It is well known how selective attention biases information processing in real time, but few work investigates the aftereffects of prolonged attention, let alone the underlying neural mechanisms. To examine perceptual aftereffect after prolonged attention to a monocular pathway, movie images played normally were presented to normal adult’s one eye (attended eye), while movie images of the same episode but played backwards were presented to the opposite eye (unattended eye). One hour of watching this dichoptic movie caused a shift of perceptual ocular dominance towards the unattended eye. Interestingly, the aftereffect positively correlated with the advantage of neural activity for the attended-eye over unattended-eye signals at the frontal electrodes measured with steady-state visual evoked potentials. Moreover, the aftereffect disappeared when interocular competition was minimized during adaptation. These results suggest that top-down eye-specific attention can induce ocular dominance plasticity through binocular rivalry mechanisms. The present study opens the route to explain at least part of short-term ocular dominance plasticity with the ocular-opponency-neuron model, which may be an interesting complement to the homeostatic compensation theory. Our brain can selectively process a subset of input that is most relevant to behavioral goals. This confined sensory processing is known as selective attention . A pioneTo date, it is known that selective attention can bias information processing by enhancing processing of attended stimulus  and suppFor this goal, we developed a “dichoptic-backward-movie” adaptation paradigm enlightened by the selective-looking paradigm . During Note that the movie plot of backward movie images was illogical and hard to follow. Subjects were thus instructed that their primary task was to try their best to follow the logic of the regular movie and ignore the superimposed backward movie. Therefore, we believe that goal-directed selective attention was predominantly focused on the regular movie. Here the eye to which the regular movie images were presented was called the “attended eye,” and the opposite eye was called the “unattended eye.”As voluntary attention can increase the proportion of time of perceiving inputs to the attended eye in binocular rivalry , we hypoTo test this hypothesis, in experiment 1, we measured perceptual ocular dominance before and after 1 h of watching the dichoptic movie by using a binocular rivalry task, which had been frequently adopted in monocular deprivation studies . ExperimTwenty-two adult humans  participated in experiment 1a. Sixteen subjects  participated in experiment 1b. In all the experiments, all subjects had normal or corrected-to-normal vision, were unaware of the experimental hypotheses, and gave informed consent. All experimental procedures were approved by the Institutional Review Board of the Institute of Psychology, Chinese Academy of Sciences. The numbers of subjects were predetermined based on the sample sizes for published studies in this field.2). While for other subjects and other conditions, a gamma-corrected 21-inch Sun GDM 5510 CRT monitor (at the refresh rate of 75 Hz and the mean luminance of 42.8 cd/m2) was used. The spatial resolution of both monitors was 1,024 × 768 pixels. Subjects viewed the stimuli through a mirror stereoscope from a distance of 70 cm, with their heads stabilized in a chinrest. Experiments were conducted in a dimly lit room.The visual stimuli were programmed in MATLAB using the Psychtoolbox extensions . For 8 sBinocular rivalry stimuli were composed of 2 orthogonal sine-wave grating disks, oriented ±45° . They were presented dichoptically and foveally with a central red fixation point (0.07° in diameter) and a high contrast checkerboard “frame”  to facilitate stable binocular fusion.Each binocular rivalry test was composed of sixteen 60-s rivalry trials. Each trial started with a 5-s blank interval. Then, the rival stimuli were presented for 55 s. Subjects were required to hold down 1 of the 3 keys  to report their perceptions . The orientation related to each eye was kept constant within a trial but randomly varied across the trials.http://www.aijianji.com/medownload.htm), concatenating the 3 segments to produce a backward movie file. That is, for each 20-min segment, the backward version of the movie was formally identical to the original one except for the absence of a logical movie plot.During 1 h of adaptation, subjects passively viewed dichoptically presented movie images  surroundFor the first 6 subjects, the audio track always synchronized with the regular movie images (i.e. the “Synchrony” condition). To examine the potential contribution of audiovisual integration, we added another 2 different adaptation conditions for the 16 later recruited subjects. In the “Asynchrony” condition, the audio track was 5 s ahead of the movie image. Since the time window of audiovisual integration is only several hundred milliseconds wide , 5 s is Before the formal experiment, all subjects practiced 3 binocular rivalry tests per day (with a 10-min break in between) for 3–7 days, to ensure a stable performance of binocular rivalry . BecausePerceptual eye dominance was determined by the last 3 sessions of the “binocular rivalry practice” stage, with the dominant eye being the one that showed the longer summed phase durations.The formal experiment included 4 phases: (1) 5 warm-up binocular rivalry trials , (2) a preadaptation binocular rivalry test, (3) 1 h of dichoptic-backward-movie adaptation, and (4) a post-adaptation binocular rivalry test.Subjects completed 4 sessions for each adaptation condition, with each eye attended in 2 sessions and the sequence counterbalanced. This resulted in totally 12 adaptation sessions. The order of the 3 adaptation conditions was balanced across subjects.TPost + TMpost/2)/(TPre + TMpre/2), where TPost and TPre represented the summed phase durations of the exclusive percept for one eye during the post- and preadaptation tests and TMpost and TMpre represented the summed phase durations of the mixed percepts during the post- and preadaptation tests. If perceptual eye dominance shifts towards one eye after adaptation, the duration ratio would be greater than 1, and vice versa.To quantify the perceptual dominance of each eye, we calculated the summed phase durations of the exclusively monocular percepts and the mixed percepts across all the trials, respectively. Then, we computed the “duration ratio” for each eye using the formula  to ensure that watching movies was still the primary task. To determine the timing of the blobs, we divided the total adaptation period (60 min) into 24 segments (each 150 s long). Twelve of them were randomly assigned to each eye. A blob was presented at a random time within the intermediate interval (50 s long) of a segment, which would fade in within 0.2 s and after 5 s fade out also within 0.2 s. Before and after adaptation, binocular rivalry tests were not conducted in this experiment.Thirty-four volunteers received a screening test first . Among them, 22  passed the screening and then completed the formal experiment.2. Other apparatuses used in the experiment were the same as in experiment 1.In the EEG experiment, Stimuli were presented on a gamma-corrected 21.5-inch LEN LS2224A LCD monitor at a resolution of 1,024 × 768 pixels and a refresh rate of 60 Hz. The mean luminance of the display was 43.9 cd/mWe used the frequency tag technique to obtain the SSVEP signal for each eye throughout the experiment . Movie images in each eye were contrast-reversed flickering. The flickering frequency was 3 Hz for the unattended eye and 3.75 Hz for the attended eye. Each subject completed an experimental session and a control session . During the adaptation phase  to maximize the shift of ocular dominance. To avoid the potential frequency-dependent variances (for details see Section 3.3.3), each subject completed a control session in addition to the experimental session. The experimental and control sessions were conducted at least 24 h apart, with the order counterbalanced across subjects.EEG data were collected in a dark and confined laboratory using the Neuroscan Synamps2 system. Whole-brain signals were recorded by means of 64-channel (Ag-AgCl) electrode caps placed according to the International 10-20 system. The EEG signals were filtered from 0.05 to 100 Hz and an additional notch filter at 50 Hz and digitized at a sampling rate of 1,000 Hz. Impedances were kept below 5 kΩ. All electrodes were referenced online to the left mastoid (M1) electrode. Vertical electrooculogram was recorded by 2 electrodes placed above and below the left eye, with horizontal electrooculogram beyond the lateral canthi of both eyes.UAET, AET, and MT represented the summed phase durations for perceiving the stimulus in the unattended eye, stimulus in the attended eye, and mixed percepts, respectively.We calculated the eye-ratio index for the pre- and post-test using the formula Off-line analysis was conducted using customized MATLAB codes and FieldTrip . The M1 Fast Fourier transform was applied to extract the power spectra. The SNR was computed as the ratio of the power at the tagged frequency  to the average power of the 20 surrounding  frequency bins . We usedt-test  passed the screening and completed the formal experiment.To minimize binocular rivalry as much as possible during adaptation, we dichoptically presented the regular and backward movie images to the 2 eyes in alternation . Thus, aFor this goal, each subject first conducted an interval test for estimating the shortest appropriate interval . The intBased on a pilot experiment, we selected 3 candidate intervals: 250 ms, 400 ms, and 550 ms. If no binocular rivalry occurred for a subject at 250 ms or binocular rivalry occurred even at 550 ms, the interval was reduced or increased accordingly (100 ms or 700 ms). The interval test contained 3 blocks, with each corresponding to one of the 3 intervals. Each block consisted of 3 trials.The ideal interval for each subject was determined by choosing the shortest interval at which the switch rate of responses was basically equal to the alternating rate of gratings. This was also verified by the subjective report that binocular rivalry did not occur. For 4 subjects, the ideal interval was 400 ms, while 700 ms for 3 subjects and 550 ms for the rest of all subjects. In the formal experiment 3, the dichoptic movie was presented in alternation across the 2 eyes at each subject’s ideal interval.It should be noted that using the ideal interval one would perceive the 2 video streams alternating roughly twice per second . Because only the regular movie was logic and synchronized with the sound, one would be able to keep watching the regular movie stream while ignoring the frequent and short interruptions by the backward movie stream. We had verified this by ourselves before running experiment 3. This was also confirmed by all subjects’ subjective reports. Moreover, all subjects were able to retell the movie logic. All this evidence can demonstrate that subjects could pay attention to the regular movie during adaptation. We could not include a blob detection task in experiment 3 because the ideal interval was too short (~500 ms), making it very difficult to present a monocular blob as in experiment 1b in which the duration of a blob was 5.4 s in total.Other stimuli and procedures were similar to those in experiment 1.Experiment 3 consisted of 3 phases: (a) 3–7 days of practice on the binocular rivalry task, (b) interval test, and (c) 2 adaptation sessions. In each adaptation session, 5 binocular rivalry trials were first completed as a warm-up. A binocular rivalry pre-test  was then conducted, followed by a 1-h adaption to the alternating version of dichoptic movie . During F = 25.06, P < 0.001, η2 = 0.63) showing a higher duration ratio for the unattended eye than for the attended eye. This indicated that the balance between the 2 eyes was shifted towards the unattended eye after adaptation. There was also a significant main effect of Eye Dominance  = 10.19, P = 0.006, η2 = 0.41), but not Adaptation Condition  = 1.86, P = 0.19, η2 = 0.11, Greenhouse–Geisser corrected). The 2-way interactions were all nonsignificant . Interestingly, the 3-way interaction was significant  = 4.37, P = 0.022, η2 = 0.23).Here we focus on the 16 subjects who completed all adaptation conditions (see the t(15) = 2.73, P = 0.016, Cohen’s d = 0.68, 95% confidence interval (CI) = [0.01 0.08], PFDR = 0.048, false discovery rate (FDR) correction); whereas the duration ratio for the attended eye in both the “No-sound” and “Synchrony” conditions was smaller than that in the “Asynchrony” condition  = −2.50, P = 0.027, d = 0.63, 95% CI = , PFDR = 0.048; “Synchrony” vs. “Asynchrony,” t(15) = −2.33, P = 0.032, d = 0.58, 95% CI = , PFDR = 0.048).As shown by the simple effect analysis, when the dominant eye viewed the regular movie during adaptation, the duration ratio for the unattended eye was significantly larger in the “No-sound” condition than in the “Asynchrony” condition (t(15) = 2.73, P = 0.015, d = 0.68, 95% CI = [0.03 0.25], PFDR = 0.045). No significant difference was found between other paired comparisons (PsFDR = 0.236).To further understand the 3-way interaction, we calculated an “adaptation score.” Specifically, for each Eye Dominance level (Dominant vs. Nondominant), the duration ratio for the unattended eye was divided by that for the attended eye, yielding an eye-status change. The eye-status change for the Dominant level was further divided by that for the Non-dominant level to produce an adaptation score for each adaptation condition. Thus, larger adaptation score means that ocular dominance shifts towards the unattended eye to a larger extent when the dominant eye views the regular movie than when the nondominant eye does. We found that the adaptation score in the “No-sound” condition was significantly larger than that in the “Asynchrony” condition  = 134.91, P < 0.001, η2 = 0.90) showing a substantially higher detection accuracy for the attended eye (M = 62%) than for the unattended eye  = 0.24, P = 0.631, η2 = 0.02) nor the Eye status × Eye dominance interaction  = 0.46, P = 0.510, η2 = 0.029) was significant. These results indicated that the regular movie images dominated the subject’s perception in most of the time, regardless of whether they were presented to the dominant eye or to the nondominant eye. Therefore, we believe that subjects mostly paid attention to the attended eye when watching the dichoptic movie.For each experimental condition, the summed number of detected trials across sessions was divided by the total number of trials (24), yielding a detection percentage. A 2 (Eye Dominance: Dominant vs. Non-dominant) × 2 (Eye Status: attended vs. unattended eye) × 3  repeated measurements ANOVA was performed. As expected, we found a significant main effect of Eye Status  = 7.02, P = 0.003, η2 = 0.32) showing better performances for the “Synchrony” (t(15) = 2.69, P = 0.017, d = 0.67, 95% CI = [0.02 0.16], PFDR = 0.026) and “Asynchrony” (t(15) = 3.37, P = 0.004, d = 0.84, 95% CI = [0.04 0.20], PFDR = 0.013) conditions as compared to the “No-sound” condition. This suggested that the detection performances were worse when subjects watched the silent movies. Though we are not certain, this result pattern presumably reflects subject’s lower arousal level in the “No-sound” condition.In addition, we found a significant main effect of adaptation condition  = 73.60, P < 0.001, η2 = 0.83, F = 4.66, P = 0.017, η2 = 0.24). Paired t-test indicated that the eye-status difference (attended-eye minus unattended-eye) of the normalized detection percentage was significantly larger in the “Synchrony” condition than in the “Asynchrony” condition (t(15) = 3.75, P = 0.002, d = 0.94, 95% CI = [0.17 0.62], PFDR = 0.006). We did not find a significant difference of “Synchrony” vs. “No-sound” (t(15) = 1.48, P = 0.160, d = 0.37, 95% CI = [−1.07 0.59], PFDR = 0.202) and “No-sound” vs. “Asynchrony” (t(15) = 1.33, P = 0.202, d = 0.33, 95% CI = [−0.09 0.41], PFDR = 0.202). Other main effects and interactions were not significant .To reduce the influences of arousal by sound, we normalized the detection percentage by dividing it by the mean detection percentage in each adaptation condition, then conducted the same repeated measurements ANOVA. We found a significant main effect of Eye Status  than in the pre-test  = 4.16, P < 0.001, d = 0.89, 95% CI = [0.06 0.19]), again suggesting that adaption shifted the perceptual ocular dominance towards the unattended eye  than to the unattended eye  = 6.49, P < 0.001, d = 1.38, 95% CI = [0.26 0.51], As expected, detection percentage was higher when the blob was presented to the attended eye  = 33.00, P < 0.001, η2 = 0.61) showing higher SSVEP amplitude for the unattended-eye condition than for the attended-eye condition. No other effect was significant .A 2 (Test Phase: pre- vs. post-test) × 2 (Eye Status: attended vs. unattended eye) repeated measurements ANOVA on the average SSVEP amplitude of all EOIs revealed a significant main effect of Eye Status . Accordingly, the significant main effect of Eye Status should not be explained by ocular dominance. In fact, as shown in Restricting the analysis on the occipital EOIs did not show any significant effect  were presented to both eyes, and the SSVEP amplitude for the unattended-eye condition was higher than that for the attended-eye condition , it is ut-tests showed that the amplitude index was larger for the attended eye than for the unattended eye in the occipital EOIs (t (21) = 2.50, P = 0.021, d = 0.53, 95% CI = [0.01 0.10], t (21) = −1.30, P = 0.207, d = 0.28, 95% CI = [−0.03 0.01]), suggesting that the regular movie images produced significantly greater visual responses than the backward ones.To avoid the potential frequency-dependent variances, we divided the SSVEP amplitudes for the experimental session by those for the control session to obtain a normalized amplitude index for the occipital and frontal EOIs, respectively. Paired Ps > 0.122).We then explored if there was any functional connectivity between the frontal and occipital EOIs. The adaptation phase included three 20-min blocks. Within each block, the low-level inputs for the 2 eyes were identical (except for the image sequence). Thus, for each of the 4 conditions, i.e. 2 (tagged frequency: attended eye vs. unattended eye) × 2 , we computed the SSVEP amplitude for each block in each subject, yielding 66 data points which were then subjected to the subsequent analysis. All the data followed normal distribution as evaluated by Kolmogorov–Smirnov tests . Moreover, for all possible combinations, the frontal SSVEP amplitudes positively correlated with the occipital SSVEP amplitudes . On the premise of linear relationship between these data  and occipital sites . The local connectivity within the same electrode could be caused by some trivial artifacts (e.g. fluctuations in impedance), which is not of our research interest. More important, we found a fronto-occipital connectivity for the attended-eye frequency . These results indicated that the frontal site played a key role in coordinating the occipital signals elicited by both the regular and backward movie images, which will be discussed in detail later.The results showed a local connectivity between the attended- and unattended-eye conditions within the frontal , no significant correlation was found between the frontal and occipital EOIs (PsFDR > 0.977), thus ruling out the account of flickering frequency.To exclude the possibility that the flickering frequencies caused the fronto-occipital connectivity pattern, we did the same analysis for the control session. Despite significant correlations between the attended- and unattended-eye conditions within the same EOIs . Because the frontal activity should be more related to higher cognitive functions (e.g. attention) than lower visual processing, these results further support the account that selective attention participates in reshaping ocular dominance in the present adaptation paradigm.Next, we investigated the relationship between perceptual ocular dominance plasticity and attention-related neural activity during adaptation. We first calculated an ocular-dominance-plasticity index (ODPI) by dividing the eye-ratio index for the post-test by that for the pre-test. An ODPI greater than 1 means a shift of ocular dominance towards the unattended eye following adaptation, with larger value indicating larger shift. To calculate a neural-activity index (NAI) related to selective attention, for each region of EOIs we divided the normalized amplitude index for the attended-eye frequency by that for the unattended-eye frequency. Although the movie images in either eye could drive SSVEP signals, selective attention to the regular movie is expected to strengthen (weaken) the signals elicited by the regular (backward) movie images. Thus, larger NAI would mean stronger attentional effect at the corresponding cortical site. Interestingly, we found a significant correlation between the ODPI with the frontal NAI  particularly in the frontal site, an area often related to high-level cognitive functions.One thing unclear is to what extent this attention-driven ocular dominance plasticity is related to binocular rivalry mechanisms, considering that dissimilar movie images in the two eyes always rivaled. Therefore, experiment 3 adopted video-alternating stimuli presented monocularly to reduce interocular competition , with tht-test did not show a significant difference of the eye-ratio index between the pre-test  and post-test  = 1.32, P = 0.206, d = 0.33, 95% CI = [−0.02 0.10] Paired The present study introduces a “dichoptic-backward-movie” adaptation paradigm. Subject’s one eye viewed the regular movie, with the opposite eye viewing the backward movie. Experimental instructions and subjects’ motivations required allocating attention to the eye that viewed the regular movie, which was further confirmed by the superior detection performances and SSVEP amplitudes for the attended eye. Importantly, 1 h of adaptation to this dichoptic movie biased the eye dominance in favor of the unattended eye.There were crucial clues pointing to a critical role of top-down attention in driving the aftereffect . Firstly, the aftereffect in experiment 1a was the strongest in the “No-sound” condition (when the dominant eye viewed the regular movie). Because of lacking auditory cues, subjects in this condition must rely solely on visual inputs to identify the more logic episode and more predictable sequence among the 2 video streams and to maintain attention on the regular video stream. Considering the correspondence between attentional effort and aftereffect, top-down attention is thought to play a key role in producing the aftereffect.Secondly, experiment 2 disclosed an interesting correlation in the frontal site between the size of behavioral aftereffect and the biased neural activity in favor of the attended eye during adaptation. This finding highlights the possible contribution of frontal attentional rather than pure visual mechanisms to the aftereffect. One may question that the frontal activity might not directly reflect the attentional effect. Here we first exclude the account of eye movement, since eye movement-related activations should not be revealed through frequency tagging. Actually, any brain activity, as long as not oscillating at the flickering frequencies as the movie images, would be missed out through frequency tagging. For instance, our subjects certainly paid attention to the audio stimuli during adaptation, yet no electrode at the temporal site showed a reliable response. Given this common sense, we admit that frequency tagging cannot exhaustively detect all attention-related brain activities, simply because not every attention-related neural response oscillates like the visual responses elicited by the movie images.Then what did the frequency-tagged frontal response likely reflect? An inspection of the frontal EOIs suggests that the frontal activity was mainly located at the frontal pole and medial frontal cortex. These brain areas have been proposed to be involved in humor processing and appreciation , semantiInterestingly, in the adaptation phase, we found a positive fronto-occipital connectivity for the attended-eye frequency and a negative connectivity between the frontal response for the attended-eye frequency and the occipital response for the unattended-eye frequency. However, in the control session, no significant correlation was found between the frontal and occipital EOIs, excluding any account of flickering frequency or trivial artifacts for the connectivity results in the adaptation session. Presumably, the frontal areas were responsible for analyzing the comedy logic based on the visual inputs; and the video information temporarily held in the frontal areas was also likely frequency-tagged . However, useful input signals were always from the attended eye. Therefore, the positive fronto-occipital connectivity might reflect an attentional enhancement coordinating the frontal response for the attended-eye frequency with the input signals from the attended eye, whereas the negative fronto-occipital connectivity might reflect a suppression on the input signals from the unattended eye. These connectivity findings together with the behavior–SSVEP correlation result strongly suggest that the prolonged eye-specific attention was responsible for the aftereffect.Nevertheless, as shown by experiment 3, attention cannot work alone to bias perceptual ocular dominance and probably has to co-work with binocular rivalry mechanisms, since the aftereffect disappeared when interocular competition was substantially reduced. Therefore, we tend to explain our findings in the framework of the ocular-opponency-neuron model of binocular rivalry . AccordiFor simplicity, suppose the right eye was the attended eye. Then in experiment 3, the R-L opponency neurons would activate whenever the regular movie was presented to the right eye and blank field to the left eye. Whenever the contrary occurred, the L–R opponency neurons would activate. Since the summed duration was equal for either type of interval, the L–R and R–L opponency neurons would adapt to the same extent. Thus, the relative mutual inhibition of the 2 eyes’ inputs would be similar in the post- and pre-tests.In experiments 1 and 2, however, dissimilar movie images were simultaneously presented to the 2 eyes, causing interocular competition. Attending to the regular movie strengthened (suppressed) the neural responses of monocular neurons for the right (left) eye, allowing the regular movie to be perceived for most of the time. Therefore, the R–L opponency neurons adapted to a larger extent than the L–R opponency neurons. This would cause the unattended eye (i.e. left eye) to receive less inhibition from the R–L opponency neurons in the binocular rivalry post-test, thus shifting the ocular dominance towards the unattended eye.Moreover, the absence of effect in the EEG post-test further supports our account. Unlike the adaptation phase and behavioral pre- and post-tests, the binocularly compatible (or fused) stimuli were presented to the 2 eyes in the EEG pre- and post-tests. Processing of such binocularly fused stimuli were least affected by opponency neurons, based on the definition of this type of neurons. Therefore, the adaptation effect of the opponency neurons in experiment 2 could not be manifested by using binocularly fused testing stimuli. As a direct comparison, our previous monocular deprivation study also adopted binocularly fused flickering stimuli in the EEG pre- and post-tests . HoweverIntuitively, larger shift of ocular dominance should be associated with more imbalanced perceptions of each eye’s inputs during adaptation, which may be associated with more imbalanced neural responses between the 2 eyes. Although we found stronger SSVEP amplitude index for the attended eye than for the unattended eye, unfortunately, the ODPI did not significantly correlate with the occipital NAI. Instead, it positively correlated with the frontal NAI. These results imply that the frontal attentional system somehow modulated the AE-UAE ocular opponency neurons . Accordingly, even if there was no large imbalance of monocular visual responses during adaptation, sufficiently strong attentional enhancement on the AE-UAE opponency neurons could still produce a large aftereffect. This bold speculation agrees with the finding in experiment 1 that the high-demanding “No-Sound” condition did not show the largest interocular difference of detection performance , yet it generated the largest aftereffect.The present study opens the route to explain at least part of short-term ocular dominance plasticity with the ocular-opponency-neuron model . Our adaMB developed the study concept. SF, LL, and JZ performed the research. SF, LL, and MB designed the research, analyzed the data, and wrote the paper. All authors approved the final version of the manuscript for submission.This research was supported by the Ministry of Science and Technology of China (2021ZD0203800) and the National Natural Science Foundation of China (31871104 and 31830037).Conflict of interest statement. The authors declared no conflicts of interest.Supplemental_Material_bhac116Click here for additional data file.


Purpose: this study aimed to examine the user satisfaction and reuse intention of the elderly for neighborhood sports facilities in South Korea during the COVID-19 pandemic. Methods: this study surveyed 386 Korean elderly individuals aged ≥ 65 years, who were users of neighborhood sports facilities, from 1 May to 31 August 2022. A total of 386 questionnaires were used for data analysis, which was carried out using SPSS 23.0 statistical software. Descriptive statistics of the mean, standard deviation, and frequency distribution were used at the descriptive level. Moreover, one-way analysis of variance (ANOVA), Scheffe’s post hoc pair-wise comparison analysis, Pearson’s correlation coefficient, and multiple regression analysis were used at the inferential level. The significance level of these tests was considered for less than 0.05. Results: the mechanistic and humanistic service factors of neighborhood sports facilities affected user satisfaction and reuse intention. Furthermore, user satisfaction of the elderly during the COVID-19 pandemic had a positive effect on reuse intention. Conclusion: this study confirmed that the service quality characteristics of neighborhood sports facilities during the COVID-19 pandemic had a positive effect on user satisfaction and intention to continue to exercise among the elderly. With rapidly rising human life expectancy , South KThis study used the SERVQUAL model to verify the hypotheses, which was developed by Parasuraman, Zeithaml, and Berry . SERVQUAH1: The service quality of neighborhood sports facilities shall have a positive effect on user satisfaction.H1-1: Mechanistic service factors shall have a positive effect on user satisfaction.H1-2: Humanistic service factors shall have a positive effect on user satisfaction.H2: The service quality of neighborhood sports facilities shall have a positive effect on reuse intention.H2-1: Mechanistic service factors shall have a positive effect on reuse intention.H2-2: Humanistic service factors shall have a positive effect on reuse intention.H3: User satisfaction shall have a positive effect on reuse intention.After receiving an explanation regarding the purpose of this study, elderly subjects living in Seoul were selected, who consented to participate, and who used neighborhood sports facilities. The minimum sample size was obtained using the G*power 3.1.9.2 program  based onThe 22-item survey used in this study consisted of 6 questions on general characteristics, 8 on the selection attribute of the neighborhood sports facilities, 4 on user satisfaction, and 4 on reuse intention. Responses were provided using a Likert 5-point scale. Referring to a previous study [After referring to a previous study , user saThe main objective of the questionnaire used in this study was to obtain relevant information in the most reliable and valid manner. Reliability and validity are concepts used to evaluate the quality of research . CronbacIn this study, six experts with a doctorate in physical education or statistics were recruited, in order to evaluate the validity of the questionnaire items. Content validity index (CVI) is the most widely used index in quantitative evaluation . The scaScores 1 and 2 were then classified as irrelevant , while scores 3 and 4 were classified as relevant . S-CVI/Ave (average variance extracted) was calculated to determine each item’s mean item level-CVI(I-CVI). The measurement of content validity depicts the degree to which an item in an assessment instrument is relevant to a representative for a particular purpose. The higher a content validity test score, the more accurate it is in measuring the target construct. I-CVI > 0.79 means that the item can be relevant, and does not need further revision; S-CVI/Ave ≥ 0.9 indicates that the items have excellent content validity . Table 2This study conducted a convenience sampling of Korean men and women aged 65 years and over who had experience at neighborhood sports facilities during the COVID-19 pandemic. In this study, random sampling was performed on the elderly living in Seoul. Written consent was obtained from the respondents, and data were collected using a structured questionnaire. A total of 386 questionnaires were used for data analysis, which was carried out using SPSS v22.0 software . Descriptive statistics of the mean, standard deviation, and frequency distribution were used at the descriptive level. Moreover, one-way analysis of variance (ANOVA), Scheffe’s post hoc pair-wise comparison analysis, Pearson’s correlation coefficient, and multiple regression analysis were used at the inferential level. The significance level of these tests was considered for less than 0.05.p < 0.001). The mechanistic service factors of “graduated from college” group (3.60) and “graduated from graduate school or higher” group (3.94) were higher than those of “graduate from high school or less” group (3.25), which was statistically significant . Scheffe’s post hoc test showed that their mechanistic service factors were statistically different, in the order of “graduated from high school or less,” “graduated from college,” and “graduated from graduate school or higher” groups. The “6 or more times per week” group (4.69) had significantly higher mechanistic service factors than those of other groups , which was verified by Scheffe’s post hoc test.p < 0.001). Furthermore, their levels of humanistic service factors were different, according to the health status of the respondents. Humanistic service factors in the “healthy” group (2.69) were significantly higher than those in the “very healthy” group (1.59) . According to the age of the respondents, they had statistically different humanistic service factors . Scheffe’s post hoc test showed that humanistic service factors were significantly higher, in the order of the 65–74 age group (2.71), the 75–84 age group (1.97), and the 85 or more age group (1.14).p < 0.01), and Scheffe’s post hoc test showed that their humanistic service factors (2.77) were higher than those of the “graduated from high school or less” group (2.44). The “4–5 times a week” group (2.97) had higher humanistic service factors than those of other groups . However, Scheffe’s post hoc test showed that the result was significant when the “4–5 times a week” group was compared with the “once a week or less” group.The “graduated from college” group had higher humanistic service factors than the groups with other educational backgrounds . Furthermore, their levels of user satisfaction were different according to the health status of the respondents. The healthy group’s user satisfaction (3.36) was significantly higher than that of the very healthy group (2.24) . The 65–74 age group (3.38) and the 75–84 age group (3.41) had higher levels of user satisfaction than the 85 or more age group (1.43) , which was verified by Scheffe’s post hoc test. The “graduated from college” group (3.39) and the “graduated from graduate school or higher” group (3.39) had higher levels of user satisfaction than the “graduated from high school” group (3.18) , which was verified by Scheffe’s post hoc test. The “2–3 times a week” group (3.71) and the “4–5 times a week” group (3.61) had higher levels of user satisfaction than the “once a week or less” group (2.98) , which was verified by Scheffe’s post hoc test. However, the marital status of the respondents did not have a significant effect on user satisfaction.p < 0.001). Moreover, their levels of reuse intention were different according to the health status of the respondents. The healthy group’s user satisfaction (3.93) was significantly higher than that of the very healthy group (2.33) . The 65–74 age group (3.34) and the 75–84 age group (3.45) had higher levels of user satisfaction than the 85 or more age group (1.43) , which was verified by Scheffe’s post hoc test. The “2–3 times a week” group (3.59) and the “4–5 times a week” group (3.59) had higher levels of user satisfaction than the “once a week or less” group (2.99) , which was verified by Scheffe’s post hoc test. However, the educational background or marital status of the respondents did not have a significant effect on reuse intention.p < 0.01). Furthermore, there was a positive correlation between humanistic service factors and user satisfaction . In particular, humanistic service factors correlated more with user satisfaction than mechanistic service factors. There was a positive correlation between mechanistic service factors and reuse intention . Moreover, there was a positive correlation between humanistic service factors and reuse intention . Furthermore, humanistic service factors correlated more with reuse intention than mechanistic service factors. Finally, there was a positive correlation between user satisfaction and reuse intention .Multiple linear regression analysis was performed, in order to examine whether mechanistic service factors or humanistic service factors significantly predicted user satisfaction.p < 0.001, R2 = 0.506). In particular, it was confirmed that humanistic service factors  had a higher influence on user satisfaction than mechanistic service factors . As a result, service factors of neighborhood sports facilities affected user satisfaction (H1 is accepted). Mechanistic service factors and humanistic service factors also affected user satisfaction .p < 0.001, R2 = 0.317). In particular, humanistic service factors  had a higher influence on reuse intention than mechanistic service factors . As a result, service factors of neighborhood sports facilities affected reuse intention (H2 is accepted). Mechanistic service factors and humanistic service factors also affected reuse intention .Multiple linear regression analysis was also performed, in order to examine whether mechanistic service factors or humanistic service factors significantly predicted reuse intention. p < 0.001, R2 = 0.753). In other words, user satisfaction had a significant effect on reuse intention . Therefore, H3 was accepted , married (3.56), healthy (3.53), 65–74 age group (3.53), “graduated from graduate school or higher” (3.94), and “2–3 times per week” (3.61). Humanistic service factors were measured to be high in variables such as male (2.73), healthy (2.71), “graduated from college” (2.77), and “4–5 times per week” (2.91). Levels of user satisfaction were measured to be high in variables such as male (3.37), healthy (3.36), 65–74 age group (3.38) or 75–84 age group (3.41), “graduated from college or graduate school” (3.39), and “2–3 times per week” (3.71). Levels of reuse intention were measured to be high in variables such as male (3.35), healthy (2.69), 65–74 age group (2.71), and “2–3 or 4–5 times per week” (3.59). Thus, it was confirmed that the elderly’s perception of service factors of neighborhood sports facilities differed according to gender, age, education level, health status, and frequency of visits to facilities.p < 0.001). Based on the above results, this study discusses the points that follow.The Pearson correlation analysis showed positive correlations among the service factors, user satisfaction, and reuse intention. Thus, this study tested three hypotheses on the relationships between service factors, user satisfaction, and reuse intention of neighborhoods. The results showed that service factors of neighborhood sports facilities affected user satisfaction. Moreover, mechanistic service factors and humanistic service factors affected user satisfaction. In addition, this study revealed that service factors of neighborhood sports facilities affected reuse intention. Mechanistic service factors and humanistic service factors also affected reuse intention. Finally, user satisfaction significantly affected reuse intention  had a higher influence on reuse intention than mechanistic service factors . In addition, humanistic service factors  had a higher influence on reuse intention than mechanistic service factors . This suggests that there is a crucial role for exercise professionals in the operation of neighborhood sports facilities [Firstly, this study revealed that the service factors of neighborhood sports facilities used by the elderly during the COVID-19 pandemic had a positive effect on user satisfaction. Previous research ,34 also cilities . TherefoFurthermore, the mechanistic service factors of neighborhood sports facilities are still insufficient in comparison to the service factors of general and private sports facilities. As a result, careful consideration, attention, and policy planning from the local and state governments are required. That is, the administrative body that is responsible for the operation and management of neighborhood sports facilities should propose plans for further improvements of service factors, in order to promote the elderly’s health and quality of life. However, most neighborhood sports facilities are operated by public institutions, which are often reported to have a lower ability to maintain customer satisfaction than private institutions . As a reSecondly, the finding that the service factors of neighborhood sports facilities directly affects reuse intention is consistent with previous studies ,37,38. TThirdly, user satisfaction in this study showed a significant effect on reuse intention, supporting the results of previous studies ,39 whichFourthly, this study has a limitation in that it was conducted on the elderly living in Seoul during the COVID-19 pandemic. The elderly who live in Seoul have many opportunities to use fitness centers and indoor gymnasiums. Therefore, when the COVID-19 pandemic is over, the elderly in Seoul can exercise indoors rather than using neighborhood sports facilities. Meanwhile, the elderly, who live in small towns or rural areas where there are not many indoor sports facilities, may have a higher possibility of using neighborhood sports facilities. In a follow-up study, it will be helpful to generalize the research results by separately approaching the elderly in rural versus urban areas. Furthermore, examining the differences in the perceptions of the elderly about neighborhood living facilities, according to gender, could provide more meaningful research results.In conclusion, this study confirmed that the mechanistic and humanistic service factors of neighborhood sports facilities during the COVID-19 pandemic had a positive effect on the elderly’s user satisfaction and reuse intention. If mechanistic and humanistic service factors are not improved, the elderly may not be satisfied with neighborhood sports facilities, or may not reuse them in the post-corona era. Therefore, this study has some implications, in that it is necessary to maintain the service quality factors of the neighborhood sports facilities to increase user satisfaction and reuse intention. However, this study has a limitation in that it did not analyze potential mediating or moderating variables in relation to service factors of the neighborhood sports facilities, user satisfaction, and reuse intention. Therefore, the need to identify potential mediating or moderating effects among the variables, using structural equation modeling in follow-up studies, is suggested.


Annona muricata as potential sources of antibiotic drugs to fight against uropathogens and reduce related oxidative stress. Crude ethyl acetate extracts from 41 different endophytic fungi were screened against three bacterial strains using the broth microdilution method, and fungi producing active crude extracts were identified using ITS1-5.8S rRNA-ITS2 nucleotide sequences. The antibacterial modes of action of the five most active extracts were evaluated using Staphylococcus aureus ATCC 43300 and Klebsiella oxytoca strains. The DPPH and FRAP assays were used to investigate their antioxidant activity, and their cytotoxicity against the Vero cell line was evaluated using the MTT assay. Out of the 41 crude extracts tested, 17 were active with minimum inhibitory concentrations (MICs) ranging from 3.125 μg/mL to 100 μg/mL and were not cytotoxic against Vero cell lines with a cytotoxic concentration 50 (CC50) >100 μg/mL. The more potent extracts  significantly inhibited bacterial catalase activity, lysed bacterial cells, increased outer membrane permeability, and inhibited biofilm formation, and the time-kill kinetic assay revealed concentration-dependent bactericidal activity. All seventeen extracts showed weak ferric iron-reducing power . In comparison, seven extracts exhibited DPPH free radical scavenging activity, with RSA50 ranging from 146.05 to 799.75 μg/mL. The molecular identification of the seventeen active fungi revealed that they belong to six distinct genera, including Aspergillus, Curvularia, Fusarium, Meyerozyma, Penicillium, and Talaromyces. This investigation demonstrated that fungal endophytes from Cameroonian Annona muricata, a medicinal plant used locally to treat bacterial infections, might contain potent antibacterial metabolites with multiple modes of action. The antibacterial-guided fractionation of these active extracts is currently ongoing to purify and characterise potential antibacterial active ingredients.Urinary tract infections (UTIs) are common bacterial infections. The global emergence of multidrug-resistant uropathogens in the last decade underlines the need to search for new antibiotics with novel mechanisms of action. In this regard, exploring endophytic fungi inhabiting medicinal plants used locally against urinary tract infections could be a promising strategy for novel drug discovery. This study investigates crude metabolites from endophytic fungi isolated from  Infectious diseases emerging throughout history have included some of the most feared plagues of the past. Recent outbreaks include Ebola, Zika, dengue, Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS), influenza, fungal infection, and more recently, COVID-19 . AlthougTesting and treatment strategies vary for the management of UTIs, with empirical therapy being the use of broad-spectrum antibiotics , 8. UnfoPenicillium notatum by Sir Alexander Fleming Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In this paper, the authors present a group of 41 endophytic fungi extracts from different parts of the Cameroonian plant Annona muricata and their effect on Staphylococcus aureus, Escherichia coli, and Klebsiella oxytoca. Seventeen extracts showed antibacterial effect and were selected for sequencing and identification by BLAST, six different fungi were identified. Finally, the five most active extracts were selected to evaluate their mode of action.The study is well conceived and conducted, and the results appear to be solid; however, some considerations for improving the work are suggested.Title. Staphylococcus aureus, Escherichia coli, and Klebsiella oxytoca are not specific bacteria of Urinary Tract Infections it would be convenient to change the more general title without specifying the disease, for example: "Crude metabolites from endophytic fungi inhabiting Cameroonian Annona muricata exhibit potent antibacterial effect".Methodology. Homogenize brand and country of reagents and equipment.Methodology: In the section "Antibacterial screening and minimum inhibitory concentrations of endophytes", the authors describe the evaluation process, but the active or non-active interpretation strategy is not clear, was it by turbidity?Line 138. Correct parenthesesLine 184-185. Change "hydrogen peroxide" to "hydrogen peroxide (H2O2)".Line 210: Is the concentration "5.10 7 CFU/mL" correct? Or does it refer to "1.5x10 7 CFU/mL"?Line 382: Change the sentence "All extracts exhibited activity against all tested bacteria..." because the AMr10 extract only had activity against one bacterium.Results. The results and figures of the effect of extracts on the permeability of outer cell membrane indicate "seven different concentrations", however, the methodology indicates three concentrations "MIC, 2MIC, and 4 MIC".Growth curve. In Figure 4, the optical density is 600 nm and in the results and methodology, it is described at 620 nm, which is correct?Line 490. (IC50 0.24-0.36 μg/mL) and , in each case refers to two different concentrations and is confused with ranges; change to (IC50 0.24 and 0.36 μg/mL) and ,The discussion does not mention the significance of the cytotoxicity result in Vero cells.Correct the size of the "MIC" symbology in Fig. 2B.The metabolites of F. waltergamsii and P. citrinum AMf6 were the most active, it is important to emphasize the discussion in this regard.In figure legends change "S. Aureus" by "S. aureus"Reviewer #2: The paper entitled " Crude metabolites from endophytic fungi inhabiting Cameroonian Annona muricata exhibit potent inhibition against the causative agents of urinary tract infections (UTIs): provide a comprehensive and significant view of the research area; I think the study is relevant and suitable for publication to the journal; however, there are some shortcoming and grammatical mistakes> It is my hope that my comments will help the authors improve their manuscript and is the intention.> One minor comment is that the title seems a little lengthy, please try a make it short and attractive2nd minor comment is that there are some writing details in the manuscript.>In title you haven’t mention “isolation” please revise it.Page, line 22, put comma after “method”Page, line 25, replace “Both the” to “The”Line 27, MIC After first appearance it should be abbreviate, In abstract it’s already mentioned.Page, line 40, “content” to “contain”Page line 47, put comma after “infection”Page, line 49, replace “spectre” to “specter”Page, line 59, delete the word “being”Page, line 60, replace the word “of”Page, line 87, remove the comma after “antibacterial”Line 91 and 318, Please revise it Use same pattern.145, put comma after “mg/mL”Line 153, Used same wording 24 h (Twenty-four) Please revise it whole manuscript.Page, line 158-159 the endophytic fungi producing more potent secondary metabolites, revise this sentence.Page, Line 161 Three bacterial species in every sentence you have used “THE” I think it should be “Extracts were screened against three bacterial species at single concentration” as well as162 line should also revise.Line 162 Mentioned CLSI after first appear, In Line 670 it should be abbreviate.169, line 169, delete the word “that were”Line 170, replace “in order to” to “to”Line 171, replace “For the determination of” to “to determine”Line 173, delete “as”Line 174, replace “where there was” to “with”Line 176, put comma after “1%”Line 179, put comma after “AMf4”Line 181, please revise it.Line 192, put comma after “buffer”Line 204, delete the word “respectively”Line 214, delete “graph of” and rewrite “graph” after “OD/450 nm)”Line 215, replace “was” to “were”Line 227, Please revise it. As well as Revise line 231.Line 233, put comma after “solution”Line 270, put comma after “min”Line 289, replace “was treated in” to “treated”Line 326 revise it.Line 399, put comma after “AMrb9”Line 399, replace “rootbark” to “root bark”Line 466, replace “that of” to “than of”Page, line 508, write “was” before “obtained respectively”Page, line 516, replace “have substantial influence on” to “substantially influence”Page, line 523, delete the word “for the ability” as well as Revise it, above in manuscript it’s already mentioned 17, so used same pattern.Page, line 581, write that before “over”Page, line 605 replace “defences” to “defenses” Please revise itPage, line 625, delete the word “with the ability”Page, line 629, replace “antibiofilm guided” to “antibiofilm-guided”Page, line 639, replace “serve as a significant indicator of” to “significantly indicate”Page, line 642, put comma after “AMf1”Page, line 659, replace “mode” to modes”**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.
 While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  3 Apr 2022Response to ReviewersReviewer 1Title. Staphylococcus aureus, Escherichia coli, and Klebsiella oxytoca are not specific bacteria of Urinary Tract Infections it would be convenient to change the more general title without specifying the disease, for example: "Crude metabolites from endophytic fungi inhabiting Cameroonian Annona muricata exhibit potent antibacterial effect". Response: Thank you very much for your suggestion. We chose the current title because this project aimed to target UTIs' causative agents specifically. Although Staphylococcus aureus, Escherichia coli, and Klebsiella oxytoca are not only involved in Urinary Tract Infections, they are among the most common agents of UTIs, accounting for about 75-80% of the case . Besides, these species are among the most isolated from patients suffering from UTIs in Cameroon and other African countries . Supporting literatureshttps://doi.org/10.1186/1756-0500-5-219Akoachere, JF.T.K., Yvonne, S., Akum, N.H. et al. Etiologic profile and antimicrobial susceptibility of community-acquired urinary tract infection in two Cameroonian towns. BMC Res Notes 5, 219 (2012). https://doi.org/10.1186/s12879-017-2743-8Bitew, A., Molalign, T. & Chanie, M. Species distribution and antibiotic susceptibility profile of bacterial uropathogens among patients complaining urinary tract infections. BMC Infect Dis 17, 654 (2017). https://doi.org/10.1038/nrmicro3432Flores-Mireles, A. L., Walker, J. N., Caparon, M., & Hultgren, S. J. (2015). Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nature reviews. Microbiology, 13(5), 269–284. Methodology. Homogenize brand and country of reagents and equipment. Response: We have now homogenized all brand and country names of reagents and equipment Methodology: In the section "Antibacterial screening and minimum inhibitory concentrations of endophytes", the authors describe the evaluation process, but the active or non-active interpretation strategy is not clear, was it by turbidity? Response: Yes, the turbidity was the strategy used to define the activity of potent extracts.Line 138. Correct parentheses. Response: Thank you, parentheses are now correctedLine 184-185. Change "hydrogen peroxide" to "hydrogen peroxide (H2O2)". Response: hydrogen peroxide is now changed to "hydrogen peroxide (H2O2)"Line 210: Is the concentration "5.10 7 CFU/mL" correct? Or does it refer to "1.5x10 7 CFU/mL"? Response: Thank you for the question. The appropriate concentration was 5x107 CFU/mL. we have now edited the methodology accordinglyLine 382: Change the sentence "All extracts exhibited activity against all tested bacteria..." because the AMr10 extract only had activity against one bacterium. Response: The sentence "All extracts exhibited activity against all tested bacteria..." is now replaced by "All extracts exhibited activity against at least one of the tested bacteria with the MIC values ranging from 3.125 to 100 μg/mL depending on the extracts and microorganisms. "Results. The results and figures of the effect of extracts on the permeability of outer cell membrane indicate "seven different concentrations", however, the methodology indicates three concentrations "MIC, 2MIC, and 4 MIC". Response: Thank you very much for this remark. Yes indeed, we tested 7 concentrations for the outer cell membrane permeability assays including 1/16 MIC, 1/8 MIC, 1/4 MIC, 1/2 MIC, MIC, 2MIC, and 4 MIC. The methodology is now revised. Thank you!Growth curve. In Figure 4, the optical density is 600 nm and in the results and methodology, it is described at 620 nm, which is correct? Response: The right optical density is 620nm. We have now edited the figure accordingly.Line 490. (IC50 0.24-0.36 μg/mL) and , in each case refers to two different concentrations and is confused with ranges; change to (IC50 0.24 and 0.36 μg/mL) and , Response: we have now changed (IC50 0.24-0.36 μg/mL) and  to (IC50 0.24 and 0.36 μg/mL) and .The discussion does not mention the significance of the cytotoxicity result in Vero cells. Response: Thank you for this remark. We have now included the discussion on the significance of the cytotoxicity result in Vero cells.Correct the size of the "MIC" symbology in Fig. 2B. Response: we have now harmonized the size of the "MIC" symbology in all figures The metabolites of F. waltergamsii and P. citrinum AMf6 were the most active, it is important to emphasize the discussion in this regard. Response: Thank you for your suggestion. We mentioned in the discussion that these findings agreed with previous reports. References 15 and 16 cited in the discussion are two comprehensive reviews articles detailing the biological activity of metabolites produced by endophytic fungi from the Fusarium and Penicillium genera. Therefore, we feel that these review articles are sufficient to support our findings allowing us to avoid unnecessary repetition of information.Supporting information: 15) Toghueo, 2020: Bioprospecting endophytic fungi from Fusarium genus as sources of bioactive metabolites. doi:10.1080/21501203.2019.164505316) Toghueo and Boyom, 2020: Endophytic Penicillium species and their agricultural, biotechnological, and pharmaceutical applications. doi:10.1007/s13205-020-2081-1In figure legends change "S. Aureus" by "S. aureus".Response: We have now replaced "S. Aureus" by "S. aureus"2. Reviewer 2One minor comment is that the title seems a little lengthy, please try a make it short and attractiveResponse: Thank you for the suggestion. We have now revised the title.2nd minor comment is that there are some writing details in the manuscript.>In title you haven’t mention “isolation” please revise it.Response: Thank you for the remark. The word isolation was replaced by “inhabiting”, which means live within. In this context, both words have identical meanings. We choose to use the less common.Page, line 22, put comma after “method”. Response: we have now put comma after “method”.Page, line 25, replace “Both the” to “The”. Response: we have now replace “Both the” to “The”.Line 27, MIC After first appearance it should be abbreviate, In abstract it’s already mentioned. Response: we have now revised the entire manuscript accordinglyPage, line 40, “content” to “contain”. Response: we have now replaced “content” with “contain”.Page line 47, put comma after “infection”. Response: we have now put comma after “infection”.Page, line 49, replace “spectre” to “specter”. Response: we have now replaced “spectre” to “specter”.Page, line 59, delete the word “being”. Response: we have now revised the sentence.Page, line 60, replace the word “of” .Response: we have now revised the sentence.Page, line 87, remove the comma after “antibacterial”. Response: we have now revised the sentence.Line 91 and 318, Please revise it Use same pattern. Response: we have now revised the sentence.145, put comma after “mg/mL”. Response: we have now revised the sentence.Line 153, Used same wording 24 h (Twenty-four) Please revise it whole manuscript. Response: we have now revised the sentence.Page, line 158-159 the endophytic fungi producing more potent secondary metabolites, revise this sentence. Response: we have now revised the sentence.Page, Line 161 Three bacterial species in every sentence you have used “THE” I think it should be “Extracts were screened against three bacterial species at single concentration” as well as. Response: we have now revised the sentence.162 line should also revise. Response: we have now revised the sentence.Line 162 Mentioned CLSI after first appear, In Line 670 it should be abbreviate. Response: we have now revised the sentence.169, line 169, delete the word “that were”. Response: we have now revised the sentence.Line 170, replace “in order to” to “to”Response: we have now replaced “in order to” to “to”Line 171, replace “For the determination of” to “to determine”. Response: we have now revised the sentence.Line 173, delete “as”Line 174, replace “where there was” to “with”. Response: we have now revised the sentence.Line 176, put comma after “1%”Response: we have now revised the sentence.Line 179, put comma after “AMf4”.Response: we have now revised the sentence. Line 181, please revise it. Response: we have now revised the sentence.Line 192, put comma after “buffer”. Response: we have now revised the sentence.Line 204, delete the word “respectively”Response: we have now revised the sentence.Line 214, delete “graph of” and rewrite “graph” after “OD/450 nm)”. Response: we have now revised the sentence.Line 215, replace “was” to “were”Response: we have now replaced “was” by “were”.Line 227, Please revise it. As well as Revise line 231.Response: we have now revised the sentenceLine 233, put comma after “solution”Line 270, put comma after “min”.Response: we have now put comma after “min”. Line 289, replace “was treated in” to “treated”Response: we have now replaced “was treated in” by “treated”Line 326 revise it. Response: we have now revised the sentenceLine 399, put comma after “AMrb9”.Response: we have now put comma after “AMrb9”. Line 399, replace “rootbark” to “root bark”Response: we have now replaced “rootbark” by “root bark”Line 466, replace “that of” to “than of”. Response: we have now replaced “that of” by “than of”. Page, line 508, write “was” before “obtained respectively”Response: we have now revised the sentencePage, line 516, replace “have substantial influence on” to “substantially influence” Response: we have now revised the sentencePage, line 523, delete the word “for the ability” as well as Revise it, above in manuscript it’s already mentioned 17, so used same pattern. Response: we have now revised the sentencePage, line 581, write that before “over”. Response: we have now revised the sentencePage, line 605 replace “defences” to “defenses” Please revise it. Response: we have now replaced “defences” by “defenses” Page, line 625, delete the word “with the ability”Response: we have now deleted the word “with the ability”Page, line 629, replace “antibiofilm guided” to “antibiofilm-guided”. Response: we have now replaced “antibiofilm guided” by “antibiofilm-guided”.Page, line 639, replace “serve as a significant indicator of” to “significantly indicate”. Response: we have now revised the sentencePage, line 642, put comma after “AMf1”. Response: we have now revised the sentence Page, line 659, replace “mode” to modes”. Response: we have now revised the sentenceAttachmentResponse to Reviewers 03.04.2022.docxSubmitted filename: Click here for additional data file. 6 Apr 2022Crude metabolites from endophytic fungi inhabiting Cameroonian Annona muricata exhibit potent inhibition against the causative agents of urinary tract infections (UTIs)PONE-D-22-00760R1Dear Dr. tOGHUEO,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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For more information, please contact Kind regards,Guadalupe Virginia Nevárez-Moorillón, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 8 Apr 2022PONE-D-22-00760R1 Annona muricata inhibit the causative agents of urinary tract infections Crude metabolites from endophytic fungi inhabiting Cameroonian Dear Dr. Kouipou Toghueo:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. 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Burnout occurs when people are exposed to emotionally demanding work situations over an extended period, resulting in physical, emotional, and mental exhaustion. This study adopted the personal burnout (PB) and work-related burnout (WB) scales of the Copenhagen Burnout Inventory to measure burnout levels. Musculoskeletal (MS) pain is one factor influencing burnout. Previous studies have demonstrated that male and female hormones may contribute to sex-related differences in MS pain. This was an observational and cross-sectional study conducted at a medical-university-affiliated hospital in Taichung, Taiwan, in 2021. Data were collected for demographic characteristics, family structure, living habits, occupation, physical health, Nordic Musculoskeletal Questionnaire score, and Copenhagen Burnout Inventory score. Of the 2531 questionnaires that were distributed, 1615 (63.81%) valid questionnaires remained after those with missing data were excluded. The results demonstrated neck and shoulder pain (NBSP) is commonly associated with burnout among healthcare workers, with a higher prevalence among women than among men. With confounders being controlled for, women were found to experience substantially higher levels of personal and work-related burnout than men did. NBSP is a mediating factor; therefore, it is a key reason why women are more likely than men to experience burnout. Burnout is a state of physical, emotional, and mental exhaustion that results from long-term involvement in work situations that are emotionally demanding . The devThe clinical symptom included emotional exhaustion, physical fatigue, cognitive impairments, disturbed sleep, and functional impairment ,4 and ofThe common musculoskeletal pain sites were the lower back, shoulder, and neck , and thaBoth male and female sex hormones may contribute to the marked sex-related differences in the occurrence of certain musculoskeletal (MS) pain conditions . Some stBased on the associations among women, MS pain, and burnout, whether women sustain high burnout than men due to MS pain was a noteworthy and interesting topic. Therefore, this study proposed two hypotheses to further explore: that sex differences in burnout (1) exist and (2) result from MS pain differences between women and men. Hopefully the results of this research can encourage the medical institutions to make sex-differentiated strategies of burnout to further reduce burnout, especially among female staff members.This study adopted an observational and cross-sectional design, which was conducted at a medical-university-affiliated hospital in Taichung, Taiwan, in 2021. All 2531 medical staff who have served for one year in the hospital were distributed a QR code of google forms linked questionnaires by email, in which 1633 (64.52%) were returned, and 1615 (63.81%) were determined to be valid after those with missing data were excluded. The professional fields and sex of 1615 participants were in The response options for level of education were “below high school”, “Bachelor’s degree”, “Master’s degree”, and “PhD”. The response options for marital status were “married” and “other”. Participants were also asked whether they were involved in raising children. The response options were “without child”, “one child”, “two children”, “three children”, and “over three children”. Raising at least one child was reclassified into a new variable, “parenthood”. The question of whether participants engage in leisure activities with family or friends (LAFF) during vacation time was also included in the questionnaire. The options, “always”, “often”, “sometimes”, “seldom”, and “never”, were scored as 100, 75, 50, 25, and 0 points, respectively. An item related to the presence of a listed chronic diseases was included in the questionnaire, and the presence of one or more diseases was classified as a “yes” response. Regarding smoking during the past month, responses of “never” or “have quit smoking” were classified as “no”. Regarding drinking during the past month, responses of “seldom” or “every day” were classified as “engaged in alcohol use at least once” (which was termed “ever AU”), whereas the option of “never” was classified as “no alcohol use ever”. Sleeping duration was classified as “<5 h”, “5–6 h”, “6–7 h”, “7–8 h”, or “>8 h”. These responses were reclassified as “sleeping duration <6 h” and “sleeping duration >6 h”. The participants were asked whether they exercised “at least once a day”, “at least once a week”, “at least once a month”, “less than once a month”, or “never”. Responses of “at least once a day” or “at least once a week” were reclassified as “regular exercise weekly”. For the question pertaining to overtime work, the options were “seldom”, “less than 45 h per month”, “45–80 h per month”, and “more than 80 h per month”. Because the participants seldom worked overtime work for more than 80 h, the responses were reclassified as “seldom overtime work” and “experiencing overtime work” . The options for questions on work schedule were “day shift work”, “night shift work”, “irregular shift work”, and “regular shift work”.The Nordic Musculoskeletal Questionnaire was developed by the Nordic Council of Ministers . The aim“How often do you feel tired?”“How often are you physically exhausted?”“How often are you emotionally exhausted?”“How often do you think ‘I can’t take it anymore’?”“How often do you feel worn out?”“How often do you feel weak and susceptible to illness?”The Copenhagen Burnout Inventory was developed by researchers from Denmark . In the 7.“Is your work emotionally exhausting?”8.“Do you feel burnt out because of your work?”9.“Does your work frustrate you?”10.“Do you feel worn out at the end of the working day?”11.“Are you exhausted in the morning at the thought of another day at work?”12.“Do you feel that every working hour is tiring for you?”13.“Do you have enough energy for family and friends during leisure time?”Items 7–13, which concerned WB, were as follows:The response options—“always”, “often”, “sometimes”, “seldom”, and ”never/almost never”—were scored as 100, 75, 50, 25, and 0, respectively, with the exception of item 13, which was inversely scored . Levels of PB and WB were represented by the mean of the total PB and WB scores (the sum of scores for items 1–6 and items 7–13), respectively.Regarding the procedures of statistical analyses, we adopted four steps to determined women effect on burnout and their mediating factors.Step 1: Factor analysis  was condt test or one-way ANOVA (continuous variables) to determine if the difference between survey variable and sex or PB/WB is significant in statistic. These survey variables were significant in statistic that would be as possible mediating factors between sex and burnout or risk/protective factors of burnout.Step 2: The present research adopted chi-square test/Fisher exact test  and Step 3: The risk or protective factors of burnout would be added in linear regression models of women effect on burnout to determine whether controlling for covariates significantly affected associations of the independent variables (IVs) with dependent variable (DV).a, first-stage effect), (2) the IV significantly affects the DV in the absence of the mediator, (3) the mediator has a significant unique effect on the DV , and (4) the effect of the IV on the DV  weakens upon addition of a mediator to the model. Among these strategies, item (2) is recommended but not required [X is an IV; a is a logistic or linear regression coefficient of X against X are a DV and an IV, respectively; b is the logistic or linear regression coefficient of c is the logistic or linear regression coefficient of X against c′ is the logistic or linear regression coefficient of X against a and b are represented by as and bs, respectively.Step 4: The mediation effect would be tested on the basis of the strategy proposed by Baron and Kenny , in whicrequired . The med in 2012 ; the forIf If The results exceeding |1.96|, |2.57|, and |3.90| (for a two-tailed test) are significant at α = 0.05, 0.01, and 0.0001, respectively. p < 0.05.Analyses were performed using SAS Enterprise Guide 7.1 software , and significance was set at First, the research must determine the underlying variables that could maximum explain the Nordic Musculoskeletal Questionnaire. As indicated in (p = 0.003), parenthood (p = 0.002), weekly exercise habits (p < 0.0001), monthly alcohol use (p < 0.0001), level of education (p < 0.0001), shift schedule (p = 0.029), and profession (p < 0.0001). The proportions for marriage (56.15% vs. 46.42%), parenthood (51.50% vs. 41.70%), regular exercise weekly (70.10% vs. 54.95%), ever alcohol use (52.16% vs. 34.40%), master’s degree or above (28.57% vs. 16.06%), DS work (72.09% vs. 64.23%), and profession of physician (30.56% vs. 3.50%) were higher among men than women. As noted in p = 0.000).Second, the present study would determine the sex-related difference on survey variables for determining possible mediating factors. As indicated in p < 0.0001), sleeping duration < 6 h , experience overtime work , presence of chronic diseases , NBSP , BAP , and BKP  were risk factors for PB. PB was significantly correlated with shift schedule (p < 0.0001) and profession (p < 0.0001). Regular exercise weekly , LAFF , and work experience  were protective factors for PB.Confounders of burnout were next identified and used as control variables in a multiple linear regression model of women’s effect on burnout. p < 0.0001), sleeping duration < 6 h , master’s degree or above , experience overtime work , presence of presence of chronic diseases , NBSP , BAP , and BKP  were risk factors for WB. WB was significantly correlated with shift schedule (p < 0.0001) and profession (p < 0.0001). Marriage , parenthood , LAFF , and work experience  were protective factors for WB.As shown in 0, M1, M2, M3, M4, and M5. Men and women did not significantly differ in PB and WB when no adjustments were made for any variable (M0). The effect of being a woman was significantly correlated with PB  and WB  in model M1, which had controls for weekly exercise, monthly alcohol use, sleep duration per day, level of education, overtime work per month, shift schedules, profession, presence of chronic diseases, LAFF, and work experience. The effect of being a woman was still significantly correlated with PB  and WB  in the M2 (M1 controlled for marriage) and M3 (M1 controlled for parenthood) models, respectively. The effect of being a woman was significantly correlated with PB  and WB  in the M4 model (M1 controlled for BAP and BKP); however, according to M5 model, the residual effect of being a women for PB  and WB  could be fully explained by NBSP, respectively.a = 0.21; p < 0.01) for women to NBSP was statistically significant. The second stage effects  for NBSP to PB and WB were also statistically significant. According to the Sobel test results, NBSP mediated  the relationships of gender with PB and WB. Mediation effect of NBSP that women sustain high burnout would be further identified by mediation models. As shown in These results verify the two hypotheses of this study: sex differences in burnout exist, and these differences are the result of MS pain differences between women and men.p < 0.0001 for both, First, we must review our findings and compare with the past study’s results to further confirm the two hypotheses proposed in the introduction. Burnout can be influenced by family, living habits, sleep, work-related factors, and health problems. For example, family members and friends play a vital role in preventing the development of burnout , and heap < 0.0001 for both); this is consistent with the results of previous studies.A positive dose-response relationship was noted between physical activity and emotional well-being . In addip < 0.0001 for both), which demonstrates that alcohol use is an unfavorable factor for burnout and does not effectively reduce burnout.Whether alcohol reduces stress is debatable . Previoup < 0.05) and WB  than those who slept more than 7 h per working day [p < 0.0001 for both). This is consistent with the results of previous studies.The development of burnout is closely related to lack of sleep (<6 h) ,48 and dking day . Table 3.The prevalence of diseases is related to the severity of burnout. Among individuals with at least one physical illness, approximately half 54%) had no burnout, whereas 63% had mild burnout and 71% had severe burnout . Moreove% had no p < 0.0001) and WB . This suggests that LAFF is a protective factor for burnout, which is consistent with the findings of previous studies.For people with high stress levels, participating in leisure activities can relieve stress, improve one’s emotional well-being, and maintain one’s physical and mental health ,57,58. T0). Notably, there were significant difference on sex and PB or WB among marriage state, parenthood, exercise habit weekly, alcohol use habit in a month, education degree, shift schedule, and medical professional. This difference could interfere women effect on burnout. Therefore, we adopted the multiple linear regression models to control these confounders for determining if exist women effect on burnout. A significant correlation between women and burnout was really found in M1 , M2 , M3 , and M4  model. This finding determined our hypothesis 1: sex differences in burnout really exist. In addition, M3 and M4 reflected the explanatory power of marriage and parenthood on the residual effect of being a woman on burnout. The results in The effect of being a woman on the likelihood of burnout was not significant in the unary linear regression model (Mp < 0.0001 for both), BAP , and BKP . Among them, NBSP was more influential on the likelihood of burnout than other sources of MS pain.Despite the musculoskeletal system is complex, the sites on musculoskeletal pain still could be suitably classified by important anatomical locations or scales such as the Nordic Musculoskeletal Questionnaire. Regarding the occurrence of pain, the past study demonstrated the lower back (26.9%), shoulders (20.9%), and neck (20.6%) were the most reported pain sites based on point prevalence . We adopp < 0.01). In addition, M1, M2, M3, and M4 models in 4) and was as a new model M5. The new model indicated that NBSP could fully explain  the residual effect of being a woman on burnout after all confounders were controlled for. This suggests that women who reported high levels of burnout may also sustain high NBSP. Based on this, we come back the beginning second hypothesis in Introduction: sex difference in burnout results from MS pain differences between women and men. We adopted mediation model  the relationships of gender  with PB and WB, which further indicates that NBSP primary explains why women sustain high levels of burnout. According to the two results, we could determine women easily suffer from neck and both shoulders pain than men, that would be important reason that women sustain high PB and WB than men. To the best of our knowledge, this finding has rarely been mentioned in the literature. Except to this, another value on the present study is proposing an effectively strategy to medical institutions that the improving plan on burnout should more focus on the MS pain problem and supply the more accessibility resource on relieve pain for women.Compared with men, women often reported neck, shoulders, waist, or back pain ,24,25,61on model  to test The women ratio is 81.26% in all 2531 distributed questionnaires. Among them, women proportion is higher than men in valid questionnaires (81.36% vs. 18.64%), too. The unbalanced sex also reflects the situation for hospital in Taiwan. We think the unbalanced sex could interfere the results of analysis, therefore, we adopted multiple linear regression to simultaneously control possible interference factors from marriage state, parenthood, exercise habit, alcohol use habit, education degree, shift work, and medical profession. Despite this, we still ignore culture factors that gender inequality exists in Taiwan society. That should be further explored in future study of burnout.The present study did not include the work loading on individuals that could lead to interfere the sex effect on burnout despite we had controlled professional factor. The future research should adopt suitable work loading scale to measure this possible risk factor. Notably, healthcare workers could face potential crisis on medical or occupational violence that could lead to increased burnout. Therefore, the future research for burnout also should consider this issue.Due to the mediation model of an observational study could be biased , the cauNeck and shoulder pain is commonly associated with burnout among healthcare workers, with a higher prevalence among women than among men. With confounders being controlled for, women were found to experience substantially higher levels of personal and work-related burnout than men did. Notably, neck and shoulder pain are a key reason why women are more likely than men to experience burnout, a factor which has rarely been mentioned in the literature. Therefore, to mitigate the symptoms of burnout among women, medical institutions should adopt strategies that are tailored to women, such as targeting musculoskeletal pain through interventions, to prevent burnout among them.


Total mesorectal excision (TME) represents the “gold standard” of rectal cancer surgery. In locally advanced lesions neoadjuvant treatments  have been shown to improve TME oncological results, reducing local recurrences rate. Nevertheless, these treatments have significant functional consequences impacting patients’ quality of life (QoL). The resulting syndrome is known as Low Anterior Resection Syndrome (LARS). The purpose of this work was to evaluate the association between risk factors and the development of LARS in a prospective series of laparoscopic sphincter-saving TME.The study was conducted as a retrospective observational epidemiological study of a prospective database, including all patients undergoing laparoscopic anterior resection surgery for rectal cancer at our Unit from 1st January 2013 to 31st May 2018. The diagnosis of LARS was performed using the LARS Score. We classified risk factors in patient-related, pre-, intra- and post-operative factors.p = 0.03 and 0.02). Distal localization of tumor was also significantly associated with LARS .The sample included 153 consecutive patients. Forty-one were affected by “low” rectal cancer, 74 by “middle” rectal cancer, 38 by “high” rectal cancer. The prevalence of overall LARS (major LARS + minor LARS) in our series was 35.9% (55/153 cases). Association between nRT and overall/major LARS was significant (respectively In our study, neoadjuvant radiotherapy and tumor localization resulted independent risk factors for LARS after laparoscopic sphincter-saving TME. Tumor localization in the “middle” and “high” rectum resulted a protective factor compared to the localization in “low” rectum. Total mesorectal excision (TME) introduced by Heald in 1983 represents the “gold standard” of rectal cancer surgery –5. RegreThe advent of laparoscopic surgery in the treatment of rectal cancer has improved the postoperative course, reducing both hospital stay and time to resume daily activities. Nevertheless, data on function outcome of the laparoscopic rectal resections are scarce and specific data on LARS are lacking. The purpose of this work was to evaluate the association between anamnestic, pre-, intra- and post-operative risk factors and the development of LARS in a prospective series of exclusively laparoscopic sphincter-saving TME.The study was conducted as a retrospective observational epidemiological study of a prospective database, including all patients undergoing laparoscopic anterior resection surgery for rectal cancer at our Unit from 1st January 2013 to 31st May 2018. The diagnosis of LARS was performed using the LARS Score, resulting from a questionnaire which consists of 5 questions, whose answers are associated with a score, the sum resulting in the final score. A score from 0 to 20 indicates absence of LARS, 20 to 29 minor LARS, 30 to 42 major LARS . The ScoThe aim of the study is to estimate the prevalence of LARS in our Center and to define LARS risk factors in patients undergoing laparoscopic surgery for rectal cancer.Patients undergoing laparoscopic surgery converted to open technique, patients who did not give their consent, patients who developed post-operative complications requiring a permanent stoma, deceased patients whose post-operative course could not be reconstructed and patients lost to follow-up were excluded.We distinguished between patient-related risk factors and pre-, intra- and post-operative factors. Patient-related risk factors are: gender, age > 65 years, smoking, body mass index (BMI), cardiovascular disease , diabetes, neurological/neurodegenerative disorders, chronic obstructive pulmonary disease (COPD), chronic corticosteroid therapy, dyslipidemias, previous abdominal and/or pelvic surgery, collagenopathies, immunodeficiency syndromes. Pre-operative factors are: nutritional status , hemoglobin level, nRT, localization of the tumor expressed in cm from the anal margin at rigid rectoscopy . Variants of surgical technique are: colorectal/colo-anal anastomosis type , TME, diverting stoma. Post-operative factors are: surgical complications, need for re-intervention, blood transfusions, adjuvant chemotherapy (CTa), adjuvant radiotherapy (RTa), timing of closure of diverting stoma (earlier or later than 3 months after the index surgery).p values reported were obtained by the 2-sided method, at the conventional significance level of 5%. Univariate and multivariate binary logistic regression was performed. Data were analyzed starting from September 2020 by R 4.0.2 .Data were analyzed using descriptive and inferential statistics. Categorical variables were expressed as absolute/relative frequencies. The impact of all risk factors was calculated using Fisher’s Exact Test for categorical variables and Mann–Whitney’s test for continuous variables. All 2 in 11 (7.2%) patients. The most common comorbidity was cardiovascular disease (56.9%), compared to dysmetabolic , respiratory (8.5%), neurological (3.9%) and auto-immune (collagenopathies 2%) diseases; none of these factors were significant associated with LARS. Forty-six (30.1%) patients received long course nRT of which 24 patients had “low” rectal neoplasia, 18 patients had “middle” rectal neoplasia, 4 patients had “high” rectal neoplasia. All the patients underwent mechanical bowel preparation. One hundred and sixteen (76.8%) TMEs were performed, the predominantly colorectal anastomosis was SCR (94.8%), transanal mechanical (89.5%), and in 47.7% of cases (73 patients) a diverting stoma was performed. Sixty-four (43.2%) patients performed CTa and 3 (2%) patients performed RTa. All baseline patients’ characteristics are shown in Table The sample included 153 consecutive patients with a median age of 66 years (range 34–91). Forty-one patients were affected by “low” rectal cancer, 74 by “middle” rectal cancer, 38 by “high” rectal cancer. Eighty 52.3%) patients were over 65 years of age. Sixty-three (41.2%) patients were female and 90 (58.8%) male. BMI was > 30 kg/m.3% patiep = 0.03 and 0.02), while the association with the minor form was not significant (p = 0.799). Distal localization of tumor was also significantly associated with LARS . All risk factors and the association with the development of minor, major LARS and overall (minor + major) LARS are summarized in Table The prevalence of overall LARS (major LARS + minor LARS) in our series was 35.9% (55/153 cases), minor 13.7% (21 cases) and major 22.2% 34 cases). In our study nRT and tumor localization resulted to be significantly associated with development of LARS. Association between nRT and overall/major LARS was significant (respectively  cases. Ip = 0.02), as well as the localization of tumor (p = 0.003). The localization of the tumor in “middle” and “high” rectum were protective factors compared to the localization in “low” rectum , while the localization in “middle” and “high” rectum resulted as protective factors, if compared to the localization in “low” rectum   , and long-term oncological outcomes . In particular, recognized advantages of a minimally invasive approach derive from the better vision and, as a consequence, preservation of anatomical structures such as nerve plexus and anal sphincter function, all contributing to a reduction of surgical trauma –29. At oTo the best of our knowledge our study is currently the only one in the literature that analyzes the association between LARS and exclusively laparoscopic TMEs and the aim of the study was to estimate the prevalence of LARS at our Center and to define LARS risk factors in patients undergoing laparoscopic surgery for rectal cancer.LARS rates after rectal surgery are quite heterogeneous, influenced by different post-operative evaluation strategies, different surgical approaches  and different rates of risk factors . Croese et al. in 2018 published a meta-analysis, in which they analyzed 11 articles, reporting variable prevalence of LARS from 17 to 65.4% . We repop < 0.001) , in order to select the most suitable treatment for each patient, as already proposed by Martellucci with a treatment algorithm in a multimodal approach . FurtherIn conclusion LARS is a complex, multifactorial syndrome with high prevalence after rectal surgery. In our laparoscopic series, LARS rate resulted consistent with the open TME literature. Neoadjuvant radiotherapy and tumor localization resulted independent risk factors for LARS. Tumor localization in the “middle” and “high” rectum resulted a protective factor compared to the localization in “low” rectum.