One hallmark feature of consolidation of episodic memory is that only a fractionof original information, which is usually in a more abstract form, is selectedfor long-term memory storage. How does the brain perform these differentialmemory consolidations? To investigate the neural network mechanism that governsthis selective consolidation process, we use a set of distinct fearful events tostudy if and how hippocampal CA1 cells engage in selective memory encoding andconsolidation. We show that these distinct episodes activate a unique assemblyof CA1 episodic cells, or neural cliques, whose response-selectivity ranges fromgeneral-to-specific features. A series of parametric analyses further revealthat post-learning CA1 episodic pattern replays or reverberations are mostlymediated by cells exhibiting event intensity-invariant responses, not by theintensity-sensitive cells. More importantly, reactivation cross-correlationsdisplayed by intensity-invariant cells encoding general episodic features duringimmediate post-learning period tend to be stronger than those displayed byinvariant cells encoding specific features. These differential reactivationswithin the CA1 episodic cell populations can thus provide the hippocampus with aselection mechanism to consolidate preferentially more generalized knowledge forlong-term memory storage. The hippocampus plays a crucial role in converting recent episodic events intolong-lasting memories, a process termed memory consolidation Since the hippocampus is well known for its crucial role in converting an episodicmemory from short-term into its long-term form, it is of great interest to useepisodic memory paradigms for the identification of memory traces in its networksTo our knowledge, however, there is no report aimed at addressing the followingimportant question: how and why does the hippocampus only convert a fraction oforiginal information into long-term memory? In the present study, we set out toinvestigate how the distinct cell populations in the CA1 region of the hippocampusmay engage themselves during the post-learning consolidation of episodicexperiences. We used a set of fearful episodic events, coupled with large-scaleneural ensemble recording methods To investigate CA1 neural activity patterns during and after learning period, weexposed naive mice to four types of the fearful episodic events: 1) A suddendrop of animal in a small elevator (Elevator-Drop); 2) A sudden air-blow toanimal's back (Air-Blow); 3) A sudden earthquake imitated by shaking theanimal in its cage via vortex machine (Quake); and 4) A startling acoustic sound(Sound). Using a 96- or 128-channel Plexon neural data acquisition system, werecorded bilaterally from the dorsal region of hippocampus using a microdrivewith adjustable stereotrodes as described previously To efficiently deal with the large datasets, we employed hierarchical clusteringanalysis to examine the firing responsiveness of those units among all of therecorded mice. Our analysis shows that each specific event is represented by aset of CA1 cell assemblies, or neural cliques, that respond with a range ofselectivity, from the general response to all four events to the specificresponse to a single event . In the While many CA1 cells changed their firing rates in response to external inputs orexperiences, it is not clear to what degree the activations of hippocampal cellsby such episodes reflect memory encoding or merely represent sensory inputs. Ourhypothesis is that the neural responsiveness should reflect changes in inputintensities (intensity-sensitive neurons) if it is mere representation ofsensory inputs. On the other hand, some neurons in CA1 may exhibit fairly equalfiring responses despite changes in event intensities (Intensity-invariantneurons) because they may be more geared towards the encoding of invariantimportant features.Thus, we conducted a series of parametric experiments and varied the level ofintensity of the two episodic events (drop and air blow). We set the droppingheight of at 5 cm, 13 cm and 30 cm, respectively, for the drop; adjusted airflowat 200 ms, 400 ms and 800 ms, respectively, for the air blow. Our parametricexperiments reveal the existence of two major types of responsive cell groups inCA1, namely, intensity-sensitive responsive neurons and intensity-invariantresponsive neurons. The input-sensitive group contains units that eitherincrease or decrease their firing rates in a monotonic fashion with changes inthe amount of stimulus inputs responded to various startling stimuli. Of them, 284 units belong to theintensity-invariant cell group and 299 units exhibit intensity-sensitive changesof their firing rates, close to 1∶1 distribution ratio. We have furtheranalyzed the interaction between the intensity-sensitivity categories vs.event-response selectivity categories. We found that percentages of invariantneurons that belong to general to specific categories are: 54% for thegeneral-responsive category , 26% for the sub-general responsive category (75/284), and20% for the specific event-encoding cells (57/284). Similarly, thepercentages of intensity-sensitive neurons that belong to general, subgeneraland specific modulated neurons are 45% (134/299), 25% (75/299) and30% (89/299), respectively .To seek a statistical description of ensemble neural activity patterns from therecorded large datasets, we employed Multiple Discriminant Analysis (MDA) whichhas been shown to be an effective method for statistical pattern classificationsof large neural data collected from well defined event categories To dynamically monitor the population firing patterns, we applied asliding-window technique to the MDA method which enabled us to directlyvisualize the real-time network-level memory encoding dynamics By scanning through the recorded CA1 neural activities in the post-event period,we observed that these transient encoding patterns triggered by startlingstimuli reactivated spontaneously as indicated by dynamical trajectories withsimilar geometric shapes but at smaller amplitudes . These rTo ensure that our MDA analysis truly captured the CA1 encoding traces as well asreactivation traces, we carried out a set of control and validation tests.First, we determined the class membership for these test data points, which wasbased on the proximity to the clusters corresponding to the training datapoints. For example as shown in As expected, when the neural activities collected during events were shuffledamong different units, our cluster structure collapsed see . As a reBecause of the statistical classification that was used, the directionality andshape of the trajectories in MDA subspaces now provide a highly valuable meansto compare and visualize the encoding patterns with any other random eventsThe second method we used for shuffling reactivation data was to switch theidentity of the neural units randomly during this putative reactivation. Asillustrated in In addition, we further analyzed the relationship between event intensity and theamplitudes of reactivation traces. Interestingly, we have noticed that themagnitude of ensemble reactivations tends to remain more or less constantregardless the original event intensity. For example, we saw that high drop andlow drop events produced similar magnitudes of reactivation trajectories of theepisodic stimuli .To further confirm and explore the nature of the pattern reactivation, we appliedtwo widely used methods, namely, pair-wise correlations Second, we also applied the Explained Variance analysis While these two cross-correlation based methods confirmed our MDA observation onthe preferential reactivations by intensity-invariant cell population, weperformed two additional tests: 1) We compared the cross-correlations betweenthe time points at which MDA analysis detected putative reactivations (during a2 sec before and 2 sec after the reactivation event) vs. the post-event timepoints at which trajectories were absent; and, 2) We shuffled the spike dataspecifically associated with transient reactivations of MDA trajectories. In thefirst test, it is expected that pair-wise correlations between cells should bemore similar between the events and the subsequent “reactivations”time points. In contrast, these correlations should become much less similarduring randomly chosen time periods of similar length chosen from thenon-reactivation time points during the same post-event period. Indeed, ouranalysis showed that correlations within the invariant cells during thepost-event non-reactivation periods showed little increase . FurtherSince we showed earlier that intensity-modulated cells can be divided into a setof subpopulation based on their response selectivity to multiple events or aspecific event, we further investigated the levels of firing correlations amongthe intensity-modulated cells encoding general, subgeneral, and specificfeatures during the reactivation periods . As expeThe trends observed for the correlations among intensity-invariant neurons aswell as among intensity-sensitive neurons are consistently manifest in the restof our data sets. More specifically, using pooled data from 7 data sets, weshowed that despite the increase in correlations for all groups ofintensity-sensitive neurons from basal levels  to eventFurther analyses of various subclasses of the invariant units suggest that thedegree of correlation is strongest for the general-responsive units incomparison to the sub-general cells and the specific neurons ; Again,A hallmark feature of long-term memory consolidation is that only a portion oforiginal information about various episodic events becomes long-term memory. Suchinformation stored in the domain of long-term memory tends to be more general andabstract, and many specific details seem to be no longer available. Despite the manyemerging studies which explore consolidation mechanisms at the molecular level By taking advantage of recent large-scale recording and decoding methods Our study on startling episodic events fits well with the reports that episodichippocampal cells can undergo reactivations immediately after such events To provide moment-to-moment decoding of post-event real-time ensemble activitypatterns, we have employed MDA/sliding window method to monitor and detect thetemporal evolution of CA1 ensemble patterns during and after the startling episodicevents. This dimensionality-reduction method has proven to be highly useful forintuitively visualizing the real-time transient dynamics and patterns associatedwith learning tasks Reactivations patterns of episodic experiences revealed by MDA/Sliding-window methodwere further corroborated by both pair-wise cross-correlations, and explainedvariance. In addition, we have found that preferential reactivations of distinctsubgroups of intensity-invariant neuronal population seem to be responsible forensemble pattern reactivations. More importantly, our analysis further revealed thatduring the reactivation time periods invariant, cells encoding general features ofall or multiple events seem to be more correlated the cells encoding for specificevents. This preferential reactivation mechanism may position the hippocampus towardextraction of generalized features from individual experiences of these events forlong-term consolidation and storage. On the other hand, relatively weakcross-correlation among specific invariant cells may be a contributing factor inexplaining why details of a given event tend to be more difficult to retain. Anothercontributing factor may be that such specific cells were detected in smallerpercentages in our experimental data. This suggests that these cells may be prone toweakening of synapses over time during storage. This hypothesis needs to be testedin future experiments.Our finding of preferential reactivations of cells encoding general and subgeneralfeatures provides a strong experimental validation for the computational modelingwork which simulates that the finer distinctions of specific events or knowledge maybe more easily lost than the more general ones during the consolidation stage Furthermore, our present study reveals a novel aspect of hippocampal consolidationprinciples unparalleled to those implied by the previous studies of place cellreplays. Here, we show that hippocampal reactivations of episodic cell ensemblespatterns are more than mere ‘imitation’ or ‘repetition’ oforiginal patterns. In other words, the preferential and stronger reactivations ofthe invariant cells encoding general features enables the hippocampus to extractthese major features of a given episodic experience and subsequently integrate theminto the brain's general knowledge structure and semantic memory. Thisexplanation may explain why patients with damaged hippocampi have great difficultyin forming episodic memories as well as concepts and general knowledge about theseevents.Cognitively, episodic memory refers to memory of episodic events, and it is the majortype of memory we encode in our daily life. In contrast, semantic memory refers tomemory of facts and knowledge that are no longer ascribable to a particular occasionin life (without necessarily remembering where and when a person acquires it) In summary, our present study describes the subclassification properties among CA1episodic cell assemblies encoding robust episodic events, and reveals their varyingdegrees of dynamic participation in post-event pattern reverberation andconsolidation. Specifically, the event-intensity invariant CA1 neurons are largelyresponsible for the post-learning pattern reactivations. Moreover, during thesetransient reactivation periods, intensity-invariant cells encoding general featurestend to exhibit stronger cross-correlations than do those cliques which encodespecific features. Such post-event preferential reactivation of thesegeneral/subgeneral cell cliques may provide a key neuronal population-levelmechanism for achieving the consolidation and storage of general information andknowledge in the brain.All animal work described in the study were carried out in accordance with theguidelines established by the National Institutes of Health in the US regardingthe care and use of animals for experimental procedures, and was approved by theMCG Institutional Animal Care and Use Committee at Georgia Health SciencesUniversity .We employed 96 and 128-channel recording arrays to record from the hippocampalregion of freely behaving mice We subsequently recorded the ensemble activity from a large number of individualneurons during freely behaving states. The recorded spike activities from thoseneurons were processed in the manner as previously described Principal component analysis was used to extract defining features from the spikewave shapes that were then used as part of the input for the MClust3.3 spikesorting program. The first two principal components, as well as the peak height,valley value, FFT and total energy of spike waveform parameters were calculatedfor each channel, and units were identified and isolated in high-dimensionalspace through the use of an autoclustering method (KlustaKwik 1.5) We exposed mice to four types of robust episodic events: 1) a short and loudacoustic startle , 2) A sudden air-blow to theanimal's back ; 3) A sudden drop of the animalinside a small elevator ; and 4)A sudden shake-like cage oscillation . Tomaintain the consistency of stimulus inputs and yet minimize possible predictionof upcoming stimuli, the stimuli were triggered using a computer and deliveredfor seven times at randomized intervals within a few minutes. We previouslyshowed that seven repetitions are sufficient for obtaining an adequate samplingof the neural responses, while minimizing the risk of habituation to the noxiousstimuli In our experiments we started recordings 30 minutes before a series of startlingepisodes were delivered to the mice. Each given type of startling event (e.g.Drop with a fixed height or Air puff with a fixed duration) was delivered in asingle session for seven times with pauses ranging from 1-to-3 minutes atrandomized intervals . A single event session lasted forabout 20 minutes and the mice were then brought back to home cage for a briefrest for 5-to-10 minutes. This was followed by a different session consisting ofeither different startling events or the same event but at a different intensity. All together, the mice would undergo three sessionsof the same events (say Drop events with three chosen heights), plus twoadditional and distinct event sessions (e.g. Airpuff and Shake). Our typicalexperiments thus consisted of five event sessions which lasted for about 2.5 to3 hours. The randomized inter-trial-intervals (1–3 minutes) were intendedto minimize possible habituation and reduce the animal's ability to predictits upcoming event. We recorded population activity patterns in the CA1 regionof the hippocampus from seven freely-behaving mice that were subjected to thefollowing set of episodic stimuli: acoustic startling sound, cage shake,air-blow and elevator drop.startle if and 0frepresent average frequency responses during startles of type i orrest states, and 0g is the average population activityduring rest states. We maintained only the units which have high scores based onthis metric. Note that this transformation allows for uniform quantification ofthe significant changes in firing patterns for units with both low- and highbaseline firing rates. More precisely, changes in responses of low-firing unitsare proportional to absolute firing rate changes (since f0 ≪g0), while response differences for the high-baseline unitsbecome proportional to the relative changes from the baseline activities (sincef0 ≫ g0).For our data analysis we selected only clearly separated single units thatremained stable throughout the duration of the whole experiment. To select theresponsive units, we first evaluated the changes in the firing frequencies intime bins of 500 ms immediately after the event start. The width of the timebins used here is appropriate to characterize the overall frequency changesafter a startle event. To facilitate comparison between neurons that exhibitdifferent increases/decreases over baseline activities, we used thetransformationSimilar to our previous research We then used Multiple Discriminant Analysis (MDA) projection methods to classifythe neural responses corresponding to different episodes into different classesin is thenumber of elements in each class,N is the number of classes, im is themean vector for each class, m is the global mean vector and thesymbol t indicate the transpose operator. To take into account the variationsoccurring for each class we also computed the within-class scatter matrixWS, which is defined as:iD represents the set ofpopulation responses triggered by the ith startle type.Using these two matrices, it follows that a set of at most N-1discriminant projection vectors can be determined by computing the eigenvaluedecomposition of the matrixTo account for transient changes that may occur immediately after the startleevents, we computed firing frequencies (f) in two 500 ms time bins immediatelyafter the delivery of the stimuli. Baseline activities were characterized bycomputing the average firing rates during time intervals preceding the startlestimuli. We set aside randomly chosen population activities from one of eachtype of startle stimuli; this constitutes our test data set. The rest of thesampled population activities were then used to train our MDA statistical model.The matrix of mean responses during each category (rest and startle states) werethen computed and used to compute the between-class scatter matrixWS werenon-invertible, which is a direct consequence of data under-sampling, since thenumber of recorded neurons is much higher than the number of repeated trials. Inpractice, the matrix WS can be rendered invertible usinga regularization technique which changes each class covariance matrices based onthe following formula: i'  = (1 - λ) Ωi + λ IΩ, whereiΩ is the covariance matrix for theith class, λ is a regularizationparameter between 0 and 1, and I is the identity matrix. Wedetermine the parameter λ automatically for each data set basedon the optimization procedure we developed previously; each particular choice isdetermined by the particular distributions within each data set For our data sets, the class covariance matrices N – 1 discriminant dimensions iscomputed, we projected the neural patterns during startle episodes in thislow-dimensional encoding subspaces. We then used the multivariate Gaussiandistribution probability functions . To ensure that noisy correlation values are not included in thedata sets, we set a low threshold (e. g. 0.05 or 0.1) and excluded them from theanalysis.To rule out the possibility that correlation results can be attributed to randomchanges in the neural population, we again used data sets where the spikeactivities were shuffled among neural units at all times during the experiment.Not surprisingly, all correlations among all neural units decrease to valuesclose to zero in this case.We used the measure of Explained Variance (EV)


Nehalennia irene. As fitness-related physiological traits, we investigated investment in immune function (phenoloxidase activity), energy storage and fecundity (abdomen protein and lipid content), and flight muscles (thorax protein content). In the first part of the study, our aim was to identify selective agents maintaining the large-scale spatial variation in morph frequencies. Morph frequencies varied considerably among populations, but, in contrast to expectation, in a geographically unstructured way. Furthermore, frequencies co-varied only weakly with the numerous investigated ecological parameters. This suggests that spatial frequency patterns are driven by stochastic processes, or alternatively, are consequence of highly variable and currently unidentified ecological conditions. In line with this, the investigated ecological parameters did not affect the fitness-related physiological traits differently in both morphs. In the second part of the study, we aimed at identifying trade-offs between fitness-related physiological traits that may contribute to the local maintenance of both colour morphs by defining alternative phenotypic optima, and test the spatial consistency of such trade-off patterns. The female morph with higher levels of phenoloxidase activity had a lower thorax protein content, and vice versa, suggesting a trade-off between investments in immune function and in flight muscles. This physiological trade-off was consistent across the geographical scale studied and supports widespread correlational selection, possibly driven by male harassment, favouring alternative trait combinations in both female morphs.The contribution of adaptive mechanisms in maintaining genetic polymorphisms is still debated in many systems. To understand the contribution of selective factors in maintaining polymorphism, we investigated large-scale (>1000 km) geographic variation in morph frequencies and fitness-related physiological traits in the damselfly  Polymorphisms are attractive model systems for understanding fundamental processes related to the origin and maintenance of genetic variation Littorina obtusata, shell colour morph frequencies changed gradually in accordance with environmental temperature regimes within and between estuaries Tito albaMegalagrion calliphyaAgouti mice Timema cristinae show spatial morph frequency variation related to the presence of host plants and the survival of a given morph is highest at the host plant where its frequency is highest Many polymorphisms show large-scale geographic variation in morph frequencies While spatially varying selection may contribute to the maintenance of different morphs at large spatial scales, alternative fitness optima linked to trade-offs between fitness-related traits may underlie local coexistence of different morphs A classic and much debated example of intra-specific polymorphism can be found in many species of damselflies Nehalennia irene, a species with large geographical variation in morph frequencies In the current study, we used the polymorphic damselfly In a first part of the study, our aim is to identify selective agents maintaining the large spatial variation in morph frequencies. If a female morph relative to the other, is favoured in local populations that differ in ecological parameters, we then expect (1) morph frequencies to co-vary with these ecological parameters N. irene is a small non-territorial damselfly , which inhabits marshy or boggy waters and is common throughout most of Canada and the Northern parts of the United States Nehalennia irene has one generation per year, with the winged adult life stage and reproduction typically occurring between early June and mid-August. After locating a potential mate, a male will attempt to grasp the female in the so-called tandem formation, where the male attaches his anal appendages to the female's prothorax The sedge sprite et al.Frequencies and densities of males and female morphs were determined at 58 populations during the reproductive seasons of 2009 and 2010. This was done along a linear and continuous 1100 kilometre transect in the central to Eastern part of the species' distribution range, in Ontario and Quebec, Canada . Along tet al.N. irene netted was noted. We aimed to catch at least 30 females at each site (mean ± SE = 62±3). This allowed calculating six population parameters either based on matures or matures plus immature Frequencies and densities of males and female morphs were determined at each site as described by Van Gossum http://www.climate.weatheroffice.ec.gc.ca). The average distance between our 89 study populations and the closest weather station was 16.8±1.0 km. Long-term annual and seasonal  climate normals were derived from the weather stations closest to each site. These climate normals are the arithmetic averages of weather elements over the 30-year interval (1971–2000). Our extracted dataset includes annual and seasonal temperature  and precipitation averaged over this thirty-year period. To obtain short-term weather data we derived seasonal mean precipitation and mean, minimum and maximum temperature, during the preceding year (starting with the summer of 2009). Additionally, on a very short-term time scale we derived daily precipitation, and mean, minimum and maximum temperatures for four periods: the day of capture, the day of capture plus the preceding day, the preceding two days and the preceding three days.Given that female morph frequencies and physiological parameters (see further) can be influenced by long-term as well as short-term weather conditions To quantify physiological parameters, individuals were collected at a subset of eight sites along our transect . We seleWe studied three key physiological parameters related to investments in immune function (phenoloxidase (PO) activity), in energy storage and fecundity (abdomen lipid and protein content) and in flight muscles (thorax protein content) that are strong candidates to affect fitness in damselflies et al.N. irene. Specifically, the thorax was homogenised using a hand-held pistil and 0.3 ml cacodylate buffer was added . The cell walls were removed via centrifugation . Each well of a 96-well microtiterplate was filled with 100 µl sample supernatant, 35 µl PBS buffer, 5 µl chymotripsine (5 mg/ml) and after five minutes 60 µl L-Dopa . The reaction proceeded for 30 minutes at 30°C. Readings were taken every 10 seconds on a temperature-controlled microplate reader at 490 nm. Enzyme activity was measured as the slope during the linear phase of the reaction when the enzyme catalyses the transition from L-DOPA to dopachrome.To quantify PO activity, we closely followed the protocol by Stoks Protein content was quantified separately in thorax (mainly flight muscles) and abdomen (mainly eggs) using the Bradford method 2SO4 and was incubated for 15 minutes at 200°C. Then 2 ml milli-Q-water was added and subsequently 200 µl of each sample was read at 405 nm on a microplate reader. Concentrations were calculated from standard curves of tripalmitine . Thorax lipids could not be quantified because all homogenised thorax sample was used to measure PO activity and protein content.Abdomen lipid content was assayed using the protocol described in Bligh & Dyer All physiological parameters were assayed twice per individual and the mean of both readings was used in all further analyses  are considered to have equal explanatory value. In such cases, the variable that best corresponded with the CART analyses was selected as best predictor. CART and PLS both selected the same best predictors in our dataset  and weather parameters . As expected, several of these parameters were strongly correlated and may therefore not be included together in one regression model, because of multi-collinearity problems aset see . TherefoThe ecological variables that could best explain variation in morph frequencies were also used to analyse variation in the physiological parameters. Therefore, we performed ANCOVA models, including morph, mate status and the selected ecological variables, plus interactions with morph. Source population was treated as a random variable. To correct for individual and morph-specific differences in size, protein and lipid contents were divided by wing length Considerable spatial variation in the frequency of andromorph females was identified ranging from 0.0 to 62.7% .The CART analysis put forward adult male density  as most important variable for explaining the spatial variation in morph frequencies, followed by maximum spring temperature normals (11.0°C). These results exactly correspond with the selected predictors of the PLS analysis see . OverallAndromorphs had significantly, ca. 10% higher PO activity levels compared to gynomorphs . The revOur results indicate large spatial variation in morph frequencies in a geographical rather unstructured way, which could be explained only weakly by numerous ecological parameters. These ecological parameters did not affect physiological fitness-related traits of both female morphs in contrasting ways. However, we showed a geographically consistent morph-specific trade-off between investment in immune function and in flight muscles. In what follows, we will discuss the above findings in a broader evolutionary context.Ischnura elegansMegalagrion calliphyaN. irene, Wong et al. I. senegalensisI. elegans at a large spatial scale I. elegans' distribution area N. irene could in part be explained by such stochastic effects While most of the investigated ecological parameters showed spatial autocorrelation, this pattern was not present in female morph frequencies. Moreover, and contrary to our expectations, we found generally little proof for weather and population parameters being important in explaining biogeographic patterns in female morph frequencies. Our results may therefore indicate the importance of stochastic effects causing spatial frequency fluctuations. Alternatively, female morphs may be adapted to highly variable and currently unidentified small-scale environmental conditions. The latter alternative explanation may seem less likely as we included most of the known weather and population parameters likely to differentially affect the colour morphs, although we acknowledge that some factors may be overlooked. Considerable variation in morph frequencies in a spatially structured manner has been reported in taxonomically diverse systems, such as birds 2 = 0.04; see Nonetheless, and in accordance with a number of studies, we found, admittedly weak (REnallagma cyathigerum were differentially affected by short-term ambient temperature in both morphs To point at selection maintaining spatial variation in the polymorphism, relative fitness components of the morphs should also differ in relation to the supposed selective agent Uta stansburiana: Podarcis muralis: Anolis sagrei: N. irene gynomorphs display more refusal behaviour when they are the majority morph I. elegans, female morphs differ in behavioural tactics in order to escape from excessive male harassment With regard to trade-offs between fitness-related physiological traits contributing to maintaining the polymorphism at a local scale, our physiological data suggest a morph-specific and geographically consistent trade-off where andromorphs, relative to gynomorphs, tend to invest more in immune function and less in flight muscles. Because traits related to immune function and flight muscles are costly to produce and maintain Whatever the underlying mechanism, our data and other studies suggest correlational selection favouring successful and alternative trait combinations among female morphs Despite many studies, the mechanisms underlying female polymorphism in damselflies are still under debate and this study area urgently needs to open its perspective and accept multiple non-exclusive explanations


Early diagnosis of Oral Squamous Cell Carcinoma (OSCC) increases the survival rate of oral cancer. For early diagnosis, molecular biomarkers contained in samples collected non-invasively and directly from at-risk oral premalignant lesions (OPMLs) would be ideal.In this pilot study we evaluated the potential of a novel method using commercial PerioPaper absorbent strips for non-invasive collection of oral lesion exudate material coupled with mass spectrometry-based proteomics for oral cancer biomarker discovery.Our evaluation focused on three core issues. First, using an "on-strip" processing method, we found that protein can be isolated from exudate samples in amounts compatible with large-scale mass spectrometry-based proteomic analysis. Second, we found that the OPML exudate proteome was distinct from that of whole saliva, while being similar to the OPML epithelial cell proteome, demonstrating the fidelity of our exudate collection method. Third, in a proof-of-principle study, we identified numerous, inflammation-associated proteins showing an expected increase in abundance in OPML exudates compared to healthy oral tissue exudates. These results demonstrate the feasibility of identifying differentially abundant proteins from exudate samples, which is essential for biomarker discovery studies.Collectively, our findings demonstrate that our exudate collection method coupled with mass spectrometry-based proteomics has great potential for transforming OSCC biomarker discovery and clinical diagnostics assay development. Oral cancer occurs most commonly (~90%) in the form of oral squamous cell carcinoma (OSCC) and develops in stages starting with healthy oral epithelium progressing to an Oral Pre-Malignant Lesion (OPML) and on to OSCC. The survival rate of OSCC has remained static over the last 30 years at about 50%. However, where malignancy is detected soon after the transition from OPML, treatments are more effective and survival is as high as 80% . DespiteAn ideal alternative to scalpel biopsy would be a non-invasively collected sample rich in molecular biomarkers which distinguish OPML and OSCC, and potentially predict the transition from pre-malignancy to malignancy. One such alternative is the use of protein or nucleic acid biomarkers in saliva that are secreted or shed from the oral lesions ,6. HowevGiven the ongoing need for improved oral cancer detection, we describe here a promising alternative method for the direct and non-invasive sampling of oral lesions, which can be coupled with mass spectrometry (MS)-based proteomics. Our method uses commercially available PerioPaper Strips, traditionally used for oral fluid sampling relevant to periodontal disease ,11, to dExudates and brush biopsies were collected from three patients diagnosed with a dysplastic OPML, at the University of Minnesota Dental School. For each OPML patient, exudate samples were first collected from the oral lesion, followed by collection of the brush biopsy from the same lesion. Exudates and brush biopsies from buccal mucosa, and whole saliva were also collected from three healthy volunteers. The healthy volunteers had no major risk factors for OSCC  and were free of oral lesions. All samples were collected with written consent using an IRB protocol approved by the University of Minnesota. Three different lesion and three healthy samples were analyzed to provide some statistical significance for measurements of differential protein abundance between tissue types while balancing the time and cost of large-scale proteomic analysis of individual patient samples.To collect the exudate we first used rolled cotton to swab away ambient saliva around tissue to be sampled (e.g. OPML). The rolled cotton was then moved adjacent to the area to be sampled to block flow of additional saliva onto the tissue. A PerioPaper strip  was left in position for ~30 seconds. Immediately after collection the strip was placed in a microcentrifuge tube, on ice, and then transferred to a -20°freezer within minutes. The PerioPaper strips were subjected to on-strip trypsin digestion in which each PerioPaper strip containing exudate was submerged in 100 μl buffer containing 5 mM DTT, 100 mM Tris pH 8.0 and boiled for 5 min. After cooling to room temperature, 2 μg of sequencing grade trypsin  was added and the microcentrifuge tube was placed in 37°C water bath to digest for 12 hours. The protein digest was then purified and concentrated using Waters Sep-Pak 3cc cartridges as described  drying tTo collect brush biopsies we first dried the tissue to be sampled as with exudate collection. Next, we collected transepithelial cells from the tissue using an OralCDx brush test kit  and following manufacturer's suggested procedure. After collection of cells, the brush head was cut off from the handle and submerged in 250 μL of 2× SDS cell lysis buffer  and 1× protease inhibitors  in a 2 mL microcentrifuge tube. In order to extract proteins from the cell lysate while removing detergents and minimizing other impurities, the proteins were precipitated with acetone added at a 5:1 ratio and left overnight at -20°C. Precipitated protein was centrifuged at 6000 rpm for 10 min at 4°C, then rinsed and re-centrifuged with pure acetone twice. Proteins were redissolved in trypsin digestion buffer, quantified using the BCA protein assay , and digested with trypsin as described above for the exudate samples.To collect the PerioPaper saliva samples we placed the PerioPaper strip at a location in a healthy volunteer's oral cavity where ambient saliva had pooled (the back of the lower lip). The strip was allowed to saturate with saliva (< 20 sec) before being removed. Once collected the PerioPaper saliva samples were immediately placed on ice. On-strip digestion, as described above, was implemented within several minutes of sample collection.Peptide digests from exudates, brush biopsies, and the whole saliva samples were subjected to offline SCX HPLC fractionation essentially as in previous studies . A UV chThe overall workflow used for data analysis is shown in Additional File Mascot output .dat files were subjected to statistical validation and protein inference using Scaffold Q+ v 3.0 . For peptide identification the false discovery rate threshold was maintained at 1%. For quantification using spectral counts, total spectra identified in a dataset were normalized with spectra identified in dataset that was to be compared. This normalization, which is achieved by using a display option called "Quantitative value" in Scaffold v 3.0, is used to determine relative abundance of proteins within datasets.Relative abundance levels of identified proteins in healthy and OPML exudates were determined via normalized spectral counting , using tOur objective was to determine whether exudate collection from oral lesions coupled with MS-based shotgun proteomics is a viable option for oral cancer biomarker discovery.To achieve our objective three fundamental questions needed to be answered: 1) Are non-invasively collected tissue exudates compatible with MS-based shotgun proteomics? 2) What is the composition and extent of contamination by saliva of the exudate proteome? 3) Can the differential abundance of protein within exudates collected from different tissue  be measured?To answer the first question, we initially explored methods for isolating intact proteins from the PerioPaper strip. For these experiments, we used representative exudate samples collected from healthy oral tissue using the PerioPaper strip as described in Experimental Methods and shown in Figure We next employed offline SCX HPLC peptide fractionation  in orderWe then moved on to answering our second question: What is the composition of the OPML proteome and extent of contamination by whole saliva? One initial concern was that the exudate strip would simply absorb saliva, despite our attempts to remove excess ambient salivary fluid from the tissue prior to exudate collection. The high abundant salivary proteins would potentially obscure the identification of lesion-associated proteins. To explore this issue, we compared the protein composition of exudates collected from three different OPML patients, to the composition of whole saliva collected from three different individuals. For this comparison, each whole saliva sample was collected and processed in a similar manner to the exudates, by absorbing saliva onto a PerioPaper strip, followed by on-strip digestion, SCX HPLC fractionation and LC-MS/MS analysis.We first compared all proteins identified from three saliva samples to all proteins identified from the OPML exudates Figure . These rTo further elucidate the composition of the exudate proteome, we compared it to the proteome derived from the epithelial cells collected from the OPMLs. Here, we collected OPML cells via brush biopsy  from the same three patients that we collected exudates, and analyzed the isolated protein using shotgun proteomics. The proteins identified from the three brush biopsy samples were then compared to the proteins identified from the three exudate samples Figure . The resFinally, we sought to answer the question of whether we could measure differential abundance of proteins within exudates collected from different tissue types. Here we decided to compare two distinct tissue types: healthy oral tissue and OPML tissue. Our objective in these experiments was to provide proof-of-principle for conducting quantitative proteomic studies in exudate samples, rather than discover new biomarker candidates. Therefore we focused on expected protein abundance differences within the samples compared that could serve as a benchmark to determine whether we could reliably measure differential protein abundance in exudate samples. Based on prior studies of OPML and similar inflammatory epithelial lesions -19, therIn this study, we first addressed the fundamental question of whether exudates collected via PerioPaper strips can be analyzed using MS-based proteomics, as new sample types such as these may not contain ample protein amounts to facilitate their analysis using MS. Although isolating intact proteins from the strips proved difficult, our on-strip digestion method liberated ample amounts of peptides for large-scale shotgun proteomic analysis.We next investigated the composition of the OPML exudate proteome. One concern was absorbance of ambient saliva, and the high abundance proteins contained therein which may obscure the identification of lower abundance exudate proteins. However, comparison of exudate samples to whole saliva samples revealed that high abundance salivary proteins were greatly decreased in their relative amounts within the exudate samples. Thus, our exudate collection procedure sufficiently removes ambient saliva, which should enable identification of proteins sampled directly from the lesion tissue.Interestingly, the exudate proteome was highly similar to the proteome of epithelial cells collected directly from the lesions via brush biopsy. This indicated that the PerioPaper strip also absorbs cells from the surface of the lesion, whose protein contents can be detected using our processing method. The presence of cellular proteins is in keeping with the accepted definition of an exudate, which is a mixture of fluid, cells and cellular debris on the surface of inflamed tissue . Thus, eFinally we investigated the feasibility of quantitative proteomic analysis of exudate samples collected from different tissue types. By comparing exudates from healthy oral tissue to OPML tissue we expected to identify inflammation-related proteins at increased abundance in the lesion exudates. Indeed this was the case, demonstrated by the proteins listed in Table We demonstrate here a promising new method for the non-invasive, direct sampling of oral lesions, compatible with MS-based proteomics. In future studies, we envision application of this method to comparative analysis of OPMLs and malignant oral lesions. This should provide a powerful means to identify protein biomarkers distinguishing these lesion types that may be useful for early detection of malignant transformation. Given our findings, lesion exudates should be amenable to the full suite of proteomic analysis tools, including those aimed at identifying post-translational modifications or sequence variants  that mayThe authors declare that they have no competing interests.JAK: Conceived experiments, carried out analysis of exudate samples, analyzed and interpreted data, wrote the manuscript. NLR: Conceived experiments, collected samples. CT: carried out analysis of brush biopsy samples, analyzed data. PDJ: Conceived data analysis workflow, analyzed and interpreted data.BJH: carried out collection and analysis of saliva samples. TJG: Conceived experiments, analyzed and interpreted data, wrote the manuscript. All authors read and approved the final manuscript.Workflow for MS-based proteomic analysis.Click here for fileProteins with significant differential abundance in healthy tissue versus OPML.Click here for file


To present the state-of-the-art of subthreshold diode laser micropulse photocoagulation (SDM) as invisible retinal phototherapy for diabetic macular edema (DME).To review the role and evolution of retinal laser treatment for DME.Thermal laser retinal photocoagulation has been the cornerstone of treatment for diabetic macular edema for over four decades. Throughout, laser induced retinal damage produced by conventional photocoagulation has been universally accepted as necessary to produce a therapeutic benefit, despite the inherent risks, adverse effects and limitations of thermally destructive treatment. Recently, SDM, performed as invisible retinal phototherapy for DME, has been found to be effective in the absence of any retinal damage or adverse effect, fundamentally altering our understanding of laser treatment for retinal disease.The discovery of clinically effective and harmless SDM treatment for DME offers exciting new information that will improve our understanding of laser treatment for retinal disease, expand treatment indications, and improve patient outcomes. Information theory states that the amount of information available from a particular event is directly proportional to the unlikelihood of that event occurring . It can We will examine this invisible retinal phototherapy, currently epitomized by subthreshold diode micropulse (SDM) laser treatment, in the treatment of diabetic macular edema (DME). DME is the most common cause of visual loss in persons under 50 years of age in the developed world. Diabetes mellitus (DM), the cause of diabetic retinopathy and thus DME, is increasing in incidence and prevalence worldwide, becoming epidemic not only in the developed world, but in the developing world as well. Diabetic retinopathy may begin to appear in persons with type I (insulin-dependent) DM within 3 – 5 years of disease onset. By 20 years, nearly 100% will have some degree of diabetic retinopathy. While the retinal complications of diabetes can be attenuated by long-term intensive glycemic control, the prevalence of diabetic retinopathy increases with duration of disease. By 10 years, between 14 – 25% of patients will have DME -13. UntrUntil the advent of thermal retinal photocoagulation there was no generally effective treatment for diabetic retinopathy. Using photocoagulation to produce photothermal retinal burns as a therapeutic maneuver was prompted by the observation that the complications of diabetic retinopathy were often less severe in eyes with preexisting retinal scarring from other causes. Photocoagulation, an effective means of producing retinal scars, was initially directed at treatment of proliferative diabetic retinopathy, using first the xenon arc photocoagulator, followed by ruby pulsed-laser and argon continuous-wave lasers (CWL). Spurred by success treating proliferative diabetic retinopathy, thermal laser photocoagulation was soon adapted to treatment of DME as well , 15. The Early Treatment of Diabetic Retinopathy Study (ETDRS) demonstrated the efficacy of argon laser macular photocoagulation in the treatment of DME. While a small percentage of treated eyes enjoyed improvement in visual acuity (VA) following treatment, the main clinical benefit of macular photocoagulation was to reduce by 50% the rate of moderate visual loss (≥ 15 letters measured on a logarithmic VA chart) in eyes with mild to moderate nonproliferative diabetic retinopathy and “clinically significant” DME .B) Although providing a clear advantage compared to no treatment, the inherent retinal damage and inflammation associated with suprathreshold macular photocoagulation  was associated with a number of significant risks and adverse effects. These include early and late visual loss due to inadvertent foveal photocoagulation, incitement or aggravation of macular edema (ME) due to treatment-associated inflammation, pre- and sub-retinal fibrosis, choroidal neovascularization, visual field loss, loss of color vision, metamorphopsia, and progressive expansion of the laser scars into the fovea. Laser-induced retinal damage limited treatment density and repeatability. The ability to treat safely near the foveal center, the location of the most visually disabling form of DME, was limited as well [The method of laser treatment employed in the (ETDRS) came to set the technical standard for macular photocoagulation for DME. Full-thickness retinal laser burns in the areas of retinal pathology were created, visible at the time of treatment as white or light grey retinal lesions . With time, these lesions developed into focal areas of chorioretinal scarring and progressive atrophy.  leakage correlated poorly with macular thickening, VA, and treatment response; and that DME often responded to photocoagulation placed in a low-density grid-fashion throughout the area of macular swelling without direct laser treatment of macular microaneurysms. In recognition, there began steady movement toward reducing the intensity of the treatment endpoint. Longer laser wavelengths, such as the 647 nm krypton red and the 810 nm diode laser, not absorbed by macular chromophores, were employed to minimize damage to the neurosensory retina. Low-density grid-pattern applications of macular photocoagulations with less emphasis on higher-intensity focal photocoagulation of microaneurysms was increasingly employed and found to be effective -33. HoweIn 1990, Pankratov reported development of a new laser modality designed to deliver laser energy in short pulses (“micropulses”) rather than as a continuous wave. Retinal lesion sizes produced by experimental thermal retinal photocoagulation with continuous wave (CW) and with pulsed laser energy were compared. For a same retinal spot size, pulse energy and pulse duration, the main determinant of retinal thermal lesion size from micropulsed laser photocoagulation was found to be the duty cycle (the frequency of the train of micropulses) and thus the length of the thermal relaxation time in between consecutive pulses. The lower the duty cycle and the longer the OFF time between pulses (lower repetition rate), the less heat build-up, resulting in less thermal retinal damage and smaller retinal laser lesions. By contrast, the higher the duty cycle, the more lesions produced by the micropulsed diode laser approximated those produced with a conventional CW laser. Using a longer wavelength near-infrared 810 nm diode laser, energy absorption by and thermal diffusion to the neurosensory retina could be minimized. By micropulsing the 810nm diode laser and reducing the frequency of laser micropulses by extending the “off-time” between micropulses (thus lowering the “duty cycle”) within the exposure envelope, higher laser energies and photothermal effects could be applied selectively to the retinal pigment epithelium (RPE), increasingly understood to be the source of potent extracellular factors acting as disease mediators, with less thermal retinal damage ,35. Beginning with Friberg in 1997 , a numbePrecisely, “subthreshold” retinal photocoagulation is defined as retinal laser applications biomicroscopically invisible at the time of treatment. Unfortunately, the term has often been used to describe several different clinical scenarios reflecting widely varying degrees of laser-induced thermal retinal damage . In geneD).“Classical” subthreshold photocoagulation describes the earliest attempts at laser intensity reduction using conventional CW argon, krypton, and diode lasers. As the retinal burns were notably less obvious than the white, full-thickness retinal burns produced according to ETDRS standards they were described as “subthreshold” or even “invisible” by comparison. Actually “threshold” (photocoagulation damage confined to the outer retina and thus less visible at the time of treatment) or even milder suprathreshold , the lesions of “classical” subthreshold photocoagulation were uniformly visible both clinically and by FFA at the time of treatment and thereafter , 36, 37 F).“Clinical” subthreshold photocoagulation describes the next step in the evolution of laser-induced retinal damage reduction. This describes lower-intensity but persistently damaging retinal photocoagulation using either a micropulsed laser or short-pulsed (10 – 30ms) CW laser that better confines damage to the outer retina and RPE. In “clinical” subthreshold photocoagulation laser lesions may in fact be ophthalmoscopically invisible at the time of treatment. However, as laser-induced retinal damage remains the intended endpoint of treatment, laser lesions are produced which generally become increasingly clinically visible with time, and many, if not all, laser lesions can be seen by FFA, fundus autofluorescence photography (FAF), and / or Spectral-domain (SD) optical coherence tomography (OCT) at the time of treatment and thereafter , 36 – 45K). “True” subthreshold photocoagulation expands the definition of “invisible by biomicroscopy” to include laser treatment non-discernable by any other known means such as FFA and newer high-resolution retinal imaging methods including FAF and SD-OCT. “True” subthreshold photocoagulation is therefore defined as laser treatment which produces absolutely no retinal damage detectable by any means at the time of treatment or anytime thereafter. Thus SDM, as a subtype of MPL, represents the fullest embodiment in the evolution of “subthreshold” laser treatment for retinal vascular disease .Inconsistency in use of the term “subthreshold” and transcendence of the original clinical definition by SDM suggests that newer descriptors such as “invisible retinal phototherapy” or “retinal photostimulation” may prove more meaningful and useful descriptors of SDM and future forms of truly invisible laser treatment for retinal disease  . In 2000 a pilot study was begun employing a new approach to laser treatment for retinal vascular disease. Using an 810nm diode laser in the micropulse emission mode, DME was treated for the first time with the intent of avoiding any laser-induced retinal damage (“low-intensity” treatment). Strict avoidance of laser-induced retinal injury facilitated a second fundamental change, that of complete and confluent treatment of all areas of macular thickening with numerous contiguous low-intensity laser applications (“high-density” treatment). No attempt was made to focally treat angiographically leaking microaneurysms  , 33, 47.In the absence of retinal damage, how might SDM work? First, in the absence of laser-induced retinal damage there is no loss of functional retinal tissue and no inflammatory response to treatment. Adverse treatment effects are thus completely eliminated and functional retina preserved rather than sacrificed. The tissue destruction and subsequent inflammation inherent in conventional photocoagulation may explain the superior VA results seen in studies using drug therapy alone vs. drug therapy combined with conventional laser for treatment of DME . Secondly, SDM spares the neurosensory retina and is selectively absorbed by the RPE. Current theories of the pathogenesis of retinal vascular disease especially implicate cytokines, potent extracellular vasoactive factors produced by the RPE, as important mediators of retinal vascular disease such as DME , 51. SDMThirdly, low power red and near-infrared laser exposure is known to affect many cell types, particularly altering the behavior of cells in pathologic environments (such as diabetes) through a variety of intracellular photoacceptors. Cell function, and thus cytokine expression, is normalized and inflammation reduced . This main-vitro studies of laser-tissue interactions, increasing irradiance may simply increase the risk of thermal retinal damage without improving the therapeutic effect [Fourthly, it has been noted that the clinical effects of cytokines may follow a “U – shaped curve”, where small physiologic changes in cytokine production (denoted by the left side of the curve) may have large clinical effects comparable to high-dose (pharmacologic) therapy (denoted by the right side of the curve) . Using sc effect , 49. 2). Laboratory studies suggest that the therapeutic alterations in RPE cytokine production elicited by conventional photocoagulation come from cells at the margins of traditional laser burns, affected but not killed by laser exposure [2A), the therapeutic effect of conventional argon laser retinal photocoagulation may derive from a ring of affected but surviving RPE cells at the margin of each retinal burn. By increasing burn intensity, the width of this therapeutic ring would enlarge , consistent with the observation that increased burn intensity is associated with an enhanced therapeutic effect, but hampered by increased loss of functional retina and inflammation. The converse would be expected with reduced intensity conventional argon laser photocoagulation.  This may explain the inferior clinical results from lower-intensity / lower-density (“mild”) argon laser grid photocoagulation compared to higher-intensity / higher-density mETDRS treatment for DME [2D) Thus, despite complete ablation of the directly treated RPE and outer retina, the rim of therapeutically affected and surviving tissue is scant. Recent reports finding superiority of conventional argon laser panretinal photocoagulation over PASCAL for diabetic retinopathy may reflect this minimization of the effectively treated retinal surface area by short-pulse CWL [2E) Permitted by the absence of tissue damage, SDM (low-intensity / high-density MP laser) is contiguously performed over all areas of retinal pathology (the “maximized effective surface area”), amplifying the therapeutic effect by maximizing therapeutic recruitment of the RPE  A recent randomized clinical trial by Lavinsky, et al. demonstrated this phenomenon, finding SDM superior to both conventional mETDRS photocoagulation and low-density MPL in reducing macular thickening and improving VA in DME [Finally, high-density SDM amplifies all of the above effects by maximizing therapeutic recruitment of the RPE through the concept of “maximized effective surface area” -6, 54 , strict and absolutely reliable avoidance of laser-induced retinal damage is imperative. It is equally imperative to perform clinically effective treatment. To this end we have three independent and corroborating sources that inform us regarding optimal SDM treatment parameters.Because of the high treatment density employed by SDM and the ability to safely treat to the edge of and through the foveal center using correct treatment parameters  absolute safety would be expected and retinal exposure to laser radiation at this level would be expected to have no biologic effect. Based on ANSI data, a 50% risk of suffering a barely visible (threshold) retinal burn is generally encountered at 10x MPE for conventional CW laser exposure. For a low-duty cycle MP laser exposure of the same power the risk of threshold retinal burn is approximately 100x MPE. Thus, the therapeutic range – the interval between doing nothing at all and the 50% likelihood of producing a threshold retinal burn – for low-duty cycle MP laser irradiation is 10 times wider than for CW laser irradiation with the same energy. Safe and effective SDM treatment has been reported at laser exposure levels as low as 18x and as high as 55x MPE [In the pilot study of SDM, it was noted that the American National Standards Institute (ANSI) has developed standards for safe work-place laser exposure based on a combination of theoretical and empirical data , 59 – 62 55x MPE . Thus, using the ANSI xMPE laser exposure model, the therapeutic window for SDM, within which effective treatment without thermal tissue injury would be expected, is broad . Laser eThe long-term safety of SDM has been reported examining the retinal burn risk determined by FAF and FFA in 252 eyes treated for DME and macular edema due to branch retinal vein occlusion followed as long as 10 years (median 47 months) postoperatively. Inadvertent retinal burns were noted in 7 eyes. All were all evident at the first postoperative visit and all burns occurred in eyes treated at a 10% or 15% duty cycle (P=0.0001). No laser-induced retina injury was found in any eye treated with 5% duty cycle, despite the use of equivalent laser exposure levels of approximately 50x MPE for both groups [F).In this same report, computational tissue temperature models were used to examine the risk of lethal cell injury at the SDM laser parameters used clinically. These calculations corroborated both clinical experience and ANSI MPE predictions. SDM performed with a 5% duty cycle demonstrated adequate thermal rise at the level of the RPE cell to stimulate a biologic response, but remained far below the level expected to produce lethal cell injury even in darkly pigmented fundi. In contrast, use of a 10% duty cycle adjusted to deliver MPL at similar irradiance and xMPE levels significantly increased the risk of lethal cell injury, particularly in darker fundi. Echoing extensive clinical experience, even a small increase in duty cycle from 5 to 10%, doubles the energy and reduces the molecular thermal relaxation time between micropulses, increasing in the risk of lethal thermal retinal injury 10-fold , 36-45  for SDM. The longer the laser exposure, the more important the center-spot heat dissipating facility toward the unexposed tissue at the margins of the laser spot and toward the underlying choriocapillaris, favoring use of small retinal laser spot sizes for SDM. This is illustrated by one study of use of a large-spot CW diode laser for subthreshold transpupillary thermotherapy treatment of DME which was abandoned following catastrophic visual loss in one study eye resulting from macular infarction. This was attributed to uneven heat distribution and insufficient heat dissipation within the large retinal laser spot . Synthesizing the above information we can derive rational and evidence-based recommendations for SDM laser parameters expected to be both clinically effective and strictly and reliably safe, despite absence of a visible treatment endpoint or laser-induced retinal damage. Key parameters appear to be the following: a) a low (5% or less) duty cycle; b) a small spot size to minimize heat accumulation, assure uniform heat distribution within a given laser spot, and maximize heat dissipation; c) sufficient power to produce retinal laser exposures of between approximately 18x – 55x MPE producing an RPE temperature rise of 7 – 14⁰C; and d) high-density treatment of the pathologic retina with contiguous laser spots to maximize the therapeutic benefit by maximizing the effectively treated area. These are the essential ingredients of “low-intensity / high-density” SDM as invisible retinal phototherapy for DME. The agreement between ANSI MPE predictions, computational tissue temperature modeling and extensive clinical observations thus provides rational bases and useful tools for evaluating invisible retinal phototherapy protocols .In another departure from conventional retinal photocoagulation, the wide therapeutic window of SDM and physical characteristics of the 810nm diode laser allow use of a single set of safe and effective laser parameters in all eyes with DME . (5) The 810nm diode laser is minimally absorbed and negligibly scattered by intraretinal blood, cataract, vitreous hemorrhage and even severely edematous neurosensory retina. Differences in fundus coloration result primarily from differences in choroid pigmentation, and less with variation in the target RPE. Treatment with SDM is thus simplified, requiring no adjustment in laser parameters for variations in macular thickening, intraretinal hemorrhage, media opacity or fundus pigmentation, reducing the risk of error.K). The parameters required for safe and effective SDM at wavelengths shorter than 810nm are currently under investigation.We caution that the preceding analysis of SDM dosimetry is based on extensive clinical experience using the 810nm diode laser. Micropulse emission has recently become available in lasers with shorter wavelengths, such as 577nm yellow and 532nm green. The higher energies and different tissue absorption characteristics of shorter wavelength lasers may increase retinal burn risk, effectively narrowing the therapeutic window. In addition, shorter 532 and 577 nm wavelengths are more scattered by opaque ocular media,, retinal hemorrhage and macular edema, potentially limiting usefulness and increasing the risk of retinal damage in certain clinical settings. Although these wavelengths are effective in the closure of microaneurysms, such focal treatment is generally unnecessary, requiring higher laser irradiances that increase the risk of thermal retinal damage and is not a strategy of SDM .With the advent of OCT and newer, higher resolution iterations such as SD-OCT, we are now able to detect the very earliest stages of DME when the macular thickening may be only a few microns. Unless constrained by treatment risks, we know that earlier treatment of chronic and progressive diseases generally improves clinical outcomes. Many patients with early DME may be asymptomatic, with excellent VA. The risks of conventional photocoagulation are generally unacceptable in such eyes. By the same token, while often effective short-term, injection of intraocular drugs is such eyes is not without risk, and thus problematic as well. Currently, treatment of “sub-clinically significant” DME would appear to be a niche uniquely suited to SDM. This constitutes the largest number of patients with diabetic macular disease, as eyes with more severe “clinically significant” DME constitute only a subgroup of eyes at the extremity of diabetic maculopathy. The unique safety prolife of SDM permits us to offer earlier treatment of DME, when it is more likely to prevent visual disability and irreversible visual loss to a greater number of patients  .Modern pharmacologic therapy, particularly use of intravitreal anti-VEGF inhibitors and anti-inflammatory drugs such as steroids, has been a boon to the management of DME and other retinal vascular disease. The benefits of drug therapy, particularly on VA, can be significant compared to conventional photocoagulation, particularly in the short-term. However, the long-term benefit from drug therapy compared to even conventional laser for DME is questionable , 50-52. The two most significant changes offered by invisible retinal phototherapy are the expansion of treatment indications permitted by the unique safety profile of SDM, and the new insights to be gained from the information that laser-induced retinal damage is not necessary to perform clinically efficacious retinal laser treatment for retinal vascular disease such as DME. With regard to the expansion of indications, we have discussed above the unique place SDM may have in treating sub-clinically significant DME diagnosed early, prior to symptomatic and irreversible visual loss, by new high-resolution imaging techniques such as SD-OCT. What of application to other retinal disorders? As of this writing SDM has been reported effective in the treatment of ME due to branch retinal vein occlusion, central serous chorioretinopathy, and proliferative diabetic retinopathy , 70-73. In the pilot study of SDM panretinal photocoagulation, SDM appeared to reduce the progression of severe non proliferative to proliferative diabetic retinopathy without complication or adverse effect. These observations await confirmation by other investigators. However, what might be the benefit of harmless early intervention to reduce the risk and rate of progression of diabetic retinopathy? What of application to other forms of retinal vascular disease? If low-power laser exposure reduces inflammation and improves the “health” of the RPE, might SDM reduce progression and the risk of visual loss due to age-related macular degeneration? , 74.in vitro and in vivo. SDM allows us to ask better, more informed questions, increasing the likelihood of better, more informative and useful answers. As stated at the outset, information theory indicates that the very unlikelihood of SDM as effective treatment for DME opens the door to vast new opportunities for improvement of our understanding of retinal disease and the effects of laser - retinal interactions. Characterization of the products and pathways of the various therapeutically advantageous cytokines and other factors and effects elicited by invisible retinal phototherapy may lead to new treatment strategies and pharmacologic therapies to improve our ability to manage and even prevent retinal disease in entirely new ways. Knowledge that we no longer need to destroy our study subjects – RPE cells – enhances our ability to examine, detect and characterize these laser-induced cellular changes both via cellular feedback mechanisms? Combining therapies can lead to inhibition (a net effect less than the sum of its parts), complementarity , or synergy . How can we avoid the former, and achieve the latter? While SDM produces clinical effects akin to pharmacologic therapy, the slower onset and lasting effect suggest that the effects of SDM, like conventional photocoagulation, are mediated by secondary laser-induced changes in RPE cellular function . ProgresFinally, the high-density treatment paradigm of SDM and use of small spot applications requires the application of many more laser spots than conventional photocoagulation. This is time consuming and can present a challenge to the busy clinician and the most cooperative patient. Automation is one answer to this problem. By virtue of its unique safety profile, SDM is ideally suited to incorporation in automated delivery systems and is an active area of development. SDM, as invisible retinal laser phototherapy, has been found to be effective in the treatment of DME. Upending nearly a half-century of universally held belief in the therapeutic necessity of laser-induced retinal damage, SDM offers exciting new information that will improve our understanding of retinal laser effects and the pathophysiology of retinal diseases, expand retinal laser treatment indications, ease case management and improve patient outcomes. A field in its infancy, the opportunities and possibilities for future research remain vast, exciting, and important.


Plant–pathogen interactions feature complex signaling exchanges between host and microbes that ultimately determine association outcomes. Plants deploy pattern recognition receptors to perceive pathogen-associated molecular patterns, mount pattern-triggered immunity (PTI), and fend off potential pathogens. In recent years an increasing number of defense-signaling components have been identified along with a mechanistic understanding of their regulation during immune responses. Post-translational modifications (PTMs) are now thought to play a crucial role in regulating defense signaling. In a bid to suppress PTI and infect their host, pathogens have evolved large repertoires of effectors that trigger susceptibility and allow colonization of host tissues. While great progress has been made in elucidating defense-signaling networks in plants and the activities of effectors in immune suppression, a critical gap exists in our understanding of effector mechanism-of-action. Given the importance of PTMs in the regulation of defense signaling, we will explore the question: how do effectors modify the post-translational status of host proteins and thus interfere with host processes required for immunity? We will consider how emerging proteomics-based experimental strategies may help us answer this important question and ultimately open the pathogens’ effector black box. Within their natural environment, plants are continuously challenged by a diverse array of pathogens such as viruses, bacteria, fungi, and oomycetes as well as nematodes and insects. In most cases, infection or disease is limited upon the perception of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) by pattern recognition receptors PRRs; . RecogniProtein post-translational modifications (PTMs) are ubiquitous in cell signaling networks and enable rapid alterations to the protein complement of the cell without need for new protein synthesis. In addition, PTMs provide enormous diversity to the proteome, allowing cells to respond with flexibility to a stimulus. PTMs regulate a wide array of processes within plants including growth, development, flowering, and defense . PhosphoIn a select few cases, plants are successfully invaded and colonized by microbes, which in turn can lead to the manifestation of disease. Disease development ultimately results from the perturbation of immune signaling networks, suppression of defense responses and modulation of metabolism in the host . PathogeFigures 1A,B).The last decade has seen the identification of pathogen encoded secreted proteins (effectors) that upon secretion, manipulate host processes, perturb signaling, and induce effector-triggered susceptibility ETS; . During In recent years great progress has been made in elucidating defense-signaling networks in plants and identifying those pathogen effectors that can suppress immunity. However, an understanding of the mechanism-of-action of most effectors remains elusive. Given the lack of models describing effector mode-of-action and the importance of PTMs in defense signaling, we will explore the question: how do effectors modify the post-translational status of host proteins and thus interfere with host processes required for immunity? In this context, we will aim to consider how emerging proteomics-based experimental strategies may help us answer this important question, and ultimately open the pathogens’ effector black box.Figure 1C) that help explain effector-triggered target modification. Both simplified models will be discussed and explored here and examples supporting both models are also provided (Table 1).Effector-triggered susceptibility is achieved through the interaction between a pathogen effector and its host target that eventually impinges on immune signaling. Mechanistically, this interaction results in a modification of the target and its cellular fate . The hosPseudomonas syringae pv. tomato DC3000 (Pst) effector AvrPtoB features an N-terminal kinase binding motif that aids binding to host receptor like kinases FLS2, BAK1, FEN, and CERK1 (Pst infection of tomato (Pst on Arabidopsis (Pst on both tomato and Arabidopsis (Pst infection where it is activated by the eukaryotic host factor cyclophilin (The first model (which we have named direct effector-triggered modification or DETM) assumes that a direct interaction between effector and target, combined with an enzymatic activity carried by the effector, ensures modification of the target. In recent years a number of plant pathogen effectors have been shown to modify host targets by direct interactions combined with a catalytic activity carried by the effector. The nd CERK1 . The Avrf tomato . AvrPtoBbidopsis . AvrPtoBbidopsis , providilophilin . Activatlophilin .Pst effector HopAI1 interacts with the Arabidopsis MAP kinases MPK3 and MPK6. During PTI, both MPK3 and MPK6 are activated by the phosphorylation of a threonine residue by upstream MAPKKs. HopAI1 phosphate lyase activity however, removes the phosphate group from these residues, preventing MPK3 and MPK6 activation by PAMP induced MAPKKs. Since phosphate group removal cannot be reversed, both MPK3 and MPK6 are effectively inhibited, leading to dampening of PTI activated MAPK signaling cascades . Examples of such mechanisms are sparse but one early report emanated from studies on the human papilloma virus oncoprotein E6. E6 was identified in oncogenic HPV strains and detailed studies of this protein led to the observation that in host cells, E6 recruits an E3 ubiquitin ligase that in turn, ubiquitinates the tumor suppressor protein p53. E6 induced p53 ubiquitination was found to mark the tumorigenesis suppressor for proteasomal degradation, providing a molecular explanation for the onset of cancer in infected cells .P. syringae effectors AvrB and AvrRpm1. Delivery of either AvrB or AvrRpm1 results in phosphorylation of its molecular target RIN4. Attempts aimed at demonstrating kinase activity of either effector have failed. However, the host kinase RIPK (RPM1-induced protein kinase) has been found to form a complex with RIN4 and AvrB, and this observation has helped to explain the effector dependent phosphorylation of RIN4. Delivery of AvrB inside host cells leads to recruitment of RIPK to an AvrB–RIN4–RIPK complex. RIPK phosphorylates RIN4 threonine residue 166 presumably suppressing PTI in the absence of RPM1  of effector-induced PTMs raises a range of issues which should be considered when examining effector–target interactions. The indirect modification model assumes that the effector binds a host target protein and recruits host machinery which then catalyzes target PTM. Detecting the interaction between three partners  is a challenge since the stoichiometry of each partner may not be equal. Conventional protein–protein interaction experiments using yeast-2-hybrid or tandem affinity purification may only detect the most abundant partners within this interaction. The target protein may be expressed at relatively low levels and the stoichiometry of the PTM often means that the modified protein represents a small proportion of the total pool for that protein. Enrichment strategies are therefore desirable if one wishes to survey the proteome for targets with a specific PTM. A range of strategies are now available for enriching proteins or peptides for specific PTMs, including antibody-based affinity enrichment and ionic interaction-based enrichment . Nühse eThe ability of pathogens to modify the post-translational state of host proteins represents a powerful means for the pathogen to tip the balance from immunity to susceptibility. Since most effectors have yet to be assigned a precise function, and given the enormous diversity that exists among those effectors identified to date, the expectation is that the future will see the identification of alternative effector-triggered PTMs and their substrates. However, a bottleneck is now forming in the characterization of effector activities, and is likely to build further as whole genome sequencing projects identify ever increasing numbers of putative effectors. The use of proteomics to monitor effector localization and host proteome dynamics is likely to emerge as a crucial tool that will enable effector activities to be linked with host PTM signaling pathways. An improved understanding of the mechanisms by which pathogens use their effector repertoire to manipulate host defense signaling, will prove invaluable for developing plant lines with improved pathogen resistance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A 36 year-old man with Wolff Parkinson White syndrome due to a left-sided accessory pathway (AP) was referred for catheter ablation. Whether abolition of antegrade and retrograde AP conduction during ablation therapy occurs simultaneously, is unclear. At the ablation procedure, radiofrequency delivery resulted in loss of preexcitation followed by a short run of orthodromic tachycardia with eccentric atrial activation, demonstrating persistence of retrograde conduction over the AP after abolition of its antegrade conduction. During continued radiofrequency delivery at the same position, the fifth non-preexcitated beat failed to conduct retrogradely and the tachycardia ended. In this case, antegrade AP conduction was abolished earlier than retrograde conduction. A 36 year-old man with Wolff Parkinson White syndrome was referred for catheter ablation. The electrocardiogram during sinus rhythm showed positive delta wave in precordial and inferior leads and an isoelectric delta wave in lead I and aVL, suggesting a left-sided accessory pathway (AP). A normal echocardiogram ruled out any structural heart disease.Via right femoral vein we placed a multipolar catheter in the coronary sinus and a quadripolar catheter in right ventricular apex. A right femoral arterial puncture was made and, through a transaortic approach, a 4mm-tip ablation catheter was placed in left ventricle. We mapped the ventricular aspect of the mitral ring during sinus rhythm and in an antero-lateral position the local electrogram showed a negative delta wave-to-ventricle interval. One second after beginning delivery of radiofrequency (RF) energy, loss of preexcitation was observed , followeWe present the case of a patient with Wolff Parkinson White syndrome due to a left-sided AP, successfully treated with catheter ablation. When treating AP with both antegrade and retrograde conduction, RF energy can be delivered, as in our case, during antegrade conduction (i.e. sinus rhythm with ventricular preexcitation) or during retrograde conduction (i.e. supraventricular tachycardia or continuous ventricular pacing). Completion of RF application usually results in abolition of AP, proved by absence of its antegrade and retrograde conduction. However, whether abolition of both antegrade and retrograde conduction over the AP during RF catheter ablation occurs simultaneously, is unclear.In our case, ventricular preexcitation was abolished soon after the beginning of RF delivery. The first non-preexcitated beat showed a LBBB morphology followed by eccentric retrograde atrial activation and a short run of orthodromic supraventricular tachycardia. It can be noted that the ablation catheter recorded a continuous VA electrogram at the time of orthodromic tachycardia. During continued RF delivery at the same position, the fifth non-preexcitated beat failed to conduct retrogradely and the tachycardia terminated.A delay in left ventricular activation and, hence, in retrograde AP penetration produced by the occurrence of LBBB led to the beginning of a short run of orthodromic tachycardia, demonstrating persistence of retrograde conduction over the AP after abolition of its antegrade conduction. This tracing shows that, in this case, antegrade AP conduction was abolished earlier than retrograde conduction.Tai et al reported a case with abolition of antegrade AP conduction with persistence of retrograde conduction . HoweverAnother possibility to explain the short lasting wide QRS tachycardia is AP automaticity secondary to a rise in tissue temperature due to RF energy -4. ThereFinally, the short lasting wide QRS tachycardia could well be catheter induced atrial tachycardia arising from the region of lateral mitral annulus occurring due to catheter movement just after elimination of preexcitation. However, position of ablation catheter on the ventricular aspect of the mitral ring argues against this possibility though it can not be ruled out completely.The development of LBBB was considered of traumatic origin during catheter manipulation, since it spontaneously disappeared five minutes after RF delivery.Various anatomic and histopathological factors  may influence in the diverse electrophysiological characteristics of AP . The anatomic disposition of the AP in our patient could have resulted in functional but not anatomic longitudinal dissociation of its antegrade and retrograde conduction, since complete AP ablation was achieved with a single RF application.