One of the fundamental goals in neurosciences is to elucidate the formation and retrieval of brain's associative memory traces in real-time. Here, we describe real-time neural ensemble transient dynamics in the mouse hippocampal CA1 region and demonstrate their relationships with behavioral performances during both learning and recall. We employed the classic trace fear conditioning paradigm involving a neutral tone followed by a mild foot-shock 20 seconds later. Our large-scale recording and decoding methods revealed that conditioned tone responses and tone-shock association patterns were not present in CA1 during the first pairing, but emerged quickly after multiple pairings. These encoding patterns showed increased immediate-replay, correlating tightly with increased immediate-freezing during learning. Moreover, during contextual recall, these patterns reappeared in tandem six-to-fourteen times per minute, again correlating tightly with behavioral recall. Upon traced tone recall, while various fear memories were retrieved, the shock traces exhibited a unique recall-peak around the 20-second trace interval, further signifying the memory of time for the expected shock. Therefore, our study has revealed various real-time associative memory traces during learning and recall in CA1, and demonstrates that real-time memory traces can be decoded on a moment-to-moment basis over any single trial. What are neural population-level memories in real-time? Can we decode neural populations' memory traces as the subjects form and recall memories? How meaningfully can real-time memory traces in the brain be related to a behavioral readout? These enduring questions have attracted a keen interest of many philosophers, psychologists, neuroscientists and beyond The CA1 region of the hippocampus is well known to be a crucial site for processing associative memories which typically contain information about what, where, and when major events occurred. One of the classical associative memory tasks widely used by researchers is the trace conditioning memory paradigm As a prevalent phenomenon in the brain, large response-variability in single neuron activity within any single trial typically makes it difficult to reliably decode the neural activity patterns on a moment-to-moment temporal basis in a given single trial. That is, the trial-to-trial variability of single neuron responses (activity changes of a single neuron) does not allow the reliable prediction of, and consequently, understand whether and/or how brains form snap-shot memory representation of events at any given moment in time. In another words, spike raster plots, while being quite useful to determine neurons' response-selectivity, are not a very effective approach to decoding real-time memory patterns. Moreover, data-averaging practice over trials intrinsically limits the analysis to a selected single time point . This would inevitably lead to loss of most crucial information about memory traces if they are reappeared in tandem with variable intervals across different trials. Therefore, one of the major goals for the memory field is to understand how memory is represented as transient dynamic patterns at the neuronal population level on a moment-to-moment basis and then to demonstrate the explicit relationship between those neural patterns with a behavioral readout during learning and recall.Since brains are likely to achieve real-time representation of memories through the coordinated activity of ensemble neurons We used the trace fear conditioning paradigm to train ten adult, wild-type male mice (5–6 month old). Our trace conditioning contained a neutral tone  which was followed by a delayed mild foot shock  delivered via a metal grid floor in a fear conditioning chamber . The fixAfter the contextual retention test, the mice were returned to the home cage for a brief break and then were placed into a novel chamber  for the trace retention test. As expected, the animals showed active exploratory behaviors in the recall chamber during the 3-min pre-tone period . Howeverin vivo recording technique that we have recently described for monitoring neural ensemble activity in the brain of freely behaving mouse Using the large-scale An example of spike rasters for a subset of simultaneously recorded responsive units is shown . While tThus, we employed a MDA method, a supervised dimensionality reduction-based statistical pattern classification method, to uncover the neuronal population level-encoding patterns from large-scale datasets In order to monitor the transient dynamics of CA1 ensemble activity patterns, we combined the MDA method with a sliding-window technique for assessing the temporal dynamics of neural processing. By using the fixed matrix coefficients produced by the MDA method, we computed the transient projection of neural ensemble responses throughout the recorded datasets with a 10-msec sliding window As the pairing between the CS and the US was repeated over learning trials, the subsequent CS began to produce distinct ensemble patterns that were often not so obvious from the mere visual inspection of spike rasters . For exast pairing) across all recorded mice, but began to emerge during the second or third CS/US pairing  at this stage elicited the associative form of the ensemble trajectories that differed from the first US response: namely, the trajectory first visited the US cluster, but then shot directly to the CS cluster before returning to the Rest ellipsoid . This trb), CS during the pairing (CSd), CS after the pairing (CSa), as well as US]. Some cells (6.5%) exhibited sub-general responsiveness ; yet some cells changed their firing rates to CSd and US (19.1%). Another set of cells responded to CSa as well as US (1.7%). Interestingly, a small percentage of cells appeared to only respond to CSb (2.2%), or CSa (4.5%). We also observed that some CA1 cells were selective to the foot shock (18.2%) but not to the tones. Again, it is worthy note that variability of single neurons during the rest period and in response to stimuli . This behavioral freezing was accompanied by the prominent presence of significant ripple oscillations  within the 20-sec CS-US time interval increased in proportion to the numbers of conditioning trials . Cross-cDuring recall tests, we first measured contextual fear memory using a one-hour contextual retention test which engages both the hippocampus and amygdala For example, mouse #1 entered the freezing state 8 seconds after re-introduced to the conditioning chamber. The observed first retrieved pattern emerged 360 mini-seconds before the animal froze  frequency band . As evidAfter the contextual retention test, the mice were subjected to the one-hour trace memory recall test by placing the animals in a non-conditioned chamber which had a different floor, color, context, and geometric shape see . In our Since the tone during trace recall was presented to the animals in a novel environment which was different from the original training chamber, we first examined whether the major information carried by conditioned tones would change as the contextual environments differed. We approached this question by assessing various categories of distinct events in the global MDA analysis. Our analysis shows that tone-triggered responses during both learning and the recall test could still form a single CS cluster that was well separated from all other events . This inWith this sensitive 2-step MDA method, we assessed how ensemble patterns were retrieved during trace recall. We found that the cue (tone) triggered a string of ensemble pattern retrievals (9 transient trajectories) within the 1-min period . As a result, the direct link between place cell firings and behavioral readout in memory acquisition and retention tests has not been demonstrated so far. For example, this is no evidence in explicitly linking place cell replay to cue-induced behavioral memory readout at memory retention tests .To overcome the above technical or experimental limitations, the present study employs the classic Pavlov fear conditioning paradigm, coupled with large-scale recording and decoding methods to investigate the real-time population-level activity patterns in the hippocampus in relation with trial-to-trial memory scores. The choice of Pavlov conditioning offers several advantages: 1) the hippocampus is widely known to be crucial for such associative function, especially with longer CS-US interval and context; 2) the well defined cues allow researchers to measure the behavioral readout for memory formation and then to correlate behavioral readout with transient ensemble patterns, thereby fulfilling the definition for the observed transient dynamics as actual memory traces.What do real-time memory traces in the hippocampus look like over the course of learning and recall? How do the associative memory traces differ from unconditioned stimulus-evoked responses? Does the brain retrieve a single memory trace or multiple memory traces upon a cued signal? If a train of memory traces are retrieved, what are they? And are there any temporal structures or relationship in its recall sequence? How do memory traces correlate with behavioral performances during the recall tests?These fundamental questions are fascinating, and the trace conditioning task is an ideal paradigm to examine these issues since it is well known to produce a variety of memories (at least in the human brain) that can include a shock or air-puff event, the causal and temporal relationship between a tone and a shock, as well as the environment in which an event occurs. It is conceivable that rich collection of various memory components can all contribute to and/or influence behavioral outputs.It is often acknowledged that behavioral readout represents a good, but not very precise, assessment of the internal memory processes in the brain. By using large-scale ensemble recording techniques and statistical pattern recognition algorithms First, CA1 ensemble patterns evoked by the CS-US pairing change rapidly from unconditioned forms to conditioned associative forms. That is, the CS-US pairing resulted in the rapid appearance of robust CS ensemble traces after one or two trials in the mouse CA1 region. Interestingly, as the conditioned tone trace emerged, US-triggered ensemble responses which originally evoked only US-specific transient trajectories began to turn into the US-to-CS association trajectories. The emergence of these new types of association trajectories suggests that circuitry-level dynamics have succeeded in capturing the CS-US causal relationship. Interestingly, the temporal sequence of association trace is reflected by its movement direction: these transient trajectories moved from the US cluster to the CS cluster in the MDA encoding subspaces. These associative patterns required the repetition of CS/US pairings, and can appear as early as the second pairing and become prevalent during the late stage of learning phase in all animals. The formation of such associative memory traces is likely the product of synaptic plasticity. It would be of great interest to examine the molecular basis of these associative patterns in using NMDA receptor conditional knockouts or other genetically modified animals with enhanced or impaired memory or in which memory can be rapidly erased Second, the encoding patterns produced by learning replay immediately within seconds during the trace conditioning acquisition phase, and more importantly, they correlated tightly with learning scores. The reappearance of temporal trajectories often retained their original directional movement and geometric shapes and subspace plane. Importantly, the immediate spontaneous reactivations following CS/US stimulations exhibited a clear trial-dependent property, that is, the numbers of reactivations increase in proportion to trial numbers. While there are few reactivations at the beginning of the trials (e.g. the first pairing), reactivations become more prevalent at late trials. The near linear correlation between the trace reactivation numbers and the levels of behavioral learning (immediate freezing) suggests that post-learning immediate reactivation may be an important physiological indicator for assessing network-level trace formation in the hippocampus. Our finding collaborates well with the previous findings showing that neurons in the brain also undergo reactivations after spatial running Third, to meet the primary criterion that transient neural ensemble patterns represent true memory traces, one need to demonstrate that those patterns observed during learning are to be retrieved upon the recall cues and they would again correlate with memory scores. In line of this criterion, we have indeed observed that these CA1 dynamics patterns reappeared at the time of both contextual and traced fear retention tests. Using contextual cues or trace cue (tone) in the absence of shock is important feature for demonstrating classic associative memories. Importantly, this well defined, retention test has offered us to link the physiological ensemble patterns to cued-induced memory recall performances that so far have not been demonstrated in place cell model experiments We find that on average, ensemble traces were retrieved at a rate of 6-14 times per minute in the mouse hippocampus during the retention tests. Importantly, the numbers of retrieved patterns in the context recall tests were tightly correlated with behavioral performances in the memory retention tests at both the individual and group levels, thereby meeting the criteria of functional readout.It is also important to note that the time scale for retrieved memory patterns in our recall test is similar to that of the memory encoding patterns in the learning phase. Furthermore, as predicted, we have observed the causal relationship between appearances of the first memory trace and the recall-induced freezing. That is, the first recalled memory trace consistently precedes freezing behavior on the average of 1.4 seconds. Given the fact that we calculated the time delay between the off-set of the first memory trace and the onset of freezing, the causality should be beyond questionable. Thus, our present experiments provide a piece of crucial evidence that neural ensemble patterns formed during learning indeed reappear and precede behavioral recall, thereby fulfilling the major criterion for those transient dynamics as population-level memory traces.Intriguingly, some ensemble traces or trajectories observed during recall were not seen during the learning phase . Emergence of richer ensemble patterns at the time of recall may reflect not only the successful retention of various associative relationships at the time of learning but also the possible on-going re-modification or updating of existing memories by the recall experience itself Fourth, the observation that the significant amounts of ensemble pattern retrievals were accompanied by sharp-wave ripples provides us a unique opportunity to examine the correlation of memory processing with hippocampal ripples that are often reported in the literature Fifth, our decoding method has revealed the memory of time for the expected shock events. The unique retrieval peak for the US shock traces at the time point of expected shock suggests that the animals retained not only the memory of the correct event, but also the memory of its timing. This level of decoding power is particularly interesting to us since at the behavioral level the animals were in the heightened freezing state through the most of the recall time . This persistent freezing reduces the sensitivity of behavioral readout, and thereby, unable to allow the distinction of various memory components . For example, the presentation of a recall tone would easily cause the animals to freeze right away because the mice retrieved tone/shock association memories, this can easily mask the behavioral readout for the memory for time for the expected shock event. This level of decoding power is highly useful given the fact that many of our own memory thought processes can occur without necessarily being expressed at behavioral level intentionally or unintentionally.From our analyses, it is quite clear that CA1 ensemble traces that had undergone trace memory recall are rich and diverse. Quite often, the various ensemble trajectories were retrieved in tandem . Although our visual inspection has not revealed any obvious simple-order temporal sequences in their emergences, it remains possible that some types of high-order temporal relationship may still exist. On the other hand, cognitive literatures often emphasize that memory contents/or sequences seem to always differ slightly at each recall, even when the human subjects were presented with the same question. Thus, it would be a great topic for more detailed investigations.It has been known that many brain regions are engaged in processing associative memories In conclusion, the population-level associative memory patterns in real-time can be mathematically described and intuitively visualized on a moment-to-moment basis at any given single trial. More importantly, our present study demonstrates the meaningful relationship of these transient neural dynamics of memory traces with behavioral scores of learning and recall not only within each animal but also across animals. The ability to decode brain's diverse memory traces in real-time from the acquisition to recall phase may have wide implications and many useful applications.All animal work described in the study have been conducted according to NIH guidelines and approved by MCG Institutional IACUC committee.96-channel or 128-channel recording arrays were constructed as previously described http://www.cbc.umn.edu/~redish/mclust, David Redish). First, the recorded data were filed as Plexon system format (*.plx). Before spike sorting, the artifact waveforms were removed and the spike waveform minima were aligned using the Offline Sorter 2.8 software . The aligned data were then saved as files in Neuralynx system format (*.nst). After that, the MClust 3.3 program was used to isolate different spiking units. Only units with clear boundaries and less than 0.5% of spike intervals within a 1 ms refractory period are included in the present analysis.The spike activity was recorded using Plexon Systems  and then sorted using the MClust 3.3 program , 258 units (for mouse#2), 242 units (for mouse#3), 232 units (for mouse#4), 308 units (for mouse#5), and 206 units (mouse#6), respectively. Isolated units were classified as either putative excitatory pyramidal cells or interneurons based on their characteristic firing activity including waveforms, inter-spike intervals, and firing rates The fear conditioning chamber was a square chamber (10″×10″×15″) made of plastic boards with a 24-bar inescapable shock grid floor, and the recall chamber was a triangular chamber made of foam boards with a smooth and opaque floor. Thus, these two chambers are distinct geometrically, contextually, and visually. Both chambers had one transparent wall, and thus freezing responses of animals in the chamber could be observed by experimenters from the small hole on the surrounding black curtain and were automatically videotaped by computers. In each test, only one animal was trained and recorded. Before training, the mouse was habituated in both chambers for five minutes per day, and three days in total . We and others have found that some CA1 cells may respond to novelty . But the neutral tone triggered responses tend to decline dramatically. The habituation protocol is used widely in the field because it gives the animals time to better differentiate CS tone with other sound or noise .On the training day, the recording began with a 1-h pre-training sleep period in the home cage (a plastic tub where the mice lived everyday) and then followed by a 3-min pre-training exploration period in the shock chamber and then a 30-min period during which a 2-sec tone (85 dB continuous tonic sound at 2800 Hz) was played ten times at random intervals. This allowed us to determine the CA1 responses to naïve tone. The animal was then brought back to the home cage for a 30-min break before trace fear conditioning began. During trace conditioning, the conditioned stimulus  was given and then followed by a 20-sec interval (after the off-set of the tone) before the unconditioned stimulus (a continuous 285-ms foot shock at 0.75 mA) was delivered in the shock chamber. This CS-US pairing was repeated for seven times, with 1–3 min random time intervals between CS/US sessions. The mouse was then brought back to the home cage for 1-h post-training sleep/rest. The immediate freezing was assessed for 30 seconds after each shock.After a one-hour rest, two kinds of memory retention/recall tests were conducted. The first one was the contextual memory recall test during which the mouse was placed back to the shock chamber again for five minutes. The other retention test was the trace memory recall test during which the mouse was put into a novel chamber and allowed to explore freely for three minutes before the onset of a 2-sec tone was played . After the completion of all retention tests, the mouse was returned to its home cage to rest for 30 minutes. After the rest, the animal was then subjected to four additional episodic events including metal sound, air-puff , earthquake-like shake , and drop (from the 13 cm height). The last set of episodic events was conducted after one-hour fear memory tests have been completed, and thereby would not affect data on fear conditioning. Inclusion of these startling episodic events was designed to provide additional information when we performed MDA analysis . Such information would permit us to ensure the statistical pattern classifications to be highly reliable in multiple MDA subspaces.Throughout all these procedures, animal behaviors were recorded simultaneously by videotapes and synchronized with spike data collection. Freezing response, defined as absence of body movement except for respiration, was counted as the measurement of fear memory. Immediate freezing responses during training were measured for 60 seconds after the foot shock. We counted ‘freezing’ by observing the animal behaviors frame by frame based on the recorded video, the temporal resolution for the video is 30 frames per second and the absolute amount of time that the mice stayed in the continuous frozen state was summed and compared to the total amount of time during which the mice were not in the non-frozen state. The behavioral data presented in f) were evaluated in two 500-ms time bins immediately after the events started. Neuron responses were defined by: f0 (2–3 Hz) is the global mean response frequency of putative excitatory neurons across the recorded neural population in an animal, excluding high firing-rate interneurons (cut-off at 20 Hz) and fpre, n is the base firing rate (computed from samples of firing rates in two 500-ms time bins before startle stimuli). This transformation emphasizes significant changes in firing patterns for units with both low- and high baseline firing rates. Effectively, responses of low-firing units are proportional to absolute firing rate changes, while responses of high-baseline units are proportional to the relative changes.A 500-ms bin was selected for sampling ensemble firing activity based on the optimization of achieving the best statistical prediction power We used Multiple Discriminant Analysis (MDA) projection method to classify and separate the neural responses corresponding to different episodes into different classes in is the number of elements in each class, N is the number of classes, im is the mean vector for each class and m is the global mean vector. To take into account the variations occurring for each class, we also computed the within-class scatter matrix WS, which is defined as: iD represents the set of population responses triggered by the ith startle type. Using these two matrices, it follows that a set of at most N-1 discriminant projection vectors can be determined by computing the eigenvalue decomposition of the matrix One set of population activity from seven repetitions of each type of stimuli was randomly chosen to constitute our test data set. The rest of the sampled population activities were then used to train our MDA statistical model. The matrix of mean responses during each category (rest and startle states) was then computed and used to compute the between-class scatter matrix WS are non-invertible, which is a direct consequence of data under-sampling, since the dimension of the number of recorded neurons is much higher than the number of repeated trials. In practice, the matrix WS can be rendered invertible using a regularization technique which changes each class covariance matrices based on the following formula: ith class, λ is a regularization parameter between 0 and 1, and I is the identity matrix. The parameter λ is determined automatically for each data set based on the optimization procedure we developed previously; each particular choice is determined by the particular distributions within each data set N – 1 discriminant dimensions, we projected the neural patterns during startle episodes in the low-dimensional encoding subspaces. We then used the multivariate Gaussian distribution probability functions Click here for additional data file.Figure S2Confirming the position of recording electrodes in the CA1 region of the mouse hippocampus. (A) Histological confirmation of electrode placement. The top panel demonstrates the position of the electrodes with orange bars in the atlas of the mouse brain. The two examples (the middle and bottom panels) show Nissl staining in the hippocampal CA1 region from one mouse. The arrows indicate the locations of electrode tips in the CA1 pyramidal layers. (B) Physiological confirmation of electrode placement by the occurrence of ripples. Local field potentials (the upper panel) and the filtered ripples (the lower panel) were shown from ten recording channels.(1.24 MB JPG)Click here for additional data file.Figure S3Stable recordings were confirmed as judged by the waveforms and inter-spike interval (ISI) histograms of recorded cells. Eight representative putative pyramidal cells are shown here. (A)–(H) The left columns are waveforms and the right columns are inter-spike interval histograms. The waveforms were plotted from a 70-sec recording before (top row), during (middle row), and after trace-conditioning trials. A 10-sec recording for each trial was plotted. The ISIs were analyzed by using the corresponding data and the bin size is 0.005 s. (A)–(D) are the data recorded from steretrodes, and (E)–(H) are the data recorded from tetrodes.(2.84 MB JPG)Click here for additional data file.Figure S4Stable recordings for putative interneurons in the hippocampus. Waveforms and inter-spike interval histogram of interneurons (eight representative units are represented here). (A)–(H) The left columns are waveforms and the right columns are inter-spike interval histograms. The waveforms were plotted from a 70-sec recording before (top row), during (middle), and after trace-conditionings (bottom row). A 10-sec recording for each trial was plotted. The ISIs were analyzed by using the corresponding data and the bin size is 0.005 s. (A)–(D) are the data recorded from steretrodes, and (E)–(H) are the data recorded from tetrodes.(3.06 MB JPG)Click here for additional data file.Figure S5A spike-raster plot of simultaneously recorded 208 CA1 units from mouse #1. (A) The sixty-sec spike-raster plot demonstrates neural activity when a tone was delivered and then followed by a foot shock in the third trial during the training. Only a set of the 208 simultaneously recorded CA1 cells were shown here. Two 500-ms black parallel windows demonstrate the MDA sliding-windows for computing dynamic trajectories in MDA subspaces. (B) The averaged histograms show neural activity during training (the left column) and during trace recall (the right column) for the corresponding colored units shown in (A) across all trials. The histograms at the first row show that this group of neurons had significant prolonged increased responses to the US presentation as well as increased responses to the CS during training. At trace recall, they also had significant increased responses to the recall tone. But the variations in spike firings of these neurons could not allow the confident assessment of the traced retrieval, although we noted a blip around 24 seconds after the recall tone was delivered. The data were pooled from all trials. The histograms at the second row show that these US-responsive neurons increased their firing rates at both onset and offset of the tone during recall. Interestingly, this group seemed to have a double peak in responding to the recall tone. The histograms at the third row show that these US-responsive neurons had a smaller response to the CS during training, and they also did not show significant firing increases when the recall tone was delivered. The fourth-row (dark blue) histograms show that this group of neurons showed a transient increase in their firing rates responding to the US during training, and they also seemed to have transient responses to the tone during recall. The fifth-row histograms show that a small set of responsive neurons decreased their firing rates both to the US during training and the tone during recall. The last-row histograms show that non-responsive neurons had no significant responses to both the US and the CS during training, and no responses to the tone during recall. X axis is time; Y axis is responsive ratio of frequency over baseline firing rates.(3.77 MB JPG)Click here for additional data file.Figure S6Representative units show neural responses to the US and/or the CS over trials. (A) Spike rasters show firing activity of a US-responsive unit. Red spike rasters show the firing activity of a representative unit in response to the US conditioning; two rasters in brown show the tone-triggered responses in the first two training trials; the five rasters in purple show the tone-triggered responses in the last five training trials; the seven rasters in green show the tone-triggered responses of this unit in the seven trials during trace recall trials; the seven rasters in blue show the naïve tone-triggered responses before paring. Time is represented in seconds on the X axis, and the trial number is listed on the Y axis. (B) The frequency responses of the unit shown in (A), obtained by smoothing the spike counts through a Gaussian kernel, indicate that this unit significantly increased its firing rate only in response to the US. (C) Spike rasters show firing activity of a US/CS responsive unit in response to various stimuli. The seven rasters in red show the US-triggered responses in this unit over trials; the two rasters in brown show the tone-triggered responses during the first two training trials; the five rasters in purple show the tone-triggered responses in the last five training trials; the seven rasters in green show the tone-triggered responses during traced recall; the seven rasters in blue show that the naïve tone did not trigger significant responses before training. Time is represented on the X axis, and the trial number is listed on the Y axis. (D) The frequency responses of the unit shown in (C), obtained by smoothing the spike count through a Gaussian kernel, indicate that this unit significantly increased its firing rate in response to the US, the tone during training, and the tone during recall. Please note that the responses to contextual recall are not listed due to the lack of objective time points for setting time zero. The need for averaging the firing responses of a single unit over trials illustrates the importance of large-scale pattern classification for revealing patterns, at any given time point, from the simultaneously recorded cell population. (E) Hierarchical clustering of the responses of 1454 recorded CA1 neurons to fear conditioning. The data were pooled from six mice. Only 9 neurons showed responsiveness to the tone before learning (CSb), the tone during CS/US pairing (CSd), the tone at trace recall (CSa), and foot shock (US); 94 neurons responded to US, CSd and CSa, which was ∼6.5% of all recorded neurons; 24 neurons responded to US and CSa; 277 neurons responded to US and CSd, which constituted 19.1% of all recorded neurons; 265 neurons responded to US but not to tones; interestingly, 32 neurons responded only to CSb, without any significant responses to tones after pairing, or to the US; 66 neurons responded only to CSa ; the other remaining 47.2% neurons had no significant responses to all four types of stimuli. The color bar on the left indicates the normalized response magnitude.(0.66 MB JPG)Click here for additional data file.Figure S7Visualization of various CA1 ensemble encoding patterns and the transient dynamics in MDA subspaces. (A) MDA analysis shows CA1 ensemble representations of various episodic events in mouse #1. Please note that the naïve tone (prior to conditioning) overlapped significantly with the Rest cluster, indicating that the naïve tone did not trigger significant CA1 responses. (B) A rotated view of the MDA subspace shown in (A) demonstrates that the ensemble representations of these events were well separated. In MDA spaces, the discrete dots in various shapes represent firing patterns in response to a give type of stimulus ; color ellipsoids were constructed for representing neural ensemble responses to various kinds of stimuli by fitting Gaussian distribution to the projected points (discrete dots) for each class. (C) Two representative US-CS association trajectory traces at the third (blue) and fifth learning trials (magenta), respectively, are shown here. At these learning stages, the foot shock triggered the patterns that moved from the Rest state to the US cluster, and then directly visited and hovered around the CS for a while before returning to the Rest. (D) A rotated view of MDA subspace in (A) shows the reliable moving paths of two activation traces despite the variability at the single neuron level.(0.45 MB JPG)Click here for additional data file.Figure S8Relationship between ripples and pattern retrievals during the freezing state of contextual recall. (A) A representative spectrogram during freezing is shown in the left panel. One typical ripple oscillation is shown on a finer time scale in the top right panel. It was filtered out from the recorded field potential using a 100–250 Hz band-pass filter (the bottom right panel). (B) The power spectral density plot shows a significant peak in 150–250 Hz range during freezing (red). It was absent when the animal was in the active exploration state (blue). (C) Three retrieved CS ensemble traces were plotted here (in different colors) during the freezing state of contextual recall. (D) Three retrieved US ensemble traces were plotted during the freezing state of contextual recall. (E) An example of co-occurrence of hippocampal ripples and a CS ensemble trajectory is shown. The red line about the upper ripple oscillations indicates the threshold line (5 s.d. above mean power). The blue curve here corresponds to the blue trajectory shown in (C). The projection distance of the CS trajectory was measured along the line through the Rest and CS ellipsoid centers. (F) Co-occurrence of ripples and a US pattern is illustrated here. The dynamic trajectory is the same as the blue trajectory in (D). The projection distance of the CS trajectory was measured along the line through the Rest and US ellipsoid centers. (G) Occurrence distribution of retrieved three major types of ensemble patterns in relation with ripples (±1-sec time window from the peak center of each ripple) during the contextual recall freezing state. (H) Occurrence distribution of large-amplitude ripples (5 s.d. above mean power) in relation with retrieved ensemble patterns (±1-sec time window from the peak of each trajectory). About 49.2% of ensemble pattern retrievals were accompanied with large-amplitude ripples.(0.53 MB JPG)Click here for additional data file.Figure S9(A) Seven colored curves show the stable ensemble trajectories during the seven trace recall trials in mouse #1. The tone-triggered the peak responses within 200 mini-seconds. The distance is computed by projecting activity trajectories onto the line through both the centers of the CS ellipsoid and Rest ellipsoid. (B) Seven circles show the time latency to freezing after offset of the recall-tone traces during each of the seven recall trials. Color corresponds to the trial number in (A). On average, the latency is 1.9857±1.5137 sec for this animal.(0.17 MB JPG)Click here for additional data file.Figure S10Diverse reactivation patterns in CA1 during trace memory recall. (A–D) Four distinct trajectories are separately shown in MDA subspaces. These patterns occurred during the 1-min trace recall period. (E–H) Rotated views of the same set of the trajectories in MDA subspaces shown in (A–D). The same trace is listed in the same row, side by side.(0.42 MB JPG)Click here for additional data file.Table S1Robustness of the MDA statistical classification.(0.03 MB DOC)<>Click here for additional data file.


Anti-tumour effectiveness was correlated with platinum accumulation in tumours and the amount of platinum bound to DNA, as determined by atomic absorption spectrometry. In tumours treated with electrochemotherapy, cell kill was increased by a factor of 20 compared with treatment with cisplatin only, as determined from tumour growth curves. The amount of platinum bound to DNA and platinum content in the tumours treated by electrochemotherapy was approximately two times higher than in cisplatin-treated tumours. Based on our results, we conclude that in vivo application of electric pulses potentiates anti-tumour effectiveness of cisplatin by electroporation that consequently results in cisplatin increased delivery into the cells. In addition, besides electroporation, immune system and tumour blood flow changes could be involved in the observed anti-tumour effectiveness of electrochemotherapy. © 1999 Cancer Research CampaignElectrochemotherapy is an anti-tumour treatment that utilizes locally delivered electric pulses to increase cytotoxicity of chemotherapeutic drugs. The aim of our study was to determine whether anti-tumour effectiveness of electrochemotherapy with cisplatin is a consequence of increased plasma membrane permeability caused by electroporation that enables cisplatin binding to DNA. For this purpose, anti-tumour effectiveness of electrochemotherapy was evaluated on SA-1 tumours treated with electric pulses 3 min after intravenous injection of cisplatin (4 mg kg


In the HPV FOCAL trial, we will establish the efficacy of hr-HPV DNA testing as a stand-alone screening test followed by liquid based cytology (LBC) triage of hr-HPV-positive women compared to LBC followed by hr-HPV triage with ≥ CIN3 as the outcome.Control arm: LBC at entry and two years, and combined LBC and hr-HPV at four years among those with initial negative results and hr-HPV triage of ASCUS cases; Two Year Safety Check arm: hr-HPV at entry and LBC at two years in those with initial negative results with LBC triage of hr-HPV positives; Four Year Intervention Arm: hr-HPV at entry and combined hr-HPV and LBC at four years among those with initial negative results with LBC triage of hr-HPV positive casesHPV-FOCAL is a randomized, controlled, three-armed study over a four year period conducted in British Columbia. It will recruit 33,000 women aged 25-65 through the province's population based cervical cancer screening program. To date, 6150 participants have a completed sample and epidemiologic questionnaire. Of the 2019 women enrolled in the control arm, 1908 (94.5%) were cytology negative. Women aged 25-29 had the highest rates of HSIL (1.4%). In the safety arm 92.2% of women were hr-HPV negative, with the highest rate of hr-HPV positivity found in 25-29 year old women (23.5%). Similar results were obtained in the intervention arm HPV FOCAL is the first randomized trial in North America to examine hr-HPV testing as the primary screen for cervical cancer within a population-based cervical cancer screening program.International Standard Randomised Controlled Trial Number Register, ISRCTN79347302 Cervical cancer screening using cervical cytology (the Pap smear) has been an extremely successful public health intervention, achieving reductions in cervical cancer incidence of up to 80% where practiced effectively. Howeversingle primary screening test with cytology reserved only for the triage of women having a positive test, especially following the advent of HPV vaccination[There is now ample evidence that infection with high-risk types of the Human Papillomavirus (hr-HPV) is a requisite intermediate step for the development of cervical cancer and its precursors,5. On thccination,13. Thissensitivity (hr-HPV testing);• Screening would be undertaken with the test having higher • 85-90% of women would be returned immediately to routine screening with a negative hr-HPV test without incurring the cost of cytology, which would be reserved only for those with a positive hr-HPV;• The high-volume screening of samples would be undertaken with a non-subjective test that can be automated, while the subjective, labour-intensive test would be restricted to high-risk samples that could be examined with greater vigilance because of the reduced number to be interpreted;• It represents a more robust screening approach that could serve the additional purpose of post-vaccination surveillance in the population;• The recommended cervical cancer screening interval can be extended, as the long term risk of CIN3 or worse in women with a negative hr-HPV test is much lower than those who have a negative cytology, thus providing greater reassurance to women and also resulting in potential cost savings.To examine these concepts, several international large randomized controlled trials (RCT) are being conducted in Europe and in Canada to evaluate HPV testing as part of primary cervical cancer screening,14-21. WThis paper describes the design and preliminary screening results of the HPV FOCAL Trial. The primary objective of the HPV FOCAL trial is to establish the efficacy of hr-HPV testing followed by liquid based cytology (LBC) triage of hr-HPV-positive women compared to LBC followed by hr-HPV triage for cervical cancer screening with ≥ CIN3 as the outcome, through a comparison of the estimated decreases in cervical intraepithelial neoplasia (CIN 2/3) that can be achieved by each screening modality in successive screening rounds. The secondary objectives of this trial are to establish the appropriate screening interval for hr-HPV negative women, using the current standard of a 2-year recall interval for cytology negative women as the benchmark of acceptable risk in British Columbia; to establish the appropriate clinical follow-up for hr-HPV positive women; and to establish the cost-effectiveness of hr-HPV testing for primary screening, all within the context of a population based Canadian cervical cancer screening program. The results of this trial will demonstrate whether or not the use of hr-HPV testing as a single primary screening test within a population based cervical cancer screening program will be able to provide further reductions in the incidence of cervical cancer and its precursor lesions, allow the screening interval to be extended, and improve the cost-effectiveness of cervical cancer screening.HPV FOCAL is a three-armed, RCT over a four year period Figure .Control arm: LBC at entry and again at two years; ASCUS cases are triaged with hr-HPV testing; combined LBC and hr-HPV at four years (exit screen) among those with initial negative results.Two Year Safety Check arm: hr-HPV at entry; LBC at two years (exit screen) in those with initial negative hr-HPV results with LBC triage of hr-HPV positives at either round.Four Year Intervention Arm: hr-HPV at entry with LBC triage of hr-HPV positives; combined hr-HPV and LBC at four years (exit screen) among those with initial hr-HPV negative results.Since the 1960s, cervical cancer screening in British Columbia has been organized as a centrally administered program. A singlWomen aged 25 to 65, registered with Medical Services Plan in British Columbia, who receive care from a participating family physician (FP) for routine cervical screening are eligible. Exclusion criteria are: a history of histologically proven CIN2 or worse requiring treatment in the last five years; a history of histologically proven invasive cervical cancer; a Pap smear within the preceding twelve months; no cervix; pregnant at time of enrolment; HIV positive or on immunosuppressive treatments; or unwilling or unable to provide informed consent.Women are invited to participate in the study when they present for cervical cancer screening and are deemed eligible to participate by their FP or when pre-identified as being due for screening from the BCCA centralized provincial cytology database. For the pre-identified, the FP office sends the woman a study package that includes an invitation letter, a study information pamphlet and the informed consent form. The invitation letter requests women to phone their FP to make an appointment for their cervical screening test and also provides them with the opportunity to contact, or be contacted by study staff to learn more about the trial and decide on participation. All participants are consented by their FP and are asked to complete an epidemiologic questionnaire. A financial implications questionnaire is also completed by a random sample of participants. Full cervical cancer screening history is available through data linkage to the cervical cancer screening program, and HPV vaccination status is self reported.In addition to the control arm and the intervention arm, a 'two year safety-check' arm is included in this trial. At the time of the trial design, there were ethical concerns identified in changing the screening interval at the same time as changing the primary screening tool in a clinical trial. To that end, we included a safety check arm, to examine the safety of hr-HPV testing within the usual screening interval. This information will be used by the data safety monitoring board to verify the safety of hr-HPV testing at the two year interval and infer the suitability of the intervention arm as the trial proceeds.® Broom-like collection device and is placed in a ThinPrep® PreservCyt vial  and is used for all the HPV FOCAL trial testing. Specimen 2 is collected in Digene STM®  and is frozen for future use. Both specimens are sent to the central laboratory in Vancouver, for trial testing (specimen 1) and for storage (specimen 2). Upon sample receipt, the woman is randomized to one of the three study arms and specimen 1 is aliquoted  is collected first with the ThinPrepn system .If the woman's test results indicate that a colposcopy referral is recommended Figure , this wiWomen referred for colposcopy fall into the following major groupings:a) Cytology: AGC (hr-HPV positive or not done);b) Cytology: ASC-H or ≥ LSIL (hr-HPV positive or not done);c) Cytology: ASC-US; .Women are managed according to the standard provincial guidelines in the province of British Columbia. ColposcPathological interpretation of biopsies is conducted at two centres which provide services to the participating colposcopy clinics. Pathologists are blinded to cytology and hr-HPV result when interpreting the slides. Histology results are stored in the same Laboratory Information System (LIS) as the cytology results.Following standard practice, if there is significant discordance between the cytologic and histologic evaluation which would potentially influence patient management, the colposcopist contacts the laboratory to request review and correlation of the histology and cytology in order to resolve the discrepancy  and determine an appropriate disease management strategy.A review of exit screen histology is an essential component of the trial. Approximately 50% of exit screen histology results will undergo second review by senior study pathologists. If the primary and review histology results agree, this will be the final study diagnosis, but in the case of disagreement between primary and review histology, the slide will be referred to another senior pathologist. If this result agrees with either the primary result or first review the agreed results will be the final study diagnosis. If all 3 results are different, the final study diagnosis will be established by consensus between all three pathologists.A database has been developed specifically for the HPV FOCAL trial. Randomization occurs through this database when samples are received at the laboratory. At the time of randomization a Study Identification Number (SIN) is allocated to the participant. Samples are stratified by age and simple equal (1/3 probability) random allocation occurs at the laboratory. Upon randomization, study staff label and batch the specimens for initial processing (hr-HPV or LBC).FPs and participants are blinded to study arm allocation. Participant results are communicated from the BCCA to the FP office as soon as they are known. If the participant's initial screening results (hr-HPV or cytology) are "negative" the report states "within normal limits". The report also states that the recommended follow-up for that participant will be communicated to the FP in two years' time. This ensures blinding is maintained for as long as possible and prevents any bias that may potentially occur from knowledge of negative results. If the screening results are hr-HPV and/or cytology positive, the results are communicated from the BCCA to the FP along with the recommended follow-up.The planned intake sample size is 11,000 women per arm . Power calculations were performed using the nQuery Advisor, version 2, software package. Rates of hr-HPV infection and associated histologically proven disease were based on results from the HART study, prevale• Control and intervention arms: Cumulative incident ≥ CIN3 detected up to and including four years in both the control arm and the intervention arm• Control and safety check arms: Incident ≥ CIN2 detected at two years. If the number of ≥ CIN2 in the safety-check group exceeds 0.8 times that in the control arm at the 2 year screen, then the trial will conclude and women in the four year intervention arm will be recalled at two years for their exit screen.• Rates of ≥ CIN2 and hr-HPV respectively at initial screen in control and safety/intervention arms;• Rates of incident ≥ CIN2 at two years in control arm and at four years in intervention arm;• The total estimated cost per woman screened and the total estimated cost per quality-adjusted life-year gained for each technology;• Clearance of hr-HPV infection in women who are hr-HPV-positive and cytology negative at initial screen.The primary outcome analysis will be a comparison of histologically confirmed ≥ CIN3 between the intervention and control arms. Rates of lesion occurrence will be calculated using person-time denominators for the different study intervals and compared via Kaplan-Meier plots. Rates whttp://www.nserc-crsng.gc.ca/NSERC-CRSNG/governance-gouvernance/ethics-ethiques_eng.asp. Ethics approval has been obtained from appropriate local research ethics boards. The trial registration number is International Standard Randomised Controlled Trial Number Register: ISRCTN79347302. All information about this trial is kept behind locked doors or in secure computer files. On interim and final reports, all data will be de-identified.This study is being conducted in accordance with the Ethical Conduct for Research Involving Humans Tri-Council Policy Statement Recruitment for trial participants commenced in December 21, 2007 through 147 FP offices. As of December 31, 2009, 37,347 women were identified as potentially eligible through the Cervical Cancer Screening Program database. Of the above identified, 28,525 women were sent invitation letters from their family physicians to participate in this study, 613 were ineligible and 2995 declined participation (data on non responders not available). 9842 women were enrolled in the trial through 147 FP clinics from Vancouver Island and Metro Vancouver and 6150 had completed both the epidemiology questionnaire (Epi-Q) and had a preliminary study specimen result. Data will be presented on women who had both a study specimen result and completed Epi-Q.Participant age ranged from 25 to 65 years, with a median age of 45 Table . As partOf the 2019 women enrolled in the control arm, 1908 94.5%) were cytology negative. Sixteen women (0.8%) had high grade squamous intraepithelial lesions (HSIL) on their cytology evaluations, and women aged 25-29 had the highest rates of HSIL (1.4%) Table . In the .5% were The HPV FOCAL study is the first RCT to examine hr-HPV testing followed by LBC triage compared to LBC followed by hr-HPV triage as the primary screen for cervical cancer within the context of a population based cervical cancer screening program in North America. This study will provide generalizable evidence to inform policy decisions concerning cervical cancer screening internationally, particularly in settings where LBC has already been adopted or is under consideration. Participants are women who are part of a long established cervical cancer screening program and represent a population based cohort of women at average risk for cervical cancer in North America. Study characteristics for the first 6,000 participants were well balanced across the three trial arms. In each arm of the trial, over 90% of women were negative on their screening examination, with the majority of abnormalities found in women in under the age of 35.Important design elements in HPV FOCAL include the use of LBC  as opposed to conventional cytology; the use of FPs as study collaborators to recruit participants; blinded analysis of cytology and pathology results with respect to hr-HPV status; use of a single laboratory for cytology analysis; inclusion of a two year safety arm and standardized colposcopy protocols. The use of LBC is of particular relevance because of the ability to use the sample for reflex testing. LBC, which involves taking a sample and placing it into a vial with liquid, and then producing a slide for examination from a suspension of the cells has been compared to conventional cytology in many studies. StudiesFindings from this preliminary analysis contribute to available literature on the prevalence of HPV in Canadian women. In this study of women recruited from a population based screening program, eight percent were positive for hr-HPV, with the highest prevalence rates being found in women aged 25-29. In the first Canadian study comparing Pap screening with hr-HPV screening, women aged 30-69 from Newfoundland and Quebec presenting for routine cervical cancer screening found to have an hr-HPV prevalence of 6.1% overall,19,25. IIn a recent joint analysis of several European cohort studies and clinical trials, Dillner found that hr-HPV results had a high negative predictive value (99%), with women who were negative for hr-HPV having a very low risk of development of ≥ CIN3 in the next six years. This stData from ongoing randomized trials comparing primary screening for cervical cancer with HPV and cytology have found that HPV-based screening offers improved sensitivity for high grade CIN lesions, but with a commensurate loss in specificity. In Dillner's review, he note* ELF has served as occasional consultant to biotechnology companies involved with cervical cytology  and HPV testing .GSO, DVN, MK, REM, TGE, LK, SP, GCES, ELF and AJC all contributed to the conception, design and secured funding for the study. DVN, MK, KC, LK, DAC, WM lead and carried out cytology and molecular studies in the laboratory. GSO, MK, REM, LS, LK, ELF and AJC developed and conducted the epidemiological and statistical analysis. GSO, MK, REM, TGE, LS, WM, DAC, ELF and AJC drafted the preliminary manuscript. DVN, LK, GCES, KC, SP made substantive revisions to the preliminary version. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/111/prepub


A synchronism exists between the respiratory and cardiac cycles. However, the influence of the inspiratory muscle weakness in chronic obstructive pulmonary disease (COPD) on cardiac autonomic control is unknown. The purpose of the present investigation was to evaluate the influence of respiratory muscle strength on autonomic control in these patients.1/FVC 59±12% and FEV1 41±11% predicted) and nine age-matched healthy volunteers (64±5 years) participated in this study. Heart-rate variability (HRV) was obtained at rest and during respiratory sinusal arrhythmia maneuver (RSA-M) by electrocardiograph.Ten chronic obstructive pulmonary disease patients . Moreover, significant and positive correlations between maximal inspiratory pressure (MIP) and the inspiratory-expiratory difference (ΔIE)  were found.Patients with chronic obstructive pulmonary disease presented impaired sympathetic-vagal balance at rest. In addition, cardiac autonomic control of heart rate was associated with inspiratory muscle weakness in chronic obstructive pulmonary disease. Based on this evidence, future research applications of respiratory muscle training may bring to light a potentially valuable target for rehabilitation. The maximal inspiratory pressure (MIP) generated by the inspiratory musculature is an index of strength that can be diminished in chronic obstructive pulmonary disease (COPD)7Alterations of the inspiratory muscle length-tension relationship in patients with COPD increase the sympathetic vasoconstrictor activity of the cardiovascular adjustment center in the bulb secondary to an altered ergoreflex response (muscle spindles and Golgi bodies) in the diaphragm.11COPD has been shown to lead to inspiratory muscle weakness that, in turn, could contribute to a negatively altered cardiovascular response during the respiratory cycle. Previous research has demonstrated that variations in the respiratory rate and tidal volume can modulate the magnitude of respiratory sinus arrhythmia, which is known as an important marker of vagal tone.Given this research gap, the objective of the present study was to evaluate the influence of respiratory muscle strength on the magnitude of respiratory sinus arrhythmia. We hypothesized that respiratory muscle weakness negatively influences HR variability during respiratory maneuvers in patients with COPD.The present study was cross-sectional. The study population comprised 10 males with a clinical diagnosis of COPD according to the Global Initiative for Obstructive Lung Disease criteria (GOLD) and nine age- and gender-matched sedentary control subjects. Inclusion criteria for COPD patients were: forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) ratio < 0.7 and FEV1 < 60% of predicted value; clinical stability for at least three months; nonsmoker status; and presentation of dyspnea under low and medium physical effort. All COPD patients used short-action bronchodilators, and six used long-action bronchodilators. Subjects in the control group were free of chronic pulmonary, cardiovascular, immune, and/or metabolic disease. All participants underwent a clinical evaluation and were screened with pulmonary function tests, functional capacity assessment according to the medical research council (MRC),2-agonists, xanthene derivates, and steroids for 24 hours before the experimental test. Prior to the initiation of the tests included in the present study, the participants were interviewed (to ensure compliance with medication instructions) and examined. Systolic and diastolic blood pressure, pulmonary auscultation, and peripheral oxygen saturation (SpO2) were all assessed. Only one data collection session was required for each subject.The volunteers were first familiarized with the experimental room environment and the researchers involved in the study. The COPD patients were instructed to avoid bronchodilators such as B® spirometer . Slow vital capacity (SVC) and forced vital capacity (FVC) were measured to determine FEV1 and the FEV1/FVC ratio. Pulmonary function tests were performed in accordance with American Thoracic Society guidelines.14Spirometric tests were performed using the Vitalograph2O) . Patients were asked to perform maximal inspiratory and expiratory efforts against an obstructed mouthpiece that had a small leak to prevent the patients from closing their glottis during the maneuver. Patients sustained their maximal effort for one second. The best of three consecutive attempts was used to determine MIP, and percent-predicted values were derived.15The MIP was obtained from the residual volume and total lung capacity. Additionally, the maximal expiratory pressure was obtained from a deep inspiration following a maximal expiration. The subjects were seated, wearing nose clips and a mouthpiece. The mouthpiece was connected to a manual shutter apparatus, with maximal pressures measured by an aneroid-gauge manometer  in the supine position in the following order: (1) for one minute at rest with spontaneous breathing; (2) for four minutes while performing the RSA-M; and (3) for one minute at rest with spontaneous breathing. During the RSA-M, the volunteers were instructed to perform a series of deep and slow inspirations and expirations to provide a pulmonary volume that varied from the total lung capacity  to the residual volume . Each respiratory cycle was performed for 10 seconds, i.e., five seconds for inspiration and five seconds for expiration, which corresponds to a breathing rate of six cycles/min. This breathing rate provides a maximal respiratory sinus arrhythmia response, according to Hayano et al.17HRV was analyzed by time and frequency domain methods using an algorithm developed in MatLab . The section of highest stability, which included a simple line comprised of at least 256 points, was selected from R-R intervals by visual inspection according to the criteria set forth by the Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology.18Time domain analysis was calculated from the RMSSD index [the square root of the sum of the squares of the differences between adjacent normal-to-normal (NN) intervals] and the SDNN .18Time and frequency analysis of the R-R intervals acquired during RSA-M was then performed. Additionally, the spectral analysis confirmed that all volunteers maintained a respiratory rate between five and six cycles/min, which corresponds to a peak spectral density frequency between 0.08 to 0.1 Hz. Another specific routine developed in MatLab was used to calculate the HR and R-R interval indices of RSA-M,Finally, the data obtained by HRV analysis in the time and frequency domains as well as the HR and R-R interval indices of RSA-M were transformed into decimal logarithms for statistical analysis.2). Due to the normal distribution (Shapiro-Wilk test) and to the variance homogeneity (Levene Test) of the data, parametric tests were selected for statistical analysis. For the inter-group comparison (COPD Group vs. Control Group), unpaired Student’s t-test was applied. The Pearson correlation was used to observe the inter-variable relationship. All statistical analyses were carried out using SPSS version 10  with the level of significance set at p<0.05. All data are expressed as means and standard deviations.The sample size was calculated using GraphPad StatMate software, version 1.01. To reach statistical significance , a sample of nine subjects in each of the groups was needed to show differences in the physiologic variables , II (n=3), and III (n=6). As expected, MIP was significantly reduced in the COPD group in comparison to the control group (p<0.001).The subject characteristics are presented in The COPD patients presented a significant reduction in LF in comparison to the control group (p<0.05) . HoweverThe primary findings of the present study were as follows: 1) COPD patients showed evidence of impaired autonomic modulation of heart rate at rest and during RSA-M; 2) the relationship between the MIP and HRV indices during RSA-M indicates that the inspiratory muscle weakness observed in this population may be associated with cardiac autonomic control.It has been observed in many chronic disease populations that the sympathetic-vagal balance of the sinus node is impaired, favoring sympathetic tone. In COPD, the coexistence of airflow limitation and the loss of the lungs’ elastic recoil results in modifications to the breathing pattern and in the volume and capacity of the lungs. For this reason, a series of adjustments through the interaction between the peripheral-central receptors and the cardiopulmonary control center, especially in the autonomic nervous system, occur to guarantee the body’s homeostasis.21In the present study, the COPD group showed a greater reduction in LF values compared to the control group. However, the cardiac autonomic control in patients with COPD is unclear. Some authorsThe HR and R-R interval indices obtained during the RSA-M provided relevant information concerning the vagal activity at the sinus node in these patients. The COPD patients showed a reduced parasympathetic response when compared to the matched control group. The results of the present study reaffirm previous findings demonstrating significant RSA-M reduction in COPDOur evaluation of the relationships between MIP and RSA-M indices and between MIP and HRV indices indicated that subjects with respiratory muscle weakness exhibited a more pronounced reduction of vagal tone at the sinus node. Therefore, alterations of respiratory parameters can profoundly influence RSA-M magnitude and HRV behavior; slow and deep breathing will amplify RSA-M magnitude and HRV behavior, whereas fast and shallow breathing may contribute to reduced RSA-M magnitude and HRV behavior.Recent studies have shown that respiratory muscle strength could affect the HRV and the RSA-M magnitude in COPD patients with respiratory muscle weakness.The present study does possess limitations. First, measurement and control of the tidal volume during respiratory control, which was not carried out in this study, could have contributed to the consolidation and interpretation of the results found herein. Second, the evaluation of complete lung function (static volumes) would be especially relevant when evaluating these patient populations. These additional measurements would have involved the use of prohibitively expensive equipment and could therefore not be included.These results could be important for identifying maladjustment in cardiac modulation and suggest the importance of rehabilitative strategies such as respiratory muscle training for patients with COPD who present significant respiratory muscle weakness. It is well documented that respiratory muscle training significantly improves pulmonary function in patients with COPD.3243 Demonstration of improved autonomic tone through respiratory muscle training would further bolster support for the implementation of this therapeutic intervention. Future research is required to clarify these issues.It is presently unknown whether an improvement in respiratory function equates to improved cardiac autonomic tone in these patient populations.


While Blood vessel epicardial substance (Bves) confers adhesive properties, the molecular mechanism of regulating this activity is unknown. No predicted functional motifs in this highly conserved integral membrane protein, other than the transmembrane domain, have been identified. Here, we report for the first time that Bves interacts with itself through an intracellular interaction domain that is essential for its intercellular adhesion activity. Glutathion-S-transferase (GST) pull-down and SPOTs analyses mapped this domain to amino acids 268-274 in the intracellular C-terminus. Site-directed mutagenesis revealed that lysines 272 and 273 are essential for homodimerization and cell adhesion. Human corneal cells transfected with wild-type Bves trafficked the protein to the cell surface, assembled junction complexes and formed epithelial sheets. In contrast, cells expressing Bves mutated at these positions did not form continuous epithelial sheets or maintain junctional proteins such as ZO-1 and E-cadherin at the membrane. A dramatic reduction in transepithelial electrical resistance was also observed indicating a functional loss of tight junctions. Importantly, expression of mutated Bves in epithelial cells promoted the transformation of cells from an epithelial to a mesenchymal phenotype. This study is the first to demonstrate the essential nature of any domain within Bves for maintenance of epithelial phenotype and function. This includes a short extracellular N-terminus with two invariant glycosylation sites, three transmembrane domains with two intervening loops and a long intracellular C-terminus Bves has the canonical structure of all predominant popdc1-null mice do not show an overt embryonic phenotype, presumably due to redundancy of expression with popdc2 and popdc3 genes, regeneration of skeletal muscle is delayed due to an inhibition of cell-cell adhesion/interaction Drosophila development results in disruption of pole cell migration X. laevis is severely restricted due to failure in epithelial morphogenesis Phenotypic analyses of this gene family are only now emerging. Due to its subcellular localization and trafficking to points of cell-cell contact during epithelial sheet formation 272 and K273) that are critical for homophilic binding. While transfection of wild type Bves promotes cell aggregation in L-cell assays, mutation or deletion of K272 and K273 abolishes this activity. Expression of these mutated transcripts dominantly interferes with normal Bves function in human corneal epithelial cells (HCE) resulting in loss of cell-cell adhesion, junction formation, TER and epithelial sheet integrity. Importantly, expression of mutated Bves leads to a change of cells from an epithelial to mesenchymal phenotype. This study is the first to identify a specific molecular mechanism by which Bves regulates cell-cell adhesion and to demonstrate that mutation of these sequences inhibits cellular functions attributed to this molecule.Here for the first time, we report a Bves-Bves molecular interaction through its intracellular C-terminus that is essential for molecular regulation of cell-cell adhesion. This domain lies within the highly conserved Popeye region of the molecule, which heretofore has no ascribed function. Further we identify two amino acids in this sequence . As seen in We next determined whether expression of mutated Bves molecules would disrupt native adhesive properties in epithelia using a human corneal epithelial cell line (HCE). Multiple stable HCE cell lines expressing WT Bves, KK-Mut Bves and KK-Del Bves were isolated and produced consistent results. As seen in Using HCE cells as a model, we have shown that transfected WT Bves is properly trafficked to and maintained at the cell membrane merge in . It shouTo detect transfected protein and endogenous Bves, cells were stained with anti-Flag and anti-Bves antibodies. Transfected WT Bves is detected at the cell membrane in these lines, and in areas of confluence, staining of both antibodies is observed around the entirety of cells. Intracellular staining of transfected and endogenous protein were also observed , panel JTo determine whether mutations of Bves at positions 272 and 273 disrupt junctional adhesive complexes, cultures were assayed for the distribution of known junctional proteins. As seen in 272 and K273 on tight junction activity, HCE cell lines stably transfected with WT Bves, KK-Mut Bves and KK-Del Bves were grown to confluence and assayed for TER ] and KK-Del Bves  are significantly reduced compared to the control parental group, while TER for WT Bves is significantly greater than those of the control parental group . Taken together, these data suggest the expression of Bves with mutation in the putative intracellular interaction domain produced marked disruption of epithelial structure and function.TER is a standard measurement of epithelial function and tight junction integrity. In order to determine the effect of mutation or substitution of K for TER . TransfeAlteration of cell-cell adhesion/interaction can lead to changes in cell phenotypes, most notably through processes involving epithelial-mesenchymal transition popdc gene family of which Bves is a prototypical member has great implications for development and disease. Still, the molecular mechanisms underlying this function are completely unknown. Additionally, no protein motif or domain has been identified to account for any activity of this gene family. Here for the first time, we demonstrate Bves-Bves interaction and identify a functional domain in the Bves molecule that regulates this process. Further, we show that mutation or deletion of specific amino acids within this domain abolishes the cell-cell adhesion mediated by the molecule and leads to predictable changes in cell phenotype. These novel studies identifying Bves-Bves interaction and its regulation have larger implications for the understanding of Bves function in development and disease.Bves is a highly conserved transmembrane protein with cell-cell adhesion function that is expressed in a variety of epithelia and muscle from flies to humans. Previous studies suggest that the function of the popdc family are highly conserved, neither Bves nor any other family member harbors a predicted protein motif that would account for its cell-cell adhesive function While members of the Expression of mutated Bves does not promote cell clustering in L cells in contrast to expression of WT Bves controls . Also, eAdditionally, immunofluorescence analysis of HCE cells expressing transfected WT Bves detects the molecule at the cell surface along with components of the adherens and tight junctions  and exprA key finding in the present study is that cells expressing mutated Bves exhibit an altered phenotype. Many, but not all, epithelial cells expressing this molecule take on varying morphologies and express vimentin, a marker of the mesenchymal phenotype 272 and K273 of Flag-tagged Bves were produced by a sequential PCR method using primers listed in Specific regions of the Bves molecule were cloned using Polymerase Chain Reaction (PCR) strategies for biochemical and cellular analysis. Primers are listed in COS-7 cells  and L-cell  were cultured in Doulbecco's Modification of Eagle's Medium  with 4mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose and 1.0 mM sodium pyruvate supplemented with 10% fetal bovine serum  and 10 µg/ml Penicillin-Streptomycin solution (Mediatech) Human corneal epithelial cells (HCE) were originally obtained from Dr. K. Araki-Sasaki , maintained as previously described GST N-terminal, GST C-terminal Bves or GST proteins were prepared using standard methods To more precisely identify the Bves-Bves interaction region in the C-terminus, a SPOTs blot membrane was generated by SIGMA-GENOSIS . Peptides were designed from the sequence of the C-terminus of Bves after the third hydrophobic membrane-spanning region. Synthesized 13-mer peptides (starting from aa 232) were covalently fixed to a cellulose membrane with ten overlapping amino acids between neighboring SPOTs . The 20 Identification of amino acid(s) critical for Bves-Bves interaction was performed on a second SPOTs membrane. On this SPOTs blot, each single amino acid of Peptide 12: TLNDKASKKIDRQ was individually substituted with L-alanine beginning with N-terminal amino acid and progressing to the C-terminal of the peptide; synthesized 13-mer peptides were fixed on a cellulose membrane. Blocking reaction, washing and development with WT Bves were identical to the protocol described above.Cellular adhesion activity of non-transfected control and stable L-cell lines transfected with Bves constructs  was compared in standard hanging drop suspension cultures Immunofluorescent analysis of protein expression and distribution was similar to previously published studies 4 cells/cm2) were seeded on a six well transwell chamber  and cultured for two weeks. TER was measured using an Epithelial volt-ohm-meter (EVOM) . TER was calculated as follows: TER = (Reading of EVOM - reading of blank)×surface area of the membrane (4.2 cm2). The data are log-transformed to stabilize variances. Analysis of variance was applied to test for overall difference, followed by Dunnett's method to compare the three experimental groups to the parental group while controlling the family-wise type I error rate at 5%. Simultaneous 95% confidence intervals of the group mean ratios were obtained using Dunnett's method. The reported p-values and confidence intervals are adjusted for multiple comparisons To examine tight junction activity in confluent cultures, the TER was measured. Non-transfected and transfected HCE cells were used in this study. Cells (2×10


Though recent advancement in proteomics has provided a novel perspective on several distinct pathogenetic mechanisms leading to preterm birth , the etiology of most preterm births still remains elusive. We conducted a multidimensional proteomic analysis of the amniotic fluid to identify pathways related to preterm birth in the absence of inflammation or bleeding.Q-profile) based on the presence of 5 SELDI peaks in the 10–12.5 kDa mass area. Women displaying the Q-profile  were more likely to deliver preterm despite expectant management in the context of intact membranes and normal amniotic fluid clinical results. Utilizing identification-centered proteomics techniques  coupled with Protein ANalysis THrough Evolutionary Relationships (PANTHER) ontological classifications, we determined that in amniotic fluids with Q-profile the differentially expressed proteins are primarily involved in non-inflammatory biological processes such as protein metabolism, signal transduction and transport.A proteomic fingerprint was generated from fresh amniotic fluid using surface-enhanced laser desorbtion ionization time of flight (SELDI-TOF) mass spectrometry in a total of 286 consecutive samples retrieved from women who presented with signs or symptoms of preterm labor or preterm premature rupture of the membranes. Inflammation and/or bleeding proteomic patterns were detected in 32% (92/286) of the SELDI tracings. In the remaining tracings, a hierarchical algorithm was applied based on descriptors quantifying similarity/dissimilarity among proteomic fingerprints. This allowed identification of a novel profile (Proteomic profiling of amniotic fluid coupled with non-hierarchical bioinformatics algorithms identified a subgroup of patients at risk for preterm birth in the absence of intra-amniotic inflammation or bleeding, suggesting a novel pathogenetic pathway leading to preterm birth. The altered proteins may offer opportunities for therapeutical intervention and future drug development to prevent prematurity. Prematurity remains the leading cause of early neonatal deaths and long term neuro-developmental handicap In prior studies we concentrated our attention on investigating the changes in amniotic fluid proteome in the context of inflammation and decidual hemorrhage induced-preterm birth The transition from the past era of hypothesis-driven research to that of a discovery-determined one stemmed from opportunities presented by the contemporaneous revolution in biomedical sciences such as the one recently witnessed in proteomics We conducted a prospective study that included 285 women recruited following admission to the Labor and Birth unit or High Risk antepartum units at Yale New Haven Hospital (YNHH). Symptoms of preterm labor, preterm premature rupture of membranes (PPROM), advanced cervical dilatation (≥3 centimeters) and/or uterine contractions unresponsive to tocolysis were considered clinical indications for ultrasound-guided amniocentesis to rule-out infection. Women were enrolled consecutively based on a protocol approved by the Human Investigation Committee of Yale University. Written informed consent for the use of biological samples was obtained prior to the procedure. All women in this study had a singleton fetus at <37 weeks gestational age. Exclusion criteria included: viral infections (human immunodeficiency virus or hepatitis), anhydramnios and presence of fetal heart rate abnormalities  requiring emergency intervention. Gestational age was established based on a second trimester ultrasonographic examination prior to 20 weeks in all instances. We defined preterm labor as presence of regular uterine contractions accompanied by cervical effacement and/or dilatation in patients <37 weeks gestational age. The diagnosis of PPROM was confirmed by vaginal amniotic fluid “pooling”, “nitrazine”, “ferning” or by an amniocentesis-dye positive test. Clinical chorioamnionitis was suspected in the presence of either fever >38°Celsius (C), uterine tenderness or fetal tachycardia. The decision to perform an amniocentesis and patient management including the decision to deliver the fetus were at the discretion of the clinical team independent of our study protocol. Patients received steroids for enhancement of fetal lung maturity if gestational age was between 24 and 32 weeks. Antibiotic therapy was initiated as per the usual PPROM protocol  Ureaplasma and Mycoplasma species by the YNHH microbiology laboratory. The clinical laboratory tests performed for the purpose of diagnosing inflammation/infection were: glucose, lactate dehydrogenase (LDH), Gram stain, white (WBC) and red blood cell (RBC) counts. Research samples were spun at 3000×g at 4°C for 20 min., aliquoted and stored at –80°C for further study.Following retrieval, amniotic fluid was cultured for aerobic and anaerobic bacteria, We used surface-enhanced laser desorbtion ionization time-of-flight (SELDI-TOF) mass spectrometry to screen for the presence of biomarkers characteristic of inflammation and bleeding in fresh samples of amniotic fluid. The results were not made available to the clinical management team. The methodology for generation of the MR score and identification of bleeding biomarkers has been previously described Q-profile  and compared the results with samples of amniotic fluid retrieved from women who presented with symptoms of preterm labor but delivered at term . 2D-DIGE was performed using the Ettan DIGE system . Amniotic fluid proteins (67 µg) were labeled with either Cy3 (Q-profile) or Cy5 (CRL). A reference pool  was labeled with Cy2 and used as internal control. For each pair, labeled samples were pooled and isoelectric focusing was performed using an isoelectric point range of 3–10. SDS-PAGE on a 12% gel was performed for the second dimension. After spot detection, automatic background correction, spot volume normalization and volume ratio calculation, dye ratios were determined using DeCyder software . Spots corresponding to ≥1.5-fold changes To clarify and expand the biological significance of the http://www.pantherdb.org) Differentially expressed proteins were classified based on the PANTHER  system  and MMP-8  were performed in duplicate by investigators unaware of clinical outcomes. Amniotic fluid samples were diluted to allow for appropriate interpolation of the results. The minimal detectable concentration for IL-6 was 1-picogram/milliliter and less than 0.02-nanogram/milliliter for MMP-8. The inter- and intra-assay coefficients of variation were <10% for IL-6 and <6% for MMP-8, respectively.p value of <0.05 was considered significant.Data is presented as mean and standard deviation (SD) or, if non-normally distributed, as median and range. Statistical analyses were performed with Sigma Stat, version 2.03 . Data was compared with one-way ANOVA followed by Student Newman Keuls tests (parametric) or Kruskal-Wallis on ranks followed by Dunn's tests (non-parametric), to adjust for multiple comparisons as appropriate. Comparisons between proportions were done with Chi-square or Fisher's exact tests. A Q-profile)  had either evidence of intra-amniotic inflammation , bleeding  or both (n = 15) . Applicaprofile)  in 32 paQ-profile we analyzed the cases that exclusively displayed this profile (n = 32) in comparison to four additional groups: those displaying the inflammation fingerprint alone (n = 64), the bleeding fingerprint alone (n = 6), the combined inflammation and bleeding profile (n = 14) and those displaying no proteomic profile who delivered at term (n = 33). In Q-profile or the inflammation profile were at an earlier gestational age at time of amniocentesis compared to the reference group , but there were no differences among the 4 groups that displayed any proteomic profile. Women with the Q-profile presented more often with intact membranes compared to the inflammation and/or bleeding groups. Among the groups with abnormal proteomic profiles, there was no significant difference in gestational age at birth. However, a significantly higher proportion of women displaying the bleeding and/or inflammation profile, underwent an indicated preterm delivery subsequent to amniocentesis results. Therefore, despite their expectant management, 84% (27/32) of the women with the still Q-profile delivered prematurely. The amniocentesis-to-delivery interval was longer for women displaying the Q-profile as compared to those displaying the proteomic fingerprint characteristic for inflammation and/or bleeding. Moreover, newborns in the inflammation groups (with or without bleeding), but not those with the Q-profile had significantly lower Apgar scores compared to all other groups.The cases displaying any type of amniotic fluid proteomic fingerprint (n = 124) had a significantly higher frequency of preterm delivery (88%) compared to the rest . To further understand the biological significance of the Q-profile group is characterized by amniotic fluid results similar to the group with no profile and delivery at term.8 All women displaying the Q-profile had negative Gram stain, negative culture results and normal levels of inflammatory cytokines. This was determined to be in sharp contrast to the women who had the inflammation profile present (with or without bleeding) who had significantly lower glucose and elevated LDH activity, WBC, RBC counts and elevated levels of IL-6 and MMP-8. The incidence of a positive Gram stain and/or amniotic fluid culture result was also significantly higher in the inflammation groups. The bleeding alone group was characterized by elevated LDH activity levels in the context of a higher RBC (but not WBC) count, and normal IL-6 and MMP-8 levels.In Q-profile had virtually no evidence of chorioamnionitis, amnionitis or funisitis, despite delivering preterm. Lastly, bleeding was more frequently confirmed histologically in the context of bleeding-induced preterm birth (with and without inflammation).The results of the pathologic examination of the placenta were available in 101 of the cases in the 5 groups . OverallQ-profile present most often with intact membranes and are managed expectantly in the context of normal amniotic fluid results. Yet, this group has a significantly higher risk for preterm birth which typically occurs in the context of normal placental histology. These findings concur with the concept that the Q-profile is representative of an amniotic fluid proteomic fingerprint which is different from that of the women with intra-amniotic inflammation or bleeding. No clinical or biochemical tests other than proteomic profiling of the amniotic fluid was able to delineate this high-risk group of women.Based on these results we conclude that women displaying the amniotic fluid Q-profile and reference samples amniotic fluid samples . Interestingly, more spots (n = 60) appeared down-regulated in the amniotic fluids displaying the Q-profile rather than up-regulated (n = 28). Of the spots picked for identification, 22 matched to a protein in the database with more than one peptide. Few spots matched to the same NCBI GenID transcript number. In fact, a total of 17 distinct protein database matches appeared and were differentially regulated at least 1.5-fold (Q-profile are characterized by up-regulation in insulin growth factor binding protein (IGFBP-1), apolipoproteins (APO A-I and APO A-IV), lumican and bikunin  and down-regulation in the anti-proteolytic factors alpha-1-antitrypsin (SERPINA1), alpha-2-antiplasmin   .Employing multidimensional proteomics analysis of amniotic fluid coupled with bioinformatic algorithms we were able to identify a novel subgroup of patients at risk for preterm birth that share common clinical, biochemical and proteomic features. These cases were distinct from those characterized by intra-amniotic inflammation or bleeding. The findings of the present study further support the notion that preterm birth is the result of several distinct pathogenetic pathways for which increased uterine contractility, cervical ripening and/or PPROM are simply common clinical features of the end product.Q-profile). By exploring the common clinical characteristics of this subgroup we determined that these women presented most often with preterm labor and intact membranes in the context of normal amniotic fluid clinical and microbiological results. However, the majority of cases in this subgroup delivered prematurely despite tocolytic and expectant management per standard protocols. To our knowledge the Q-profile is the first proteomic pattern with the ability to identify women at risk for preterm birth in the absence of intra-amniotic inflammation and bleeding.Proteomics encompasses protein separation and identification techniques along with the necessary bioinformatic tools that allow for biomarker discovery. Two current and rather opposing proteomic approaches have emerged. One relies on generating proteomic patterns from biological samples using high-throughput mass spectrometry approaches while minimizing the need to know the identity of the discriminatory biomarkers (proteomic pattern diagnostic approach) Q-profile in the context of a preterm delivery. In the current study we have determined that the differentially expressed proteins which characterize the Q-profile are primarily involved in protein metabolism, signal transduction and transport. The identities of the proteins matched in the database pointed to a metabolic rather than inflammatory abnormality since none of the protein identities had apparent inflammatory function.With few exceptions, the current stage of medical practice emphasizes disease classification based on shared clinical features which together are representative of a syndrome. Assignment of individual cases to these diagnostic categories frequently dictates treatment choices. However, while in clinical instances such as tumors or infectious etiologies the medical treatment is further conditioned based on strict histopathologic or microbiologic examinations, obstetrical practice lags seriously behind in this respect. Perhaps, our failure to recognize and prevent preterm birth rests with the inability to rapidly and accurately discern among the several distinct pathogenetic mechanisms leading to premature delivery. Personalized medicine, stands at the opposite end of this “one size fits all” approach and relies heavily on development of classification algorithms based on subclinical features and biomarkers APO A-I and APO A-IV are associated with high density lipoprotein (HDL) and chylomicrons and have important roles in cholesterol, trygliceride and phospholipid transport and metabolism in vitro model Popovici et al demonstrated that hypoxia up-regulates fetal hepatocyte IGFBP-1 mRNA steady-state levels and protein, which is the major IGFBP derived from the fetal hepatocytes Q-profile was lumican, a member of the small leucine-rich proteoglycan family (SLRP) with important roles in embryonic development, tissue repair, tumor growth, organization of collagens IGFBP-1 is the predominant insulin-like growth factor binding protein in amniotic fluid in vivoBikunin  is a Kunitz-type serin-protease inhibitor predominantly found in amniotic fluid and to a lesser degree in urine and serum where it circulates bound to the inter-alpha inhibitor proteins Q-profile  were members of serpin superfamily. The roles of these factors in amniotic fluid are not well understood. PEDF is known to be the most potent inhibitor of angiogenesis in the mammalian ocular compartment All proteins found differentially down-regulated in amniotic fluid expressing the Q-profile. Furthermore as SELDI and 2D-DIGE technologies are not equivalent, the biomarker peaks comprising the Q profile may not be part of the differentially expressed identities. Yet the SELDI diagnostic pattern itself proved essential in isolating a subgroup of preterm birth cases with unique features at the level of their amniotic fluid proteome. This will perhaps prompt a future research direction and paradigm into the prevention of prematurity.The present study demonstrates how diagnostic pattern proteomics can be used in conjunction with identification-centered proteomic techniques and bioinformatic tools to provide insight into a problem which has frustrated clinicians for decades. We acknowledge that at this time due to the limitation of the current technology we have not been able to identify the specific proteins responsible for the five SELDI peaks of the