for tailoring the mechanical properties of made-to-order nanomaterials. A tensegrity structure is one composed of members that are permanently under either tension or compression, and the balance of these tensile and compressive forces provides the structure with its mechanical stability. Macroscale tensegrity structures, which include Buckminster Fuller's geodesic domes, achieve exceptional structural integrity with a minimal use of resources. The question we address in this work is whether nature makes use of molecular-scale tensegrity in the design of proteins. Using Molecular Dynamics simulations of the protein ubiquitin, we measure the network of pairwise forces connecting the amino acid residues and show that this network does indeed have the character of a tensegrity structure. Furthermore, we find that the arrangement of tensile and compressive forces is such that hydrogen bonds in the protein's beta sheet, which are crucial for bearing mechanical loads, are compressed. This pre-compression is enough to significantly lengthen the lifetime of a bond under a given force, and thus should be an important factor in determining the protein's mechanical strength. The rational design of molecular prestress networks promises to be a new avenue for the engineering of proteins with made-to-order mechanical properties, for applications in medicine, materials and nanotechnology. The principle of ‘minimal frustration’ tensegritySuch local frustration in a protein must give rise to residual mechanical forces – thus, proteins are in some sense prestressed materials. D. Ingber has proposed that proteins and other biological structures should be understood in light of the architectural concept of It has been demonstrated recently a priori assumptions about the form of the force profile. From the effective force profiles we extracted average forces for each residue pair, thereby constructing a prestress network for the protein. We found that high residual forces exist throughout the protein, and are particularly associated with hydrogen bonds and salt bridges. The magnitude of these forces is shown to be enough to significantly influence the protein's mechanical properties, most notably its unfolding pathway. We also discover that, for some side-chains, prestress is dynamic – inter-residue mechancial coupling switches between a number of distinct regimes depending on side-chain conformations.But how is such a global elastic stress built up within a protein scaffold? We used all-atom Molecular Dynamics (MD) simulations to quantify the importance of prestress in the native state of ubiquitin. To this end, we adapted an earlier technique for measuring force distributions in mechanically perturbed proteins From 100 ns of MD trajectories, we calcuated effective force profiles for every pair of amino acid residues in ubiquitin , as descThe most obvious large-scale structures in the network are the relatively ordered bands of both tensile and compressive forces that connect neighboring beta strands: specifically, the two parallel pairs of beta strands 1/2 and 3/5, and the anti-parallel beta strand pair 1/5. In contrast, isolated cases of strongly tensed (red) residue pairs are also observed, such as Lys27-Asp52 and Lys11-Glu34, which do not correlate with neighboring residues. These high tensile forces occur only between residues with charged side-chains; as discussed in more detail below, they correspond to tensed salt bridges.To get a clearer picture of the prestress pattern associated with the main-chain interactions in beta sheets, the force network accounting for only inter–main-chain interactions is shown in Apart from the hydrogen-bonded pairs, every other residue pair in beta strand pair 1/5 experiences an average attractive force  all correspond to salt bridges between charged side-chains . The pair with the highest tensile prestress is Lys11-Glu34, which comprises a salt bridge connecting the C-end of the alpha helix with the N-end of beta strand 2. This particular salt bridge has been shown experimentally to contribute significantly to the thermodynamic stability of ubiquitin Unlike the main-chain-only prestress network, for which each residue has significant interactions with at most two others, some nodes in the side-chain network are connected to as many as four or five others, widely separated in sequence-space. In the context of network theory, these residues may be thought of as ‘hubs’ of the network; perturbing these residues may be expected to lead to a wide-spread redistribution of force in the prestress network. In fact, simulations in which two of the most obvious hub residues, Asp52 and Arg72, were separately mutated to glycines exhibited no statistically significant changes to the prestress network beyond the local neighborhood of the mutated residue. This suggests that, at least with respect to perturbations of these specific residues, redundancy in the mechanical network imbues the pre-stress distribution with a certain amount of rigidity, and that intentional engineering of a protein's prestress network may require a more sophisticated mutation strategy beyond simply perturbing individual network hubs.The connections between the hub residues Lys27, Asp52 and Arg72 form a clear triangle in Such switching between discrete states is also observed for hydrophobic residues. We have shown that forces in the native ensemble of ubiquitin, measured from all-atom MD simulations, generate a tensegrity-like pattern of prestress at the residue-residue level. This includes pre-compression of the hydrogen bonds connecting beta strands, and conversely pre-tensing of alpha helix hydrogen bonds. The differences between the pre-compression of individual beta strand hydrogen bonds are sufficient to significantly modify the kinetics of hydrogen bond breakage under force, and thus should be an important factor in determining the protein kinetic stability and unfolding pathway under mechanical perturbation. Salt bridges known to be important for ubiquitin's thermodynamic stability are found to be strongly pre-tensed, and the effective force profiles for side-chain–side-chain interactions reveal a connection between side-chain dynamics and inter-residue mechanical coupling. We propose that the observed dynamic equilibrium of multiple side-chain states contributes to the complex rupture kinetics observed in AFM experiments, since each discrete side-chain state corresponds to a different well in the rough global energy landscape. A correlation is found between tensed salt bridges and twisted peptide bonds in the protein backbone, which suggests that tension in stretched side-chains, transmitted as torque to the backbone, might play a role in determining the conformation of the protein's native state. Finally, we find the tensegrity network remarkably robust with regard to mutations at network hubs.It remains to be shown whether the observations reported here apply generally to all proteins, or are specific to ubiquitin. A preliminary study of the titin immunoglobulin [I27] domain  and 15 right.(PDF)Click here for additional data file.Figure S2Average atomic forces for main-chain inter-residue interactions between Lys6 on beta strand 1, and Leu67, His68 and Leu69 on strand 5. Atom colors: cyan (carbon), blue (nitrogen), red (oxygen), white (hydrogen). Red (blue) lines represent attractive (repulsive) forces.(PDF)Click here for additional data file.Figure S3(a) Mean inter-residue forces (mainchain only) for the alpha helix. Vertices connecting residue pairs have the same meaning as in (PDF)Click here for additional data file.Figure S4For the residue pair Leu8/Val70, a scatterplot of the angle (PDF)Click here for additional data file.Figure S5(a) The network representing the inter-residue forces for the titin immunoglobulin domain, averaged over 50 ns of molecular dynamics simulations, superimposed on the 3D structure of the protein (PDB code 1WAA). The color and width of cylinders have the same meaning as (PDF)Click here for additional data file.Figure S6(a) Average length and (b) average torsional angle ((PDF)Click here for additional data file.Figure S7biquitin .(PDF)Click here for additional data file.Text S1Backbone stress correlates with tensed salt bridges.(PDF)Click here for additional data file.Video S1Average inter-residue forces, measured from 100 ns of MD simulation, projected on the 3D structure of ubiquitin (PDB code 1UBI). Blue (red) cylinders represent repulsive (attractive) forces, and cylinder width is proportional to force magnitude, with the maximum force around 500 nN.(MPG)Click here for additional data file.Video S2As for (MPG)Click here for additional data file.Video S3As for (MPG)Click here for additional data file.


P = 0.03). The one-year graft survival was significantly reduced in positive pre-transplant HLA-DSA patients . The positive predicted value of HLA-DSA in relation to rejection reached 100% if patients lost their previous graft in the first year after transplant. Among anti-HLA antibodies present in patients before transplant, HLA-DSA were significantly associated with high risk of acute humoral and cellular rejection and reduced graft survival in posttransplant outcome. The negative impact of these antibodies was even higher when patients suffered an early loss of the previous transplant.It is well know that anti-HLA antibodies are an important obstacle in kidney transplantation. Our aim was to study the clinical impact of pretransplant donor specific anti-HLA antibodies (HLA-DSA), in highly sensitized (HS) patients. We analyzed retrospectively the day-of-transplant sera by Luminex Single Antigen Assay (LSA) in HS patients, and the results were correlated with episodes of humoral and cellular rejection as well as with graft and patient survival. All HS subjects received the same induction therapy and rejection episodes were biopsy proven. Thirteen patients (56.5%) preformed HLA-DSA, and we observed higher incidence of acute rejection in aforementioned patients than in the pre-transplant negatives DSA recipients (77% versus 30%,  A best knowledge of immune system and the introduction of several immunosuppressive agents have led to reduce the incidence of acute rejection (AR) but had a limited impact on long-term allograft survival . It is wde novo DSA has been well established, the determination of the clinical relevance of the preformed anti-HLA antibodies is today under consideration [Since the number of retransplanted patients is increased year by year, the study of anti-HLA antibodies using a method which can improve sensitivity and specificity to detect donor specific anti-HLA-antibodies (DSA) would be useful in order to avoid the acute rejection. Luminex technology is one of the solid assays which have been developed in the last few years. This technology is able to detect lower levels of alloantibody and to define them more accurately than the conventional complement-dependent cytotoxicity (CDC) assay –6. Despideration , 8. Publderation . Severalderation , 14. Howderation .Our aim was to determine among all of the anti-HLA antibodies, the effect of pre-transplant (pre-Tx) DSA in clinical outcome. For that purpose we selected HS patients and we analyzed retrospectively the day of transplant sera by Single Antigen in order to elucidate how predictive were the DSA present in the receptors before kidney transplantation in acute rejection and allograft survival. This is a retrospective study to analyze the role of preformed antibodies in solid organ rejection. In order to guarantee the presence of DSA in pre-transplant sera, we selected all HS patients  transplanted in a short period of time (26 months). The induction protocol used in all HS patients was the same: thymoglobulin (1–1.2 mg/kg pre-transplant and 4–7 doses after-transplant 0.25–0.75 mg/kg/daily), tacrolimus , mycophenolate mofetil  and steroids: methylprednisolone . No desensitization protocol was performed.All patients received steroids pulses as a first-line treatment for cellular rejection, and in the most severe cases we added thymoglobulin. In patients with AMR, the treatment was based on intravenous immunoglobulin , and when there was severe rejection (Banff grade II or III), we added plasmapheresis or rituximab (1–4 doses of 375 mg/m2 in 7/10 cases). The analysis of anti HLA class I antibodies was carried out by Single Antigen  according to manufacturer's instruction. Luminex 100 IS 2.3 system was used for data acquisition and analysis and all beads with mean fluorescence intensity (MFI) above 3000 were considered as positives. The determination of anti HLA class II antibodies was carried out by Lifecodes Class II ID, which is a Luminex assay for detection of IgG panel reactive antibodies to HLA class II molecules.t-Student or U Mann-Whitney. Level of significance was P < 0.05 SPSS v. 13.0 software was used in statistical analysis .Normally distributed variables are expressed as mean ± standard deviation (SD) and nonnormally distributed variables are expressed as median and interquartile range (IQR). Association between qualitative variables was evaluated by chi-square test or Fisher's exact test when necessary. Effect was calculated as risk ratio (RR) and 95% confidence interval. Quantitative variables by groups were evaluated with We selected 23 HS patients out of 191 subjects transplanted in our Renal Transplant Unit between 2007 and 2008 and followed during 25 months  and no differences were found between HS patients with nonpreformed DSA and non HS-patients . The incidence of AR and the death censored allograft one year survival was significantly different between non-HS group and the HS patients . InteresIn We also performed a Kaplan-Meier survival analysis, and the presence of DSA in the pre-transplant sera correlates with a pour graft outcome . In order to improve this predictive value, we analyzed the characteristics of all data which could be implied in acute rejection. Neither induction treatment  nor immunization events, 22 out of 23 had a previous transplant, could be considered as critical predictive factor. We also analyzed the number, class and MFI value of the pre-transplant DSA and no differences were found between patients with and without acute rejection. Regarding clinical parameters, we considered the number of previous transplant, elapsed time after the last transplant, whether patient had suffered AR in the previous transplant and the survival of the last graft. Interestingly, only the early loss of previous graft showed a significant statistically association with the presence of preformed DSA and improved its predictive value of rejection. In fact, when patients lost the previous transplant before first year and preformed DSA against the new graft, the presence of AR was 100% . In these cases we would be able to talk about “DSA susceptible patients” who are more sensitive to foreign HLA antigens and usually the graft loss occurs in a period of time lower than a year. We considered that the presence of both pre-transplant DSA and early loss of the previous organ is a high risk factor for rejection. In case that DSA not disappear in a reasonable period, desensitization protocols  could be necessary to transplant these patients and to avoid the almost sure nephrectomy.