Neural responses demonstrate evidence of resonance, or oscillation, during the production of periodic auditory events. Music contains periodic auditory events that give rise to a sense of beat, which in turn generates a sense of meter on the basis of multiple periodicities. Metrical hierarchies may aid memory for music by facilitating similarity-based associations among sequence events at different periodic distances that unfold in longer contexts. A fundamental question is how metrical associations arising from a musical context influence memory during music performance. Longer contexts may facilitate metrical associations at higher hierarchical levels more than shorter contexts, a prediction of the range model, a formal model of planning processes in music performance (Palmer and Pfordresher,  Whether dialing a telephone number, playing a musical instrument, or carrying out a surgical operation, people learn and remember the serial order of the actions they produce. Early theoretical accounts of sequence production focused on the role of simple event associations, which triggered the successive actions to be produced in a sequence Watson, . SequencMost modern theories of serial order in sequence production propose that mental planning or preparation of events extends incrementally during performance to a subset of sequence events  and a metrical similarity component (M). One range model component contributing to the strength of contextual event activations is the serial proximity component (S). The serial proximity component predicts a graded decrease in activation of contextual events as the distance between the current event and the contextual events increases. The most active contextual events are located at positions closest to the current event and the least active at positions furthest away. Contextual activations (xS) are modeled by the equationContextual event activations in the range model  is the number of events between the current event and another sequence event; the contextual activations are weighted by the serial distance between them. The t parameter, limited to durations typical of musical sequences, 0.1 < t ≤ 2.0 s, represents the duration of a single event and is determined by production rate , in s). As the parameters t or a decrease, the serial component predicts a faster drop-off in activation strength with sequence distance. An important implication is that events surrounding the current event are less active when events are produced at faster rates  or with higher constraints on working memory . The model assumes that event activations increase as tempo slows (larger t) because more time is available for performers to retrieve information about the surrounding context while preparing events for production, as supported by the fact that errors tend to arise from nearby contextual positions at faster tempi.where M). Metrical similarity between the current event and each contextual event at distance x within one metrical cycle of position i is computed asAnother range model component contributing to the strength of contextual event activations is the metrical similarity component  with which an event at position i is associated, the stronger the accent. The ratio in Equation 2 reflects a generalized form of Weber’s law; perceptual sensitivity to a difference between any two metrical accents depends on the absolute difference between the two events’ metrical levels. The metrical similarity computations, when averaged across current event positions, predict an alternation of high and low metrical similarity across sequence positions, which matches error proportions measured in pianists’ serial ordering errors , for example, the weights applied to the levels 1, 3, and 4 would equal ((1−w2)/(k−1)), where k represents the total number of metrical levels, to ensure that weights across all levels sum to 1. The metrical accent strength of each event in a musical sequence, im, is therefore determined by multiplying the sum of the metrical accents present at each position i and metrical level j, jig, with the weight, jw, at each level:There is often one metrical level that is more salient than other metrical levels; this level is referred to as the tactus, and is usually the level at which people clap or tap to the beat  by the metrical component (M). The nonlinear (multiplicative) relationship between serial and metrical components, shown in Equation 4, describes serial ordering error patterns in music performance better than other models, including an additive model  refers to the current event, for which distance x = 0. Contextual events  are specified by x ≠ 0. On average, the model predicts equivalent activation for contextual events before and after the current event, due to the symmetrical relationship of the serial component.This equation is based on the model’s theoretical assumption that performers use information about the surrounding context while retrieving the current event; the activation of the current event is defined by the activations of surrounding events. The range model also makes predictions related to the length of a sequence context that we test here. Figure The assumptions of the range model yield several predictions regarding the relative importance of highly accented metrical events in longer musical contexts. First, longer sequence contexts yield greater memory activation for the event currently being produced and its contextual sequence events, resulting in a larger range of planning for longer contexts. This prediction is shown in Figure We tested these predictions by manipulating the size of sequence context available to performers as they produced musical sequences at fast and slow production rates. Pianists’ serial ordering errors and tone intensities (corresponding to loudness) were compared for musical excerpts that were placed in long or short sequence contexts. We tested the prediction that the mean range  was greater when excerpts were surrounded by long contexts compared to short contexts. We also tested the prediction that longer contexts should enhance hierarchical relationships between events with similar metrical accent strength; therefore, we expected greater metrical similarity for serial ordering errors that occurred in excerpts placed in long contexts compared to short contexts. Finally, we tested the prediction that pitch error rates would show stronger effects of metrical hierarchies when the excerpts were surrounded by long contexts compared to short contexts. We fit the range model to mean and individual subject data, and tested whether the model’s metrical component accounted for increased influence of context on performance errors.M age = 22.9 years, range = 18–31, SD = 7.7) participated. Participants had a mean of 15.8 years of experience performing the piano . All participants reported playing the instrument regularly and none reported having any hearing problems. Of the 26 participants, 24 reported being right-handed and 2 were ambidextrous; 16 were female. Participants provided written informed consent, and the study was conducted in accordance with the principles laid down in the Declaration of Helsinki and approved by the McGill University Research Ethics Board.Twenty-six experienced adult pianists from the Montreal music community  in 4/4 meter. Each musical excerpt was then embedded in one long context (12 sixteenth-note events preceding and 13 following) and one short context (4 sixteenth-note events preceding and 5 following), which were composed of isochronous sixteenth notes for each hand, followed by a final whole note; thus, all sequence locations contained equivalent numbers of events across excerpts, in order to examine effects of context at all metrical positions. Figure The sequences were performed by pianists on a Roland RD-700 electronic keyboard. All auditory feedback was delivered to participants through AKG K271 Studio headphones, with an Edirol Studio Canvas SD-80. A Classical A02 Piano1d Sound Canvas timbre was used for piano tones and a classical drum timbre was used for metronomic clicks at the beginnings of trials. FTAP computer software controlled the timing of metronome ticks and recorded the pitch, timing, and MIDI velocity of all key press events Finney, .In a within-subjects design, participants were presented with 2 contexts (long/short) × 2 tempi (fast/medium) × 4 test blocks (1–4). To test the model’s predictions, each pianist performed half of the excerpts placed in long contexts and the other half placed in short contexts, and each pianist performed half of the pieces at a medium tempo  and the other half at a fast tempo . The pieces assigned to each tempo condition were counterbalanced across participants. The experiment consisted of a learning phase, in which participants learned all of the musical stimuli, and a test phase, in which each piece was performed 2 times per block over 4 test blocks for a total of 8 test performances. In the test phase, long and short contexts were grouped within each block and half of the pianists performed excerpts surrounded by long contexts first, and the other half performed excerpts surrounded by short contexts first. Presentation order of long and short contexts was manipulated in a Latin square design across blocks. Medium and fast production rates alternated every trial and their order was counterbalanced across participants. Thus, the test phase of the experiment contained a total of 4 (excerpts) × 2 (tempi) × 4 (blocks) × 2 (repetitions) = 64 trials.At the start of the session, participants completed a questionnaire about their musical background. Participants then completed the learning phase of the experiment. During the learning phase, participants were presented with the music notation for one of the musical stimuli  and were asked to practice the piece, for a maximum of 3 min, until they felt comfortable playing it at a slow tempo. Participants were then asked to perform the piece using the notated fingering at a slow tempo of 429 ms per sixteenth-note IOI (35 beats per minute), as indicated by an initial metronome beat that sounded on the quarter note. Participants were given three attempts to perform each piece without making any errors at this slow tempo before moving on to the next piece. Piece orders were counterbalanced; the same order was used for the learning phase and the first block of the test phase of the experiment. All participants were able to perform every piece without errors at the slow tempo before continuing to the test phase of the experiment. Thus, all pianists learned the 8 excerpts in their associated contexts to a note-perfect criterion (pianists were never presented with the excerpts in isolation). These slow performances ensured that errors that occurred at faster tempi were not due to sight-reading failures or incorrect learning of the musical pieces.Following the learning phase of the experiment, participants were asked to perform the pieces at the faster tempi in a test phase consisting of 4 experimental blocks. In each trial, participants first heard a metronome click to establish the prescribed tempo, which sounded at the quarter-note level throughout each trial. Participants were instructed to perform the sequence at the tempo indicated by the metronome using the notated fingering and, if they made any errors, to continue playing and not stop or try to correct them. They performed the piece twice in each trial, pausing between repetitions, with the musical notation in view. The entire experiment lasted 60 min, and participants received a nominal fee for their participation.n = 21, less than 1% of all errors produced during the test phase). Although subjects were instructed not to stop to correct errors, subjects occasionally corrected errors; corrected errors were also excluded from all analyses.Pitch errors were identified by computer comparison of pianists’ performances with the information in the notated musical score Large, , 1995. Ta-priori global alpha level was 0.05. Greenhouse-Geisser corrections were performed for analyses when necessary. Pianists’ MIDI key velocities  were analyzed in both correct and incorrect keystrokes for evidence of metrical hierarchies.The relative frequency with which contextual errors arose across sequence distances was examined in terms of a movement gradient, a distribution of error frequencies as a function of the distance of the nearest intended event from the current event  in the fast condition (prescribed rate = 187.5 ms) and 226.7 ms per tone (SD = 3.8 ms) in the medium condition (prescribed rate = 225 ms). Thus, the participants performed at tempi close to those prescribed.Participants performed a total of 7,469 errors across the entire musical sequences. The mean error rate per trial, which was based on all pitch errors that occurred within participants’ performances, was 0.10. Eighty percent of produced errors were contextual errors . The mean production rate across performances was 194.2 ms per tone . Thus, the types of errors and error rate produced by pianists within the excerpt were representative of those produced across the entire sequence. All further analyses are reported on the performances of the embedded musical excerpts.F = 68.66, p < 0.001. Tones produced at more strongly accented positions were performed with greater intensity than tones produced at more weakly accented positions , indicating that the metrical accent manipulation reliably altered participants’ keystroke intensities. In addition, there was a main effect of context, F = 8.84, p < 0.01. Tones within long contexts were produced with greater intensity  than tones within short contexts . The three-way interaction between metrical accent, context, and tempo was also significant, F = 3.20, p < 0.05. Post hoc comparisons indicated that tones in the long contexts were produced with greater intensity when they aligned with strong metrical accent strengths (3 or 4) than with weak accent strengths (1 or 2) in long contexts, for both tempo conditions . Tones in short contexts also showed a significant change in intensity between strong and weak metrical accents; this difference was reduced at the medium tempo. There was no main effect of tempo on tone intensities, and there were no other significant interactions. Thus, both the metrical accent strength of individual tones and the size of the surrounding context increased the intensities with which tones were produced.We tested the hypothesis that longer contexts would increase the salience of metrical levels by evaluating tone intensities in terms of their metrical accent strength. Figure F = 31.53, p < 0.001. As with correct events, errors produced at the most strongly accented positions were performed with greatest intensity , followed by errors at the next strongest accent positions , followed by the two most weakly accented positions , which did not differ from each other . There was a significant three-way interaction between metrical accent, context, and tempo, F = 8.34, p < 0.001. Post hoc comparisons indicated that, in the long context condition, tones receiving the strongest metrical accent strength (4) were performed with greater intensity than other tones  at the medium tempo, but not at the fast tempo . The opposite pattern was found in the short context condition: Tones receiving the strongest accent strength (4) were performed with greater intensity than other tones  at the fast tempo but not at the medium tempo. There were no other significant main effects or interactions. An analysis of only contextual errors in which metrical accent strength was coded in terms of the intruder position (as opposed to the target position) revealed that error intensities at metrical levels 3 and 4 did not significantly differ from those at levels 1 and 2 , suggesting that contextual errors adopt the intensity of the position at which they are produced. Similar to the correct events, performers produced errors with greater intensity when they were aligned with positions of strong metrical accent, and these effects were mediated by the context and tempo. Planned metrical associations may therefore differ from executed metrical accents.Context effects on the salience of metrical levels were also investigated in the tone intensities of excerpt errors. Figure We predicted that longer contexts would enhance effects of meter on performers’ error rates. Specifically, lower error rates were expected at excerpt locations aligned with stronger metrical accents compared to weaker metrical accents, and this effect was predicted to be larger for errors produced in long contexts compared to short contexts. Excerpt error rates broken down by metrical accent strength were adjusted for the number of opportunities for an error to receive that metrical accent strength  = 7.50, p < 0.05, and a significant main effect of metrical accent, F = 30.23, p < 0.001. Overall, error rates were higher at the faster tempo  than at the medium tempo . Thus, the tempo manipulation reliably altered pianists’ error rates. In terms of metrical accents, error rates were lowest at positions receiving a metrical accent strength of 3  and 4 , next lowest at positions receiving a metrical accent strength of 2 , and highest for positions receiving a metrical accent strength of 1  . The main effect of context was not significant (p = 0.06). Critically, the context × metrical accent interaction was significant, F = 13.15, p < 0.001. Error rates for excerpts in long contexts were significantly lower for positions receiving a metrical accent strength of 4  compared with all other accents, and higher for positions receiving a metrical accent strength of 1  compared with all other accents . The only significant differences seen for short contexts were reduced error rates at positions receiving an accent strength of 3  compared with accent strengths of 1  and 2 . There were no other significant interactions.Figure F = 6.24, p < 0.05, and main effect of tempo, F = 5.54, p < 0.05, but no interaction. Contrast values were generally negative, indicating a linear decrease in error probability with increasing metrical accent (errors were least likely at the most strongly accented positions), but the magnitude of contrasts was greater for long contexts  than for short contexts , and for fast tempo sequences  than for medium tempo sequences . Thus, longer contexts and slower tempi enhanced beneficial effects of metrical accent on error rates.We further explored the differences in error rates across contexts and metrical levels using a contrast analysis  = 12.98, p < 0.005. Target-intruder distances were greater when the same excerpt was placed in a longer context  than in a shorter context , when possible target-intruder distances were controlled for across context length, indicating that context facilitated a greater range of planning. There was no effect of tempo or interaction with context.We tested the prediction that error types are affected by context; we predicted that the target-intruder distance for contextual excerpt errors would increase as the size of the surrounding context increased. The mean range between each contextual pitch error and the sequence position for which that pitch was intended was calculated for all contextual errors by averaging the absolute distances between the location of contextual errors and their sources within 8 events of the target event . A two-way ANOVA on mean ranges for excerpt errors revealed a main effect of context, M = 6 tones apart) to include only those errors whose distances fell within this chance estimate; this is an important control for the distributions of pitches outside the excerpt from which the errors might have arisen. The same 2 (context) × 2 (tempo) ANOVA on mean ranges of contextual errors within the pitch repetition chance estimate replicated the main effect of context, F = 5.50, p < 0.05, with greater target-intruder distances for longer contexts than shorter contexts.To assess the possible influences of pitch repetition rates in the musical sequences on error distance measures, we analyzed the distances over which pitches recurred across the entire musical sequence and applied that criterion  = 139.49, p < 0.001, and a significant distance × context interaction, F = 4.78, p < 0.001. Post hoc comparisons revealed that a significantly larger proportion of errors arose from distance 1 than distance 2 in short contexts: This was not the case in long contexts. The tempo × distance interaction was also significant, F = 2.25, p < 0.05, but did not survive a Greenhouse-Geisser correction (p = 0.106). Importantly, and in line with model predictions, differences across contexts reflected greater accessibility of metrically similar events (even numbered positions) and lower accessibility of dissimilar events (odd numbered positions) for the long context. Differences attributable to metrical similarity across contexts were only apparent at close serial proximities, which is consistent with the range model’s prediction that metrical similarity effects are lessened at far distances due to the modulating effect of serial proximity  of contextual excerpt errors in long contexts arose from metrically similar distances, whereas only 31% (SE = 2.7%) of excerpt errors in short contexts arose from metrically similar distances, t(25) = 2.87, p < 0.005 (one-tailed). Thus, metrically similar errors  were more likely to arise in long contexts than in short contexts, similar to the model’s predicted context effects.To test whether longer sequence contexts affect performers’ conceptions of metrical similarity, we compared the proportions of contextual errors arising from metrically similar events  across long and short contexts. On average, 44%  = 79.62, p < 0.001, and a significant distance × context interaction, F = 3.92, p < 0.05. Post hoc comparisons revealed that a significantly larger proportion of errors arose from distance 1 than distance 2 in short contexts: This was not the case in long contexts , similar to the analysis of error proportions arising from distances 1 through 8. The tempo × distance interaction was not significant, F = 2.39, p = 0.08. There were no other significant effects or interactions.In a second follow-up analysis, contextual error proportions were computed for errors arising from distances 1 through 4. The same 4 (distance) × 2 (context) × 2 (tempo) ANOVA on contextual error proportions revealed a significant main effect of distance, r(62) = 0.11, p = 0.37, or for short contexts, r(62) = 0.04, p = 0.74. In a final test of whether pitch repetitions within the musical sequences differentially influenced contextual errors in long and short contexts, we correlated the distances at which pitches repeated within each excerpt across the long and short contexts; the pitch distances were highly correlated across context lengths for both the right hand part, r(62) = 0.90, p < 0.001, and the left hand part, r(62) = 0.94, p < 0.001. This finding follows from the fact that the pitch contents (4 events) on either side of the excerpt were identical across long and short contexts.Finally, we tested whether the rate at which pitches repeated in the melodic sequences differed across metrical accent strengths or context lengths. The mean distance between repeating pitches at each position in the stimulus excerpts did not correlate with the metrical accent strength at each position for long contexts, To address whether the distance effects on error proportions shown in Figure M = 74.7, SE = 0.48) than tones produced by participants’ left hands , F = 237.32, p < 0.001, similar to previous studies  or perseveratory . Proportions of all pitch errors coded in terms of finger distance did not correlate significantly with the observed movement gradients in Figure r(6) = 0.41, p = 0.32. ANOVAs on the additional factors by context and metrical accent strength yielded no significant differences.We evaluated additional factors that may influence the error patterns reported here. Previous findings , which weights different values of m in Equation 2, was fit to each experimental condition, based on previous findings that this level was weighted heavily with similar pieces possessing the same 4/4 meter  / (k−1)), where k represents the total number of metrical levels, as described in the introduction. Of the 26 pianists, 3 pianists produced no errors in the long-medium condition, 3 produced no errors in the short-medium condition, and 2 produced no errors in the short-fast condition; model fits could not be performed on these individual conditions. We fit only those combinations of participant and condition for which there were error data (n = 96).The range model was fit to individuals’ excerpt error proportions from the movement gradients separately for each context-tempo condition. Fits were carried out in two steps, following the same procedure introduced by Palmer and Pfordresher . All modp < 0.05), and the mean variance accounted for (VAF) values per individual ranged from 0.46 to 0.91 ; fits by individual were significant for 23 out of 26 individuals . The model’s parameter a, which is thought to capture individual working memory constraints, was correlated with the behavioral metric for mean range based on error distances. A significant positive correlation was found between individuals’ a parameter values (range = 0.77–0.89) and their mean range of planning , r(24) = 0.46, p < 0.05. Thus, participants with higher a values had greater mean ranges of planning, similar to previous findings  and the other without the metrical component (S). Both the meter-free model (S) and serial × metrical (S × M) model were first fit to the mean data for each tempo-context condition with a varying, and subsequent fits used a values averaged across conditions. All metrical levels were weighted equally (jw = 0.25) for fits of the serial × metrical (S × M) model so that the number of free parameters was identical to the meter-free (S) model. Figure S) and one containing the metrical component in addition (S × M). The diagonal line in Figure S) and (S × M) models to individual subject data for each of the 4 tempo-context conditions. The (S) model provided a significant fit in 58 of the 96 individual experimental condition fits, and the (S × M) model provided a significant fit in 72 of 96 individual fits (p < 0.05). Thus, inclusion of the metrical component was able to explain more variance for the majority of individual data.We tested whether the model’s metrical similarity component was necessary to explain the excerpt errors by first comparing two different versions of the model: one containing the metrical component  model, the VAF was improved for all four conditions over the serial (S) model and serial × metrical (S × M) model fits, shown in the upper half of Table S × M) model with the additional free parameter w2 provided a better fit for the 2 long context conditions, but not the 2 short context conditions. Thus, the S × M model’s additional metrical weight parameter, derived from fits of the model to the data, improved the long context fits above and beyond the introduction of the model’s metrical similarity component (column 2 of Table S × M) model with jw fixed at 0.25 provided a significant fit in 72 of the 96 individual experimental condition fits, and the (S × M) model when w2 was allowed to vary provided a significant fit in 85 of 96 individuals fits (p < 0.05). Thus, inclusion of the metrical weight on level 2  was able to explain more variance for the majority of individual data.Finally, we tested whether the model’s metrical parameter, w2 values) yielded a significant main effect of tempo, F = 6.49, p < 0.05. Excerpt errors from medium tempo conditions yielded model fits with larger metrical weights  than excerpt errors from fast tempo conditions . Fits of the model to the data from long context conditions tended to result in larger metrical weights  than fits to data from short context conditions , although the main effect of context on metrical weights was not significant (p = 0.06). The interaction between tempo and context variables was not significant. Thus, the metrical weighting parameter indicated greater emphasis on level 2 of the metrical hierarchy when excerpts were performed at a slower tempo and in a longer context.A 2 (tempo) × 2 (context) ANOVA on individuals’ fitted metrical weights  model of Brown et al.  also premore accessible than it otherwise would have been if there were less (or no) sequential context. Most models of memory retrieval have neglected to incorporate hierarchical structure in theoretical frameworks. Research on effects of hierarchical structures on recall has led to the acknowledgment that short-term memory retrieval processes must somehow interact with information stored in long-term memory (Atkinson and Shiffrin, The range model differs from other models in its claim that contextual information is not only accessible during production, but also makes the current event The current study tested range model predictions with musical pieces that contained isochronous events in order to maintain equivalent rates of musical events occurring at different metrical positions. Because strong and weak metrical accents alternate strictly in binary meters across all sequence positions, metrical similarity is not confounded with primacy or recency effects. Much of Western tonal music contains varying tone durations, which can influence chunking processes, in which retrieval of musical segments is an all-or-none process cued by longer durations occurring at chunk boundaries (Ericsson and Kintsch, The musical pieces used in the current study were characterized by binary metrical structures that contained a strict alternation of strong and weak beats. An analysis of a large corpus of musical styles has shown that the frequency with which pitches occur in different metrical locations can differ for binary and ternary meters and can differ across musical styles and genres (Palmer and Pfordresher, Metrical hierarchies may modulate influences on memory of other musical dimensions such as tonality, harmony, and timbre, which also yield similarity-based confusion errors in comprehension and performance (Crowder and Pitt, Predictions of the range model can be applied to the production of other complex sequences, such as typing and speech, whose elements hold hierarchical relationships defined within each context (cf. Crump and Logan, Sometimes more is better: Increased metrical associations in longer sequential contexts facilitate performers’ retrieval of individual sequence events. Incrementally planned representations are popular among memory retrieval models during sequence production; the range model (Palmer and Pfordresher, 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.


Listeria monocytogenes is a serious foodborne pathogen that can contaminate food during processing and can grow during food shelf-life. New types of safe and effective food contact materials embedding antimicrobial agents, like silver, can play an important role in the food industry. The present work aimed at evaluating the in vitro growth kinetics of different strains of L. monocytogenes in the presence of silver, both in its ionic and nano form. The antimicrobial effect was determined by assaying the number of culturable bacterial cells, which formed colonies after incubation in the presence of silver nanoparticles (AgNPs) or silver nitrate (AgNO3). Ionic release experiments were performed in parallel. A different reduction of bacterial viability between silver ionic and nano forms was observed, with a time delayed effect exerted by AgNPs. An association between antimicrobial activity and ions concentration was shown by both silver chemical forms, suggesting the major role of ions in the antimicrobial mode of action. Listeria monocytogenes is a foodborne pathogen responsible for severe disease in immunocompromised people and for non-invasive infections in immunocompetent individuals . This safety criterion has been set in relevant EU regulations for RTE foods during their entire shelf life. US food policy on L. monocytogenes is characterized by more stringent requirements regarding the monitoring process in processing plants, in order to avoid food contamination  has recommended, as an objective for public health, that the level of mination .Listeria spp. to grow between 0 and 4°C and its tolerance to some commonly applied preservatives, like salt, explain the challenge of applying alternative control strategies along the whole food chain  or consumer levels can result in failure to control growth of the pathogen.The shelf life for such products is often calculated based on the kinetics of bacterial growth. Temperature control is critical in the overall control of In this context, the development of new types of safe and effective food contact materials able to extend the shelf life of food products or to prevent cross contamination is of great interest. Among different chemicals with antimicrobial activity, silver is considered as a good candidate, as it is known to exert antimicrobial properties toward both Gram positive and Gram negative bacteria .L. monocytogenes has never been proved.Previous studies have shown that nanoparticles (NPs) could be used as effective bactericidal materials in antimicrobial formulations due to their enhanced reactivity, resulting from their high surface/volume ratio . In partNevertheless, data arising from studies on the effectiveness of NPs as antimicrobials are difficult to interpret due to the specific chemical and physical characteristics of these particles. Thus, it is of primary importance to provide detailed characterization of any tested NPs starting from size and shape evaluation, as this would allow the scientific community to compare the results.L. monocytogenes and to explore intra-species variability and its relation with antimicrobial resistance. Moreover, the effectiveness of AgNPs as an antimicrobial material is discussed in the light of its mechanism of action.The aims of this work were to evaluate the efficacy of AgNPs as an antimicrobial toward 3) 1000 mg L–1 stock solution was purchased from Ultra Scientific .Silver nanoparticles colloidal suspension in citrate 2 mM  and their control dispersant medium were purchased from Nanocomposix . Silver nitrate ; details are presented in Table 1. L. monocytogenes ATCC® 13932TM was also examined. The isolates were stored in Microbank™  at -80°C until needed. All strains were identified as L. monocytogenes according to ISO standard (Twenty standard .®) (120 KV) at the Department of Technology and Management of Industrial Systems (University of Padua). AgNPs were diluted with Muller Hinton Broth (MHB) culture media to reach a final concentration of 50 mg/L. TEM observations were performed at 6000X and 100KX and microphotographs were elaborated using ImageJ® software, enabling evaluation of average particle diameter and roundness for a sample of 500 particles.Morphological parameters of AgNPs were verified using a transmission electron microscope (TEM) JEM 2000 EX II  according to the manufacturer’s recommendations. The results were visually evaluated after 48 h of incubation at 37°C and the minimum inhibitory concentration (MIC) was defined as the lowest concentration able to completely inhibit bacterial growth.In this experiment a two steps susceptibility assay was performed.L. monocytogenes isolates to different concentrations of AgNPs and AgNO3 was assessed as follows. L. monocytogenes isolates from Microbank™ were subcultured in Tryptone Soya supplemented with yeast and incubated at 37°C overnight. The day after a loopful was transferred to 15 ml of Mueller Hinton Broth (MHB) and incubated at 37°C overnight to produce the inocula. MHB was used as culture medium with the objective of obtaining a good compromise between L. monocytogenes growth and NPs stability, given that this is a quite simple and non-selective liquid medium, specifically designed for susceptibility studies and having the minimum requirements for bacteria growth (L. monocytogenes in the Lag phase (OD A600 = 0.3 = 108 CFU/ml) (–1) or AgNO3 . The tubes were incubated in an orbital shaker at 200 rpm at 37°C for 4 h. A positive control (flask containing AgNPs and MHB) and a negative control (flask containing inoculum and MHB) were included. The high rotary shaking speed was selected to prevent the aggregation and settlement of the NPs over the incubation period and Ag concentration levels were selected according to previous observations with Salmonella (The different susceptibility of the 21 a growth . The fin CFU/ml)  in the plmonella .–8 of L. monocytogenes, 100 μl aliquots were spread-plated on ALOA Agar according to After 4 h of incubation, a volume of 50 μl was removed from liquid culture and, after serial 10-fold dilutions in MHB broth up to 10L. monocytogenes isolates was evaluated. A cell culturability assay was carried out as described above, with different concentrations of AgNPs (300 mg/L–1) and AgNO3  at different time points . Each experiment was performed in triplicate. Positive and negative controls were included.The time dependent evaluation of the effectiveness of silver as an antilisterial on selected Listeria monocytogenes (strain 5) liquid culture were prepared as in the antimicrobial susceptibility assay and incubated in the presence of AgNPs (300 ppm) and AgNO3 (30 ppm). After 0, 24, 48, and 72 h of incubation in an orbital shaker , 250 μl of each suspension was transferred to a centrifuge tube  and centrifuged at 5000 g for 60 min. After filtration, filtered samples were diluted with milli-Q water in the ratio of 1:500 as preparation for atomic absorption analysis.–1 (corresponding to 150–1500 μg L–1 in samples) by dilution of the AgNO3 stock solution with Milli-Q water. Data acquisition and processing was conducted using thermo solaar software to evaluate the recovery of ionic silver after filtration, and thus its potential interaction with the centrifuge tube filter, an additional parallel extraction was performed. Briefly, three solutions of 30 ppm of AgNO3 were prepared in MHB without cells. The ion concentrations were measured before and after filtration to obtain a recovery factor applicable to all ionic release data.Total silver was quantified in suitably diluted samples by Electrothermal Atomic Absorption Spectrometry (ETAAS) using an M6 mkII Atomic Absorption Spectrometer  with D2 and Zeeman background correction, equipped with a GF95 Graphite Furnace atomiser (GFAAS). Analytical conditions are reported in Supplementary Tables 3 dilution (before centrifugation) and the amount of total filtered silver (after centrifugation). A mean value was calculated.For ionic release assessment, two values per time point  were acquired and corrected by a recovery factor, obtained by calculating the percentage ratio between the total amount of silver ions released after AgNOCounts resulting from AgNPs susceptibility assay were log-transformed and then summarized as mean values and standard errors. For statistical purposes a value of 300 colonies was assigned to plates on which more than 300 colonies were observed according to ISO 7218/2007 . A Wilcoxon test was also performed to verify the accordance to the t-test.A Figure 1A). Most particles were spherical, but the sporadic presence of regular polygonal particles was also observed. Particle size distribution showed one major population, with median diameters of 23(±2) nm .As visualized by TEM, AgNPs suspension appeared as a mixture of homogeneous particles by size and shape  and AgNPs showing effectiveness only after 24 h incubation . The effect caused by AgNO3 was proportional to the dose, with 30 mg/L being the most effective, and 10 mg/L the least effective for all six L. monocytogenes isolates . Some statistically significant differences were observed among the selected strains at different time points. The maximum difference among strains was observed after 4 h of incubation both in the presence of AgNO3 and AgNPs. Conversely, no differences were detected after 24 h of incubation in the presence of AgNO3. In detail, in the presence of AgNO3 (30 and 20 ppm) L. monocytogenes ATCC and strain 20 counts were lower than the others (p < 0.05). ATCC strain exerted statistically significant lower counts also when incubated with 10 ppm of AgNO3 difference is confirmed in the case of ATCC. In the case of AgNPs a behavior similar to AgNO3 (30 ppm) could be described with statistically significant differences at 4h (ATCC and strain 20 more susceptible) and a convergence of growth curves at 24 h. In this case, however, differences among strains were also observed after 48 h with strain 20 being more sensitive than strains 4 and 7 (p < 0.05). Interestingly after 72 h ATCC, as also strain 4, showed a statistically significant recovery if compared with other L. monocytogenes strains.Results of the second step are shown in 3  and AgNPs , even though the relative extents were different. As shown in Figure 4B, in the case of AgNO3 a large amount of released silver ions was detected in the analyzed solution at the beginning of the study, when the measured silver concentration was approximately 22,000 μg/L. At this concentration, AgNO3 was able to immediately exert the expected antilisterial activity .Some correlation between ionic release and strain 5 growth curve was found both in the case of AgNOFigure 5B). As suggested by Figure 5A, the antilisterial effect exerted by AgNPs started after 24 h of incubation, and after the released silver ions reached the concentration of approximately 6,000 μg/L.The amount of silver ions released from AgNPs increased over time, with a maximum observed concentration of approximately 14,000 μg/L after 72 h of incubation  suggests that ions are the more effective elements exerting antibacterial activity. This findings agrees with scientific literature where several studies demonstrates that cations could interfere with the normal function of the bacterial electron transport chain, and thus, be responsible for ROS (Reactive Oxygen Species) formation at the cell membrane (In our study toxicity and dissolution tests were performed in parallel, as silver ions released upon dissolution of AgNPs have been proposed to be the primary contributor to the antibacterial activity of these compounds . The relmembrane . Howevermembrane . Both iomembrane .L. monocytogenes is sensitive to silver and that the efficacy is linked to ionic release. We speculate that silver-based food contact materials could play a useful role in the food industry, perhaps to reduce surface contamination, or eventually, to prolong shelf life. However, to confirm this, studies under industrial conditions are needed, to evaluate silver efficacy as well as the potential, hazardous migration of silver from food contact materials into food (Our study suggests that nto food . As persnto food . It is into food .SB, CL, AR conceived and designed the research, acquired and interpreted the data, drafted the work; IP analyzed the data; LR, DC, FG acquired the data; VC, PC, SS conceived the research and interpreted the data.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.


Objectives. The accumulation of lipids stored as excess triglycerides in the liver (steatosis) is highly prevalent in obesity and has been associated with several clinical characteristics, but most studies have been based on relatively small sample sizes using a limited set of variables. We sought to identify clinical factors associated with liver fat accumulation in a large cohort of patients with extreme obesity. Methods. We analyzed 2929 patients undergoing intraoperative liver biopsy during a primary bariatric surgery. Univariate and multivariate regression modeling was used to identify associations with over 200 clinical variables with the presence of any fat in the liver and with moderate to severe versus mild fat accumulation. Results. A total of 19 data elements were associated with the presence of liver fat and 11 with severity of liver fat including ALT and AST, plasma lipid, glucose, and iron metabolism variables, several medications and laboratory measures, and sleep apnea. The accuracy of a multiple logistic regression model for presence of liver fat was 81% and for severity of liver fat accumulation was 77%. Conclusions. A limited set of clinical factors can be used to model hepatic fat accumulation with moderate accuracy and may provide potential mechanistic insights in the setting of extreme obesity. Obesity is associated with fat accumulation in the liver, diagnosed as nonalcoholic fatty liver disease, and considered a manifestation of the metabolic syndrome , 2. AlthBoth environmental and genetic factors play a significant role in mediating risk for development of increased fat in the liver . A majorThe clinical standard for diagnosis and staging of hepatic fat is liver biopsy . Such da2, no evidence of hepatitis B virus (HBV), hepatitis C virus (HCV), or human immunodeficiency virus (HIV), evaluable liver biopsy results, and no diagnosis of alcohol abuse were included in this study. Alcohol abuse was evaluated during a required comprehensive behavioral evaluation with a clinical psychologist prior to surgery, where patients were asked about current and past substance and alcohol use. If a patient endorsed using alcohol, they were further queried based on the clinical criteria for alcohol use disorders [Patients enrolled in the Bariatric Surgery Program at the Geisinger Clinic Center for Nutrition and Weight Management were offered participation in an IRB approved research program on obesity. Clinical data were obtained during a standardized in-depth preoperative preparation program as described . Particiisorders . PatientWedge biopsies of the liver, obtained intraoperatively during the bariatric surgery from a similar anatomic location as part of clinical standard of care , were fihttp://dx.doi.org/10.1155/2014/368210). The research was approved by the Geisinger Clinic Institutional Review Board and all participants provided written informed consent.Clinical variables were obtained from an electronic database as described previously . BrieflyP values <0.10 were considered for subsequent inclusion in the model.Mean with interquartile range and overall range was used to describe the distribution of quantitative variables. Logistic regression was used to determine which of the more than 200 preoperative clinical variables were independently associated with the presence of any degree of hepatic lipid versus normal histology. In separate analyses, logistic regression was used to determine which preoperative clinical variables were independently associated with grade 1 steatosis versus grade 0 steatosis, that is, mild versus normal, and which variables were associated with grade 2 and grade 3 steatosis versus grade 1 steatosis, that is, severe versus mild. For all analyses, univariate analysis was conducted first to identify a subset of variables for consideration in model building; that is, those variables with univariate Regression modeling was conducted in two phases. First, variables were selected based on (1) whether statistical significance was achieved in univariate analysis and (2) whether published information was available implicating them in NAFLD/NASH. For the second phase, the variables significant in univariate analysis but not included in the first phase were each added individually to determine their effect on the association strength. Potential nonlinearity of continuous variables, for example, lab results, was evaluated through various transformations including the use of quartiles and categorization based on clinically relevant thresholds, for example, classifying glucose values by potentially diagnostic groups, <100 mg/dL, 100–124 mg/dL, and ≥125 mg/dL. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the equation :(1)HOMA-P values < 0.05 were considered significant.Effect modification was determined with interaction terms between variables within the final multiple regression models. c-statistics (area under the curve) and receiver operator characteristics (ROC) curves were used to evaluate discrimination of the final multiple regression models. SAS version 9.3 was used for statistical analysis and 2 who had no evidence of HBV, HCV, or HIV and no diagnosis of alcohol abuse and who had undergone primary bariatric surgery [P = 0.035) and slightly younger  but with similar levels of body mass index (BMI) of 49.5 kg/m2 versus 49.6 kg/m2 (P = 0.897) and liver function test (LFT) results .We analyzed clinical and pathology data on 3209 patients with BMI >35 kg/m surgery . PatholoOf the 2970 patients with available liver pathology results, 41 (1%) were excluded from analysis due to adverse histological findings, that is, 7 with inflammation, 33 with fibrosis, and 1 with cirrhosis, in the setting of grade 0 hepatic fat. In the remaining 2929 patients , the disμU/mL, triglycerides ≥ 125 mg/dL, HDL < 50 mg/dL, and an increasing odds ratio for increasing ALT levels. In addition, ferritin levels of more than 400 ng/mL for males or more than 100 ng/mL for females as well as red blood cell count of more than 4.38 M/uL, chloride of more than 103 mmol/L, iron binding capacity of at least 320 mcg/dL, and a serum zinc level of more than 70 mcg/dL were associated with a greater risk for hepatic fat. Use of the medications metformin, benzodiazepine anticonvulsants, and topical corticosteroids was also associated with an increased risk. Clinical variables including a waist-height ratio of more than 0.9, a clinical diagnosis of sleep apnea, and failing to lose at least 10% of excess body weight in the preoperative period also placed patients at increased risk. Only two variables, the use of estrogen and/or progestin or serum creatinine level of >1.2 mg/dL, were associated with a decreased risk for hepatic fat. Interaction terms between these measures were not significant, suggesting that the effects are consistent within selected subsets of the population.In a univariate analysis comparing the 702 patients with grade 0 hepatic fat to the 2227 patients with mild, moderate, and severe hepatic fat accumulation, 87 of the 204 clinical variables were selected for the initial multiple regression model. These 87 variables were included in stepwise model building, resulting in 19 variables that demonstrated independent association  and resuThe above analysis was repeated comparing grade 1 (mild) to grade 0, which produced essentially identical results (data not shown). A reanalysis was also conducted for the patients who were documented to have no use of alcohol. In this subgroup, the overall area under the curve (c-statistic) increased slightly to 0.826, although the no alcohol group had a higher percentage of T2D that may have also influenced hepatic fat content. Similarly, the HOMA-IR was used instead of glucose and insulin, which slightly increased the area under the curve (c-statistic) to 0.817.μU/mL), triglycerides (≥125 mg/dL), and use of metformin. Lower preoperative weight loss was again associated with increased risk. A graded increased risk was associated with ALT levels to more than 5-fold at ALT >37 U/L, while AST levels >31 U/L showed an almost 1.7-fold increased risk. Independent factors increasing the odds of moderate to severe hepatic fat included female gender (30% increase), patients with a BUN level <13 mg/dL (>50% increase), and use of tricyclic or modified cyclic antidepressants (50% increase) or fibric acid derivatives (>75% increase). Interaction terms between these measures were not significant (data not shown). We also evaluated the association in patients who were documented to have no use of alcohol. In this subgroup, the overall area under the curve (c-statistic) increased slightly to 0.785. Reanalysis using HOMA-IR increased the area under the curve (c-statistic) to 0.777.We then sought to determine what clinical factors were associated with higher levels of hepatic fat versus low levels by comparing moderate (grade 2) and severe (grade 3) to mild (grade 1). For this analysis, the 702 patients with grade 0 hepatic fat levels were excluded from the analysis, leaving 2227. In univariate analysis, the 1248 patients with grade 1 were compared to the 979 patients with grades 2 and 3. A total of 56 of the 204 clinical variables were brought forward for multiple regression analysis. These 56 variables were included in stepwise model building, resulting in 11 variables that were independently associated with the higher level (grades 2 and 3) of hepatic fat , includi2, and 75% steatosis prevalence reported in these studies (Supplementary Table S2). The overall prevalence of hepatic fat in these studies is slightly lower than previously reported [Fatty liver is a highly prevalent finding in extreme obesity that has several potentially significant clinical sequelae. The characterization of associated clinical characteristics in populations with high prevalence, such as those with extreme obesity, is thus important for identifying potentially modifiable risk factors and putative pathophysiological mechanisms. To date, most studies have been limited by small sample sizes, use of noninvasive methods to assess hepatic fat, and a focus on a relatively small number of clinical variables. We surveyed 22 published reports (Supplementary References) on hepatic fat accumulation representing 4274 patients with extreme obesity undergoing bariatric surgery and liver biopsy (Supplementary Table S2), a prime opportunity in which to assess population-based aspects since such patients generally are not documented to have, nor are suspected of having, liver disease, which is a bias for studies based on percutaneous liver biopsies. Our single center study thus represents a sample size of nearly 75% of that of the combined reports we were able to identify. Our basic cohort characteristics were similar to the 78% female, average age of 41.5 years, average BMI of 47.6 kg/mreported  in the mThe accuracy of our final multiple logistic regression model for presence of hepatic fat was over 80%. The variables contributing to this moderate level of accuracy included elevated AST and/or ALT, where risk progressively increased over a relatively restricted range (by quartile) considered “normal,” from <20 U/L to greater than 37 U/L. These data are consistent with that reported in other studies  and a prFatty liver has been independently associated with a lower level of HDL cholesterol and a higher serum triglyceride level, similar to our findings, although large studies have used imaging as a surrogate for classifying hepatic fat , 24. IntWe found that patients with a diagnosis of obstructive sleep apnea had more severe levels of hepatic fat accumulation, replicating findings from recent meta-analyses . This inHigher ferritin, iron binding capacity, red blood cell count, and serum zinc levels were also found to be associated with hepatic fat accumulation. The ferritin, iron binding capacity, and red cell count are consistent with a dysregulation of iron metabolism that has previously been described , given tFemale sex and the use of estrogens and progestins  were associated with slightly increased odds for hepatic fat, consistent with previous human and animal studies . SeveralDespite the large sample size, our data were limited to a single center with patients who were primarily of Caucasian European ancestry and were predominantly middle-aged women, limiting generalizability of the data, though allowing for comparison to other bariatric surgery studies. The extreme level of obesity limits extension of the findings to lower BMI levels. A cohort of bariatric surgery patients may also be a biased population relative to the vast majority of individuals with extreme obesity who do not undergo the procedure.We characterized histologically defined fatty liver in the context of the largest prospectively enrolled single center cohort of extremely obese adults coupled to a comprehensive and well-curated entry database of clinical data . RegressThe Supplemental materials contain Table S1 that contains the clinical variables used in regression analysis (n=204 variables) and Table S2 that contains a summary of 22 available published reports on 4274 patients with extreme obesity undergoing bariatric surgery and liver biopsy.


Objective: To study the relationship between TLR4 and NF-κB p65 protein expressions in tumor tissues after radiotherapy and clinical radiosensitivity of patients with esophageal squamous cell carcinoma.Methods: A total of 93 patients with esophageal squamous cell carcinoma first treated in our hospital by radiotherapy and surgeries from November 2010 to December 2013 were selected. They were then divided into a severe reaction group, a moderate reaction group and a mild reaction group according to the postoperative pathological examination results of tumor tissues. The expressions of TLR4 and NF-κB p65 in the tumor samples were detected by Western blotting.Results: Compared with the severe reaction group, the expression levels of TLR4 and NF-κB p65 in the moderate reaction group significantly increased (P<0.05). Similarly, the expression levels of the mild reaction group were significantly higher than those of the moderate reaction group (P<0.05).Conclusion: Reducing the expression levels of TLR4 and NF-κB p65 proteins may increase the radiosensitivity of patients with esophageal cancer. Besides, multi-center retrospective and prospective studies have verified that postoperative radiotherapy could evidently decrease regional recurrence rate and odds of lymph node metastasis.2 However, some patients are prone to resisting radiotherapy. Therefore, it is of great clinical significance to uncover the molecular mechanism of resistance of tumor tissues to radiotherapy and to propose individualized treatment plans for esophageal cancer. In this study, the relationship between TLR4 and NF-κB p65 protein expressions in tumor tissues after radiotherapy and clinical radiosensitivity of patients with esophageal squamous cell carcinoma was explored. The results provide valuable evidence for clinical treatment of esophageal cancer.China has the highest morbidity and mortality rates of esophageal cancer worldwide, of which squamous-cell carcinoma is most common. Most patients have entered the middle or advanced stage upon diagnosis, thus making the prognosis very unsatisfactory.General information: A total of 93 patients with squamous cell carcinoma in the middle thoracic esophagus , who were first treated in our hospital by radiotherapy and surgeries from November 2010 to December 2013, were selected. They all received radiotherapy before surgery. This study was approved by the institutional ethics committee of our hospital, and written consent was obtained from all patients. Exclusion criteria: The patients who had received chemotherapy and immunotherapy were excluded.Postoperative radiotherapy methods: The patients received radiotherapy at primary tumors and mediastinal lymphatic drainage areas by using SIEMENS/PRIMUS accelerator (6MXX) 6 MV-X rays. Double-spiral CT was used for targeting, and image transmission CMS XIO 4.6.4 system was employed to prepare treatment plans. Gross tumor volume (GTV) and clinical target volume (CTV) were defined by ICRU Report 50. GTV included esophageal tumors and surrounding swollen lymph nodes, and CTV included suspicious infiltrates and mediastinal lymphatic drainage areas around the esophageal tumor. In addition, a 5 mm planning target volume (PTV) outwards was generated. According to intensity-modulated radiation therapy protocol, the patients were treated with the dose of DT to 40 Gy/20 f/26 d, 2-3 weeks after which thoracotomy was performed.Diagnostic standards for radiosensitivity: Paraffin-embedded tumor tissues were subjected to HE staining according to standard operating procedure. The postoperative reactions were classified according to pathological standards and previous literature.3 (1) Mild reaction: Mild degenerative changes of tumor cells, decreased nuclear division, and infiltration of a small number of inflammatory cells and angiogenesis in tumors; (2) moderate reaction: disappearance of most tumor cells, and degenerative changes of residual tumors that were wrapped by granulation tissues; (3) severe reaction: complete disappearance of tumor cells, fibrosis in the tumor bed, reduced blood vessels, chronic infiltration of inflammatory cells, and scarring.Western blotting: The tumors resected during surgery were transferred in liquid nitrogen immediately, and stored in a -80°C refrigerator. Tumor tissues (100 mg) were collected from each case, the nuclear and total proteins in which were extracted according to the instructions of corresponding kits. The proteins were quantified by the BCA method. Protein samples (35 μg) were transferred onto a film after electrophoresis and blocked by 5% skimmed milk powders for 1 h. β-Actin, H3, NF-κB p65 and TLR4 antibodies were diluted by 5% milk (1:200), with which the film was incubated at 4°C overnight, washed with TBST and rabbit-anti goat secondary antibody diluted by 5% milk. Afterwards, the film was subjected to Western blotting according to ECL instructions by using 1:1 luminescent working solution, and gray scale values were analyzed by Image J software.Statistical analysis: All data were analyzed by SPSS 13.0. The numerical data were subjected to χ2 test, and the categorical data were subjected to univariate analysis of variance. P<0.05 was considered statistically significant.Relationship between radiosensitivity and clinical pathological characteristics: The age, gender and tumor length of the three groups were similar (P>0.05), but their differentiation degree, clinical staging and lymphatic metastasis were statistically significantly different (P<0.05). The patients with early clinical staging, high differentiation degree and without lymphatic metastasis had better outcomes  ((P<0.05) .4 endanger the patients who are not sensitive to radiotherapy. However, the mechanism for radiation resistance remains unclear. Hence, it is of great clinical significance to improve the prognosis by minimizing radiation resistance and by preparing individualized treatment plans. In this study, radiosensitivity was associated with differentiation degree of tumor tissue, clinical staging and lymphatic metastasis. After verifying the individual differences of radiosensitivity, the expressions of TLR4 and p65 were subjected to semiquantitative analyses to elucidate the molecular biological mechanism.It has been demonstrated that postoperative radiotherapy can degenerate esophageal cancer cells, decrease the proliferative activity and reduce the tumor volume, thus leading to vascular thrombosis and eliminating micrometastases. As a result, the odds of regional recurrence can be lowered, and subclinical lesions around the tumor can be controlled. Moreover, some patients may enjoy radical resections. Regional recurrence of esophageal cancer usually occurs at the periphery of the original tumor, which can mainly be ascribed to the unradical resection of tumor cells that have invaded adjacent organs and tissues. Such cells may, although highly sensitive to radiation due to high proliferative activity and oxygenation status,5 In general, the patients with high TLR4 expressions survived significantly shorter than those with low expressions and are significantly more prone to recurrence.6 Once TLR4 receptor is activated by ligand, it exerts biological effects by activating nuclear transcription factor NF-κB that can be activated by several DNA damage pathways, inhibit cell apoptosis, and dominate in the tumor radiation injury pathway.7 NF-κB mainly comprises Rel (cRel) and p65 (RelA) subunits, and p65 mainly exerts functional effects. IκB, the inhibitor of NF-κB, binds NF-κB through specific ankyrin repeats of the C-terminus. Meanwhile, IκB suppresses NF-κB from transferring to the nucleus by covering the nuclear localization region. Under resting conditions, NF-κB and IκB coexist as an inactive compound in the cytoplasm. Upon extracellular stimulation, however, IκB is phosphorylated owing to the activation of IκB kinase, which thus exposes the nuclear localization site of NF-κB. Accordingly, free NF-κB rapidly migrates to the cell nucleus and binds specific κB sequences to induce the transcription of relevant genes.8 NF-κB can bind DNA more actively in esophageal tumor tissues. In the meantime, significantly more RelA is expressed in tumor tissues than that in paracancerous tissues, while significantly less IκB is expressed. Besides, increase in the activity of NF-κB can induce the drug tolerance of esophageal tumor cells,9 so inhibiting its activity is able to enhance the radiosensitivity.10 In contrast, activation of NF-κB is bound to reduce the radiosensitivity of esophageal tumor cells. In vitro studies have shown that suppressing the NF-κB activity can remarkably augment the radiosensitivity of several tumor cells,11,12 indicating that it is crucial to suppress its activity. In this study, the expression levels of TLR4 and p65 were significantly higher in the highly sensitive group than those in the lowly sensitive group, suggesting that activation of the TLR4/NF-κB signaling pathway may play an essential role in the radiation resistance and that radiotherapy in combination with NF-κB activity-inhibiting drugs may boost the sensitivity.It has previously been confirmed that TLR4 was closely related with the lymphatic metastasis and clinical staging of esophageal cancer patients.in vitro and animal model experiments.It is well-established that the patients with severe reactions survive longer than those insensitive, so increasing the radiosensitivity of esophageal tumor tissues is important for improving the prognosis in clinical practice. In this study, the findings showed that the activity of the TLR4/NF-κB signaling pathway was closely associated with the radiosensitivity of esophageal cancer. Presumably, this pathway may be involved in the decreased radiation resistance of tumor tissues, which still requires TH and WF: Designed the study and revised the manuscript;ZXF, ZJ, CL, LJD and XEC: Collected and analyzed the data, and prepared the manuscript.


The present study aimed to determine the effects of chronic treatment with different doses of testosterone on endothelium–dependent coronary vascular reactivity in male rats. Adult male rats were divided into four experimental groups: control (SHAM), castrated (CAST), castrated and immediately treated subcutaneously with a physiological dose  or supraphysiological dose  of testosterone for 15 days. Systolic blood pressure (SBP) was assessed at the end of treatment through tail plethysmography. After euthanasia, the heart was removed and coronary vascular reactivity was assessed using the Langendorff retrograde perfusion technique. A dose–response curve for bradykinin (BK) was constructed, followed by inhibition with 100 μM L-NAME, 2.8 μM indomethacin (INDO), L-NAME + INDO, or L-NAME + INDO + 0.75 μM clotrimazole (CLOT). We observed significant endothelium–dependent, BK–induced coronary vasodilation, which was abolished in the castrated group and restored in the PHYSIO and SUPRA groups. Furthermore, castration modulated the lipid and hormonal profiles and decreased body weight, and testosterone therapy restored all of these parameters. Our results revealed an increase in SBP in the SUPRA group. In addition, our data led us to conclude that physiological concentrations of testosterone may play a beneficial role in the cardiovascular system by maintaining an environment that is favourable for the activity of an endothelium–dependent vasodilator without increasing SBP. Cardiovascular diseases (CVDs) constitute the most common cause of death worldwide, among which coronary artery disease assumes great importance . Recent Because men are more susceptible to cardiovascular events, testosterone has been associated with deleterious effects ,5. HowevTestosterone can exert rapid (“extra-nuclear”) vascular effects, including relaxation of the mesenteric bed  and meseIt has also been shown that testosterone may act through both endothelium–dependent  and endo2), and endothelium–derived hyperpolarizing factor (EDHF) [The endothelium is a cellular monolayer lining the inside of blood vessels. Previous studies conducted in the 1980s reported that the endothelium plays a functional role in vascular reactivity . This ror (EDHF) –20.While the direct action of testosterone on blood vessels has been well explored, the effects of chronic treatment on endothelium–dependent vascular function in the coronary circulation have not been fully elucidated. Claudio et al.  found thRattus norvegicus albinus) were reared at the animal facility in the Centre of Health Sciences (Centro de Ciências da Saúde–CCS) at UFES. All procedures were approved by the Ethics Committee on Animal Use (CEUA) of UFES under protocol No. 049/2012. The animals were housed in cages  and were provided food (Purina Labina) and water ad libitum. In addition, they were maintained under controlled conditions of temperature (22–24°C) and humidity (40–60%), with a 12/12 h light–dark cycle. The animals were randomly divided into four experimental groups: control (SHAM); castrated (CAST); castrated and treated with a physiological dose of testosterone (PHYSIO); and castrated and treated with a supraphysiological dose of testosterone (SUPRA).In this study, 10-week-old adult Wistar rats , the animals were placed in a supine position, fixed with surgical tape over a surgical bed. Before incision, disinfection of the testicular pouch was performed with iodized alcohol. Surgery was performed through an incision in the midline of the testicular pouch, and the testes were exposed by compression. After exposure of the testicles, the tunica vaginalis was opened, and the testes were ligated with absorbable sutures placed around the spermatic cord. Subsequently, the testes were removed, and the testicular pouch was sutured. The control group underwent sham surgery (SHAM) following the same procedures as the conventional surgery, except that the testicles were not removed. At the end of the procedure, the animals were given a dose of antibiotics  to prevent infection.Testosterone replacement  was performed for 15 days via subcutaneous injection of a dose of 0.5 mg/kg/day, mimicking a physiological concentration, as previously described , or 2.5 After a period of adaptation, the animals were placed in a heated chamber within a container and restrained with a pneumatic cuff attached to the proximal region of the tail. A sphygmomanometer was inflated and deflated automatically, and SBP was recorded using a transducer coupled to a computer, as previously described . The tem2.2H2O, 5.4 mM KCl, 2.5 mM MgSO4.7H2O, 2.0 mM NaH2PO4.H2O, 27.0 mM NaHCO3, 1.2 mM Na2SO4, 0.03 mM EDTA, and 11.0 mM glucose, heated continuously at 37°C in a water bath and pressurized with a carbogenic mixture containing 95% O2 and 5% CO2 in a saturation chamber to maintain a stable pH of 7.4. Coronary flow was maintained constant at 10 mL/min using a roller pump .At the end of treatment, the animals were anesthetized with chloral hydrate  and euthanized via decapitation for blood collection. The thoracic cavity was opened, and the heart was exposed, followed by removal of blood vessels. The heart was quickly transferred to the perfusion apparatus and isolated through cannulation of the aorta. Examination of the coronary vascular bed was conducted using the modified method of Langendorff . The isolated hearts were perfused with a nurturing solution containing 120 mM NaCl, 1.25 mM CaClThe baseline coronary perfusion pressure (CPP) was measured using a pressure transducer (AD Instruments MLT0380/A Reusable BP Transducer) connected in close proximity to the aortic perfusion cannula through which the coronary artery bed was perfused and connected to a digital data acquisition system (PowerLab System). Because the flow rate was maintained constant at 10 mL/min with a roller pump, the changes in CPP were directly related to changes in vascular resistance. A latex balloon at the end of a steel cannula was inserted into the left ventricle and connected to a pressure transducer (AD Instruments MLT0380/A Reusable BP Transducer) for measurement of the isovolumetric cardiac force. The balloon was pressurized with the aid of a glass syringe to maintain a preload of 10 mmHg.ω-nitro-L-arginine methyl ester  or 2.8 μM indomethacin , or combined inhibition using either 100 μM L-NAME + 2.8 μM INDO or 100 μM L-NAME + 2.8 μM INDO + 0.75 μM clotrimazole .After stabilization of the system for 40 min, baseline CPP was calculated, and a dose–response curve of bradykinin was generated , which was administered in bolus form (0.1 mL) at increasing concentrations  before and after perfusion with either 100 μM NAt the end of the study, the heart was removed from the equipment, and the cardiac chambers were carefully separated and weighed on a precision scale (SHIMADZU AUY 220) after 24 hours of oven drying at 100°C to obtain the dry weight.For determination of the lipid and hormonal profiles, 5 mL of blood was collected after decapitation. The blood was centrifuged (centrifuge Excelsa IV Model 280R) at 3,500 rpm for 15 minutes at 4°C, and the serum was collected and stored at –20°C. Assays for testosterone were performed based on electrochemiluminescence immunoassays using a Roche Diagnostics COBAS enzymatic kit. The assay sensitivity ranges were 0.025–15 ng/mL and limit of quantification were 0.120 ng/mL. The concentrations of total cholesterol (TC), triglycerides (TG), and high-density lipoprotein (HDL) cholesterol were measured using the enzymatic methods of the Colestat enzyme kit AA, TG Color GPO/PAP AA kit, and HDL cholesterol monophase AA Plus kit, respectively, with a Konelab model 600i spectrophotometer. The concentrations of low-density lipoprotein (LDL) cholesterol and very-low-density lipoprotein (VLDL) cholesterol were calculated using the Friedewald equation : VLDL chData analysis was performed using the statistical software GraphPad Prism 6. The data were expressed as the mean ± standard error of the mean (SEM). Comparisons between the groups were performed through one-way analysis of variance (one-way ANOVA), and the vasodilator response to bradykinin was evaluated through two-way analysis of variance (two-way ANOVA). In both cases, the post-hoc Tukey test was used, and a significance level of p<0.05 was established.Castration did not alter the weight of the right ventricle (from 1.03 ± 0.03 to 1.06 ± 0.02 mg/g) or left ventricle (from 3.99 ± 0.05 to 3.97 ± 0.05 mg/g) compared with the SHAM group. Furthermore, the weight of the heart chambers was not altered by replacement with different doses of testosterone.Unlike the changes observed for SBP, CPP was not altered in any of the study groups , as shown in Similarly, after adding the nurturing solution and inhibitors for the production of endothelium–derived relaxing factors, we observed a significant increase in CPP between the groups, although this difference was not significant, as shown in The main objective of this study was to evaluate the coronary vascular reactivity of normotensive rats after replacement treatment with different concentrations of testosterone. For this purpose, a relaxation curve was constructed using increasing concentrations of BK administered directly into the coronary vascular bed of rats .As shown in However, when NOS was inhibited with 100 μM L-NAME , the vasA similar result was observed when COX was inhibited with 2.8 μM INDO . InhibitCombined inhibition with L-NAME + INDO  further By analysing the hormonal profile , we confThe main finding of this study was the observed efficacy of treatment with physiological and supraphysiological doses of testosterone in preventing the damage caused by hormone deficiency in the endothelium–dependent coronary vasodilation induced by BK. Furthermore, castration was found to alter lipid and hormonal profiles and decrease body weight. However, replacement with testosterone restored all of the above parameters to levels similar to those found in the SHAM group. We also observed an increase in SBP in the group treated with a supraphysiological dose of testosterone.Our results indicate that body weight had decreased 15 days after castration compared with the SHAM group and that replacement therapy prevented this decrease. These data corroborate the results obtained by Bourghardt et al. , who alsOne possible explanation for the weight loss recorded in the CAST group may be related to decreased serum levels of triglycerides. Our results indicate that the concentrations of triglycerides and VLDL (responsible for the transport of triglycerides in the blood) were decreased after 15 days of castration. Moreover, testosterone decreases the activity of the enzyme lipoprotein lipase (LPL) , and modConsidering that LPL is associated not only with the hydrolysis of triglyceride molecules found in plasma lipoproteins, chylomicrons, and VLDL , but alsOur results also confirm previous results with respect to SBP, where castration and replacement therapy with physiological doses of testosterone were not found to affect SBP. However, SBP levels increased significantly in the SUPRA group. In this respect, other studies have shown that high doses of testosterone can increase blood pressure , whereasIt is well established that testosterone can stimulate the renin–angiotensin–aldosterone system (RAAS), which, through the formation of angiotensin II, can raise blood pressure . ReckelhIt has been shown that testosterone can modulate the metabolism, storage, release, and reuptake of norepinephrine in sympathetic neurons , which cAlthough SBP was modulated by testosterone, such modulation was not observed for CPP. Our results allow us to suggest that coronary vascular tone in normotensive rats may not be modulated by testosterone, in contrast to findings obtained for female hormones (oestrogen), as previous studies from our laboratory have demonstrated that castration decreases CPP in normotensive female rats .Although we did not observed changes in CPP in the experimental groups, the primary objective of this study was to evaluate endothelium–dependent vascular coronary reactivity mediated by BK. Our results clearly indicate that testosterone treatment was effective in preventing the impairment of vasodilation caused by castration compared with the SHAM group. Moreover, both the physiological and supraphysiological treatments not only prevented this decrease but also enhanced vascular coronary relaxation.The results of this study demonstrate for the first time that testosterone can modulate vascular reactivity in the endothelium–dependent coronary vascular bed in male rats. It is known that testosterone can directly modulate the CVS by relaxing vascular smooth muscle –12. NotaOther authors have shown that castration can decrease the expression of voltage–gated potassium channels , which iIn fact, our data suggest that castration somehow altered the vascular environment, possibly promoting endothelial dysfunction to the point that the animals exhibited an impaired response to BK. Previous studies have shown that sex hormones are involved in maintaining a healthy vascular environment. Accordingly, Claudio et al.  observedBecause the animals treated with testosterone experienced a degree of relaxation superior to that in the control animals, our objective was to evaluate the role of the endothelial mediators through which this relaxation occurred. For this purpose, the first step was to inhibit NOS with L-NAME (a non-specific NOS inhibitor). The result of this experiment revealed that incubation with L-NAME decreased BK–induced relaxation in the FISIO and SUPRA groups to levels similar to those found in the SHAM group, indicating the important role of NO in the dilation of the coronary arteries. In fact, testosterone can exert direct action on the coronary arteries by promoting vasodilatation, and this response often involves the formation of endothelial NO (via eNOS) and/or extra-endothelial sources of NO (via nNOS) . These f2 receptors, promoting the release of NO, PGI2, and EDHF [2 in relaxation. Previous studies from our laboratory have demonstrated the importance of vasodilator prostanoids in normotensive male rats compared with other vasodilator agonists [BK is a potent endothelium–dependent vasodilator peptide that acts via stimulation of endothelial Band EDHF –41. Accoagonists .Furthermore, as expected, the vasodilator response to BK in the coronary vascular bed of normotensive rats was completely abolished in all groups when the three endothelial mediators were inhibited. In this case, clotrimazole (an inhibitor of cytochrome P450) was added along with L-NAME and INDO. These data provide evidence of the important role of EDHF in BK-induced relaxation. The involvement of CYP metabolites, i.e., epoxyeicosatrienoic acids, as candidate EDHFs in coronary arteries has been demonstrated in previous studies from our laboratory, in both normotensive  and hypeIn summary, our data suggest that testosterone treatment can prevent damage due to hypogonadism in the studied model. Therefore, we suggest that the damage on the coronary vascular reactivity induced by extreme deficiency of male sex hormone, i.e. after castration, can be prevented by replacement of testosterone


Pelagia noctiluca (Scyphozoa) has been identified as direct causative agent for several documented fish mortality events both in Northern Europe and the Mediterranean Sea aquaculture farms. We investigated the effects of P. noctiluca envenomations on the gilthead sea bream Sparus aurata by in vivo laboratory assays. Fish were incubated for 8 hours with jellyfish at 3 different densities in 300 l experimental tanks. Gill disorders were assessed by histological analyses and histopathological scoring of samples collected at time intervals from 3 hours to 4 weeks after initial exposure. Fish gills showed different extent and severity of gill lesions according to jellyfish density and incubation time, and long after the removal of jellyfish from tanks. Jellyfish envenomation elicits local and systemic inflammation reactions, histopathology and gill cell toxicity, with severe impacts on fish health. Altogether, these results shows P. noctiluca swarms may represent a high risk for Mediterranean finfish aquaculture farms, generating significant gill damage after only a few hours of contact with farmed S. aurata. Due to the growth of the aquaculture sector and the increased frequency of jellyfish blooms in the coastal waters, negative interactions between stinging jellyfish and farmed fish are likely to increase with the potential for significant economic losses.Jellyfish are of particular concern for marine finfish aquaculture. In recent years repeated mass mortality episodes of farmed fish were caused by blooms of gelatinous cnidarian stingers, as a consequence of a wide range of hemolytic, cytotoxic, and neurotoxic properties of associated cnidocytes venoms. The mauve stinger jellyfish  In recent years, negative interactions between jellyfish blooms (JB) and marine finfish aquaculture have been reported. Such interactions have included mass fish mortalities with severe economic impacts on the aquaculture companies ,2. JellyPelagia noctiluca , also known as mauve stinger, is one of the most common stinging jellyfish species across the Eastern Atlantic and the Mediterranean Sea, producing major outbreaks with subsequently highly negative impacts on human activities, including caged finfish aquaculture  the impacts of different jellyfish densities on cultured fish health, and [III] the histological evolution of gill lesions over time following initial jellyfish sting treatment.To increase knowledge on impacts of gelatinous plankton blooms on Mediterranean caged fish species and support early monitoring of risks for aquaculture production, an experimental assay was set up to assess [I] the potential histopathological damage that This study was performed in accordance with the European Commission Directive 2010/63/EU. The experimental protocol was designed to comply with the European policy of the “3 Rs”  in aquatic animal experimentation and was approved by the Institut Supérieur de Pêche et d'Aquaculture de Bizerte (Research unit 05/ur/11-15), which is under the double supervision of the Tunisia’s Ministry for Agriculture and the Hydraulic resources, and of the Ministry for Higher education and the Scientific Research and Technology.Fish were monitored daily (early in the morning and during afternoon) over the complete experiment duration. Check-list including different humane endpoints was revised at group and also at individual level when necessary. The main established criteria were swimming behavior, skin pigmentation, frequency of opercular movements, ability of food uptake, weight loss, prostration, hyper-excitability and itching. The maintenance of animals during the experiment as well as the euthanasia procedure was monitored and carried out by trained and competent staff, in order to minimise animals’ suffering.Sparus aurata adult fish (mean weight of 200 ± 19.23 g) were obtained from “Tunisian Teboulba Fish” aquaculture facility and transported to the Institut Supérieur de Péche et d'Aquaculture de Bizerte, Tunisia (ISPA). Fish were homogeneously distributed in 8 circular tanks of 300 litres each (fish stocking density of around 9 kg m-3) and allowed to acclimate for one week before starting the experiment. All tanks were supplied by a continuous flow  of double-filtered  seawater (FSW). The water circulation flow was kept at natural sea temperature of 15.5 ± 1.0°C and 36.8 ± 0.3 salinity) with aeration to keep dissolved oxygen at 100% saturation. Throughout the experiment, the fish were fed daily with standard commercial pellets (Skretting S.A.) and maintained under a natural photoperiod .A total number of 136 Pelagia noctiluca jellyfish is not an endangered or protected species. Specimens from Bizerte gulf were collected without the need of a permit because sampling was never conducted in a restricted marine area.Jellyfish (4.5 ± 0.9 cm bell diameter) were collected by a dip net the day before the start of the experiment from the Channel of Bizerte (Tunisia) and maintained in 25 litres buckets with FSW and at low density for one day. P. noctiluca at low (LJ), medium (MJ), and high jellyfish densities (HJ): 3, 7 and 15 jellyfish per tank with 18 experimental fish . These densities were predetermined to reproduce a range of different jellyfish concentrations observed during P. noctiluca bloom periods in Tunisian waters and Sicily Channel (unpublished observations). A 1-mm stainless steel mesh was placed at the outlet of each tank preventing jellyfish pieces to spill out the experimental tanks.To simulate a realistic encounter between jellyfish that had been pressed by currents against aquaculture cages and cultured fish, jellyfish were chopped into small (≥ 1 cm) pieces immediately prior to the start of the jellyfish exposure. The four treatment groups consisted of two control tanks (without jellyfish) and six tanks with chopped P. noctiluca jellyfish in surface waters, following sunset and the diel vertical migration of their crustacean prey [The experiment began when jellyfish pieces were placed simultaneously in all treatment tanks with fish. The maximum fish-jellyfish interaction lasted 8 h; after that, all jellyfish pieces were removed using a 200-μm mesh hand net. The exposure time to jellyfish tissue of 8 h was used to represent the minimum night time with ean prey –28.Fish health was monitored nine times during the experiment: shortly before jellyfish incorporation to the fish tanks (0 h), during fish-jellyfish contact (3h), one hour after the removal of the jellyfish (9h), and six later times, 24 and 48h; 1, 2, 3 and 4wk, respectively before the end of the experiment at 4 weeks. At the highest jellyfish density sampling was not carried out at 24 h, 3 and 4 weeks because of the shortage of experimental individuals and fish mortalities. At each sampling time, 4 fish were randomly sampled from each treatment group (two per tank), anesthetised and then killed according to the current animal care rules using a lethal dose of UNICAINE 2% (lidocaine-HCl 500 ppm) . ImmediaInterpretation of the gill damage was based on a recently developed gill histopathology scoring system ,12, ratiA Shapiro-Wilk test indicated that the assumptions of normality were violated ; therefore, differences among treatments and among sampling weeks were tested using the non-parametric one-way Kruskal-Wallis test (SPSS v.20.0). Significant results were further tested by pairwise post-hoc comparisons , adjusted for type I error, and Similarity percentages analysis, SIMPER (PRIMER 6).Gills from the control fish group without jellyfish retained a normal morphology throughout the experiment. Each gill arch supported many distinct and regular filaments arranged perpendicularly in two rows and without significant lesions. In contrast, gross pathology in fish exposed to jellyfish pieces was observed throughout the experiment , with thAt 3 h after initial contact with jellyfish pieces, fish gills already showed abrasion of lamellar filaments . After 21 = 25.267, p = 0.001). Gill scores also differed significantly among the groups treated with jellyfish . The gill scores in the LJ density group showed no significant differences throughout the experiment , with average scores of 2.25 ± 0.9 (SE). For the MJ density group, significant gill lesions were observed 1 week after the start of the experiment , with scores peaking after 2 weeks (gill score 6 ± 1.5 SE). Significant gill damage was observed immediately in the HJ density group, only 3h after the exposure to jellyfish began . Those high scores continued over time with a peak after 48h (6 ± 1.3 SE)  4+). All these troubles leaded death of fish. In the MJ density group, gill scores decreased during the third and fourth week of sampling, mainly because of reduction in the percentages of hyperplasia and cellular anomalies. By contrast, fish from the LJ density group presented mild damage during the experiment, principally represented by hyperplasia and lamellar fusion  and the Strait of Messina  [P. noctiluca on finfish aquaculture farms. Our results showed that jellyfish stings can severely affect caged S. aurata fish by causing significant gill damage shortly after contact with jellyfish tissues and subsequent deterioration on fish health.Frequency of occurrence and abundance of   ,33,34. OSalmo salar) during blooms of P. noctiluca and Aurelia aurita scyphomedusae in northern Europe [S. salar to realistic A. aurita jellyfish bloom densities showed significant and increasing gill damage starting 24 h after the initial contact [Comparable gill damage was observed previously in farmed salmon , mild damage to fish gills were observed. Conversely, at higher jellyfish concentrations (≥ 25 jellyfish m-3) impacts ranged from moderate damage, leading to potential effects on the fish metabolism, to more severe consequences including death, due to high levels of lesions and respiratory distress [Here we investigated the intensity of gill damage on cultured sea bream at increasing Tenacibaculum sp. was confirmed, due to the absence of filamentous bacterial mats on the necrotic patches [Three weeks after the initial exposure to jellyfish, fish from the medium density group showed early signs of tissue repair in the gills. Recovery was characterized by significant decreases in the percentages of lamellar hyperplasia and fusion, in observed inflammatory reactions, and disappearance of cellular anomalies. At last, recovery of tissue integrity was observed in fish in the MJ density group, whereas fish from HJ density died 2–3 weeks after exposure to jellyfish. Exposure to HJ density led to intense and increasing gill damage, eventually impairing homeostatic mechanisms and adaptive physiological responses . Non bac patches . OverallPelagia noctiluca in the Mediterranean Sea occurs in large swarms reaching densities over 100 medusae m-3 for prolonged periods (up to weeks), with temporary aggregations caused by wind, currents, coastal geomorphology and jellyfish behaviour containing up to 600 medusae m-3 [-3). Furthermore, shortly after sexual reproduction—in springtime—large swarms of ephyrae and juvenile jellyfish are regularly encountered in the Southern Tyrrhenian Sea (Aeolian islands), with much higher densities, up to several thousands of individuals m-3; . Temporary paramount densities may therefore represent a key threat affecting the physiological integrity and health of fish living in sheltered areas where extremely high jellyfish aggregations occur, such as bays or fjords .Further investigations are required to clarify whether the potential rise of both temperature and jellyfish numbers in a global change scenario may exacerbate negative impacts not only on farmed fish, but also on wild fish populations ,45,46.P. noctiluca jellyfish could be detrimental to the health of caged fish, causing minor but significant gill lesions, which may progress over time and be worsened by bacterial infections. Investigation of the different effects of P. noctiluca blooms will enable estimation of the response time required by aquaculture facilities to undertake appropriate countermeasures that could differ in magnitude according to the damage level. Due to the recent and projected future growth of the aquaculture sector [The consequences of episodes of jellyfish proliferation can be of high importance for aquaculture, considering they could affect not only fish health, but also the growth and quality of caged fish ,30. The e sector  and the e sector ,48, negae sector .