Even though prejudice towards Bolivian immigrants is one of the main reasons for discrimination in Argentina, there is no valid measure to assess it. The aim of this study was to explore the psychometric properties of the subtle and blatant prejudice towards Bolivian immigrants scale. In addition, we tested correlations with right-wing authoritarianism, social dominance orientation, feelings towards Bolivian immigrants, and ideological self-placement. Data was collected through a convenience sample of 431 undergraduate students from Buenos Aires, with an age range from 18 to 45 years old (38.75% men and 61.25% women). Results showed adequate psychometric properties for the scale. Moreover, significant correlations between subtle and blatant prejudice and the other psychosocial variables tested were found. Implications of these findings are discussed. From the beginning of the nineteenth century, migratory movements to Argentina were key elements in the foundation of the nation and its subsequent development  as the covariation of three attitudinal clusters: authoritarian submission, authoritarian aggression, and conventionalism. Regardless of the factorial structure of the scale, the links between authoritarianism and prejudice have been identified in a large number of studies  is also related to different forms of prejudice   and subtle prejudice (α = 0.65). Years later, using data from a probabilistic survey with a sample of 896 subjects also from Chile, Cárdenas (α = 0.82 for subtle and α = 0.76 for blatant prejudice). Finally, more recent evidence  and for the two dimensions  was adequate. However, because of the strong correlations between subtle and blatant sub-scales , they concluded that the one-factor model solution was the most adequate. All these studies , subtles (high scores in subtle and low scores in blatant prejudice), and bigots (high scores in both forms of prejudice). They asked the participants what kind of actions do the government should have taken with Bolivian immigrants’ rights, and they found that egalitarians wanted to enlarge immigrants’ rights, subtles oscillated between restrict their rights or leave them as they are, and bigots agreed with restricting them. Besides, when testing the differences between the three categories and the emotions towards Bolivian immigrants, significant statistical differences were found between bigots and both subtle and egalitarian individuals . From the entire sample, 38.8% were men (n = 167) and 61.2% women (n = 264). Regarding participant’s ideological political self-placement, 4.2% choose right, 7.4% center-right, 52.6% center, 25.4% center-left, and 7.4% left.A total of 431 all first- and second-year undergraduate students from the psychology program at a large public university in Buenos Aires City were recruited for this study, with an age range from 18 to 45 years old  was adapted and validated for the purposes of this study. Responses were measured on a 5-point Likert-type scale with anchors at 1 = totally disagree and 5 = totally agree. The internal consistency levels for the subtle and blatant subscales in the present study were the same (α = 0.70).The 10-item subtle and blatant prejudice scale (5 for blatant prejudice and 5 for subtle prejudice) based on the originally scale developed by Pettigrew and Meertens (N = 302), being both internal consistency (α = 0.82) and construct validity  adequate. In our study, we have observed an adequate internal consistency (α = 0.91). Responses were measured on a 5-point Likert-type scale with anchors at 1 = totally disagree and 5 = totally agree. Higher levels suggest a higher social dominance orientation.The scale used was an adaptation and validation from the original scale  and its construct validity  was adequate .A local version of the RWA scale  for a right adjustment of an instrument from one language context to another , standard deviation (SD), asymmetry (S), and kurtosis (K).Table KMO = 0.860) test and Bartlett sphericity test (p < 0.001). The sedimentation graphic showed the presence of two factors with eigenvalues > 1  was conducted  arguments regarding subtle prejudice as a “new form of prejudice” or if it is just an expression of a global traditional prejudice, we contrasted the one-dimension model of prejudice with the two correlated dimensions model of subtle and blatant prejudice (Table r < 0.57), and so, the Cronbach alpha if item deleted .The results in Table ΔCFI ≤ 0.01 for the strict invariance  =  − 2.221; p < 0.05; Cohen’s d = 0.234), being women  who scored higher than men .It has studied the difference between the subtle and blatant forms of prejudice according to the participant’s gender. First, we tested the gender invariance of the scale, and all models  had an adequate fit. Following the criterion of In order to analyze the judgments about Bolivians’ rights and the feelings towards Bolivian immigrants, we constructed a typology based on Pettigrew and Meertens  recommenegalitarian (46.8%), followed by subtle (44.7%), bigot (7%), and error (1.5%) categories.The results on Table eelings towards Bolivian immigrants, perceiving statistically mean differences  = 13.309; p < 0.001). According to the post hoc Tukey b contrast, two groups were identified: Egalitarians  and subtles  on the one hand and bigots on the other . These results indicate that subtles and egalitarians negative feelings towards Bolivian immigrants were lower than those of bigots. Besides, regarding the participants’ answers about Bolivian immigrants’ rights, percentages are shown on Table Consequently, this typology was used as a grouping factor to perform one-way ANOVA analysis with the other variables assessed to test the validity of the scale. We proceeded to compare the means for the variable fχ2(2) = 35.535; p < 0.001).According to Table After testing the psychometric properties of the subtle and blatant prejudice scale towards Bolivian immigrants, we analyzed the correlations of both types of prejudice expressions with RWA, SDO, ideological self-placement (PI), and feelings towards Bolivian immigrants Table .Table 6RR2 = 0.246 was observed, and the contribution of SDO  and RWA (β = 0.304 p < 0.001) was similar. However, with subtle prejudice (R2 = 0.192), the RWA makes a greater contribution  than SDO .As it can be observed on Table r < 0.73) between the blatant and subtle subscales found in previous studies .After analyzing the psychometric properties of the subtle and blatant prejudice scale towards Bolivian immigrants in Argentina, the factorial structure identified was coherent with previous studies (Passini & Morselli, After grouping participants according to their subtle and blatant prejudice levels, most of them were classified as egalitarians — lower in both types of prejudice — compared with those on Chilean studies (Arancibia-Martini et al., As well as results obtained by Cárdenas et al. , in the The results in the present study indicated, like previous studies (Pettigrew & Meertens, As well as Passini  proposedAlso, it was observed that social dominance orientation explained partly the blatant prejudice towards Bolivian immigrants. This would indicate that, for people with higher levels of blatant prejudice, Bolivians would be perceived as a defiant social group that threats the sustaining of social inequality. As previously mentioned, both the perceived economic competition because of the scarce job offer (Valverde, One of the main limitations of this study was related to the convenience sample (Hernández Sampieri et al., From what was exposed here, it is necessary to continue assessing the variables proposed with samples that include participants from other social clusters and participants having direct contact with Bolivian immigrants or at least to include questions about immigrant background in order to know if their parents were both born outside Argentina —first-generation immigrants — or in Argentina — second-generation immigrants. Finally, we are aware that the hierarchical test performed in this study is the minimum requirement to compare a single factor with a two factor model of subtle and blatant prejudice. However, since the original scale has been adapted to assess prejudice towards Bolivian immigrants, it is important to consider that items are based on the original instrument, but the results cannot fully represent results as if the original instrument was administered.Despite these limitations, to have a version of the original scale adapted and validated for the analysis of subtle and blatant prejudice towards a social group highly vulnerable in our context and analyzing its psychological basis can contribute to develop future studies that allow the reduction of prejudice and discrimination towards that collective.


Multivariate logistic regression analysis was performed to establish radiomics model for predicting PCa. The diagnostic performance was assessed using receiver operating characteristic curve analysis. The radiomics model exhibited the best performance in predicting PCa, which was better than the performance of PI-RADS v2.1 scoring by the junior radiologist in the training group [area under the curve (AUC): 0.932 vs 0.803], testing group (AUC: 0.922 vs 0.797), and the entire cohort (AUC: 0.927 vs 0.801) (P < 0.05). The radiomics model performed well for lesions with PI-RADS v2.1 score of 3  and PI-RADS v2.1 score of 4–5  assigned by junior radiologist. The radiomics model quantitatively outperformed PI-RADS v2.1 for noninvasive prediction of PCa in men with PSA levels of 4–10 ng/mL. The model can help improve the diagnostic performance of junior radiologists and facilitate better decision-making by urologists for management of lesions with different PI-RADS v2.1 score.To develop MRI-based radiomics model for predicting prostate cancer (PCa) in men with prostate-specific antigen (PSA) levels of 4–10 ng/mL, to compare the performance of radiomics model and PI-RADS v2.1, and to further verify the predictive ability of radiomics model for lesions with different PI-RADS v2.1 score. 171 patients with PSA levels of 4–10 ng/mL were divided into training (n = 119) and testing (n = 52) groups. PI-RADS v2.1 score was assessed by two radiologists. All volumes of interest were segmented on T Serum prostate-specific antigen (PSA) is widely used as a highly-sensitive screening tool for PCa. Serum PSA level of 4 ng/mL was previously considered as the cut-off point for performing biopsy3. However, PSA level of 4–10 ng/mL is termed as the gray zone, as there is no clear consensus on the indication for performing biopsy in these patients5. In previous studies, biopsy-positive rate in men with PSA levels of 4–10 ng/mL was less than 30%, which implies that up to 70% of men were overtreated7. Unnecessary biopsy is a wastage of resources and can also lead to complications. Therefore, development of a non-invasive tool to predict PCa in patients who have PSA levels in the gray zone is a key imperative to reduce unnecessary biopsies.Prostate cancer (PCa), with an estimated incidence of 7.1% and mortality rate of 3.8%, is the most common malignancy and the second leading cause of cancer-related death among men in the world8. Currently, patients with PI-RADS score ≥ 3 are considered for prostate biopsy in clinical practice9. Of note, lesions with PI-RADS score of 3 are usually termed as "indeterminate" or "equivocal"8. Previous study has shown a low specificity of use of PI-RADS score of 3 as the cut-off point for PCa (62.1%), leading to a high proportion of false-positives10. Moreover, the PI-RADS score can be affected by the radiologists' subjectivity, and the most controversial lesions in this respect are those with PI-RADS score of 3. These limitations together with the development of increasingly powerful image acquisition and processing technologies have led to an increasing interest in new quantitative analysis methods.The Prostate Imaging-Reporting and Data System was released in 2012 and updated in 2019 (PI-RADS v2.1)12. However, in the past five years, studies16 have mainly focused on PI-RADS score, patient’s age, prostate volume, PSA level, and other clinical indicators to predict the risk of PCa in men with PSA levels in the gray zone, while radiomics has rarely been applied in this field. Thus, there is still room for improvement in non-invasive tools to identify patients who are most likely to have PCa and select these patients for biopsy.Radiomics provides a non-invasive and low-cost automated technology for objective and quantitative evaluation of tumor heterogeneityTherefore, the aim of this study was to develop MRI-based radiomics model as a non-invasive tool for predicting PCa in men with PSA levels of 4–10 ng/mL. In addition, we sought to compare the performance of radiomics model and PI-RADS v2.1, and to further verify the predictive ability of the radiomics model for lesions with different PI-RADS v2.1 score.A total of 171 patients with PSA in the gray zone were included in this study, including 66 patients with PCa and 105 patients with Non-PCa. In the evaluation of lesion location, 64 cases were located in the PZ and 107 cases were located in the TZ. Among the 66 PCa cases, 61 were clinically significant and 5 were clinically insignificant. Among the 105 patients with Non-PCa, there were 58 cases of benign prostatic hyperplasia and 47 cases of prostatitis. The characteristics of the study population are summarized in Table On univariate analysis, significant differences were observed between PCa and Non-PCa patients in the training group with respect to age (P = 0.019), lesion location (P < 0.001), PI-RADS v2.1 score by Radiologist A (P < 0.001), and PI-RADS v2.1 score by Radiologist B (P < 0.001). In addition, there were significant differences with respect to lesion location (P = 0.003), PI-RADS v2.1 score by Radiologist A (P < 0.001) and PI-RADS v2.1 score by Radiologist B (P < 0.001) in the testing group.The weighted Cohen's kappa value was 0.584 , indicating moderate overall inter-reader agreement with respect to PI-RADS v2.1 score. Among the 171 patients, 131 cases were consistently scored while 40 cases were inconsistently scored; of these 40 cases, 27 cases were over-graded by the Radiologist A. The PI-RADS v2.1 score of junior Radiologist A was more likely to be overestimated than that of Radiologist B (P = 0.030). Details of the PI-RADS v2.1 score between Radiologist A and Radiologist B are shown in the Table The diagnostic performance of radiomics model and PI-RADS v2.1 scoring by the two radiologists are shown in Table The rad-score of radiomics model showed significant differences between PCa and Non-PCa in the training group, testing group, and the entire cohort  than those by Radiologist B  for predicting PCa. And the radiomics model performed best for lesions with PI-RADS v2.1 score of 4–5 assigned by Radiologist A  and Radiologist B , as shown in Fig. In this study, we developed a MRI-based radiomics model to predict the risk of PCa in men with PSA levels of 4–10 ng/mL and compared its performance with the PI-RADS v2.1. Specifically, the radiomics model outperformed the results evaluated by the junior radiologist using PI-RADS v2.1 in the training group, testing group, and the entire cohort. A key finding was that the radiomics model also performed well for lesions with different PI-RADS v2.1 score.19. The advantage of our study was that we used the PI-RADS v2.1, which did not affect the overall diagnostic accuracy as compared to the PI-RADS v2, but helped improved the inter-reader consistency and simplified the allocation of scores21. Another interesting finding was that the diagnostic performance of PI-RADS v2.1 score by senior radiologist was better than that by junior radiologist. Moreover, the junior radiologist tended to overestimate the PI-RADS v2.1 score (P = 0.030). This may be due to the complexity of prostate signal pattern and the fact that PI-RADS v2.1 mainly relies on subjective analysis of MRI 22. It must be acknowledged that the PI-RADS v2.1 score depends on the radiologist's experience and diagnostic capability23. Therefore, there is a need for development of more quantitative analysis methods.In our study, we observed moderate inter-reader agreement of PI-RADS v2.1 , which was consistent with a previous study evaluating PI-RADS v2 scoring by experienced radiologists25, with an AUC of 0.844 and 0.941, respectively. The other previous study26 employed radiomics features extracted from 2D segmentations to predict the risk of PCa in men with PSA levels of 4–10 ng/mL. However, compared with 2D segmentations, 3D segmentations can help reduce inter-reader variability; moreover, it better captures the heterogeneity of lesions, and helps reduce the sampling errors30. It is worth noting that our radiomics model performed better in the testing group . This further demonstrated the ability of radiomics model for predicting PCa in men with PSA levels of 4–10 ng/mL, which can help decrease overdiagnosis and reduce unnecessary biopsies.Radiomics features offer the advantages of being an objective and quantitative tool. In our study, the radiomics model exhibited the best performance in both the training and testing groups, with an AUC of 0.932 and 0.922, respectively, which is consistent with the results of the two previous studies31. This is likely attributable to the fact that the PI-RADS v2.1 for predicting PCa is subjective and highly radiologist-dependent, whereas radiomics model is quantitative and relatively objective. In addition, we used a variety of methods to gradually filter the features to ensure that the selected features were valuable for predicting PCa. These encouraging results suggested that radiomics is a promising non-invasive technique for predicting PCa in men with PSA levels of 4–10 ng/mL. And in our study, we only included patients with PSA levels of 4–10 ng/mL to obtain a more precise diagnosis, thus resolving the clinical dilemma of whether biopsy should be performed.On comparing the ability of the radiomics model to predict PCa with that of PI-RADS v2.1, the radiomics model was found to perform slightly better than the PI-RADS v2.1 score by senior radiologist, but outperformed the PI-RADS v2.1 score by junior radiologist in the training, testing, and the entire cohort (P < 0.05). This finding was consistent with a previous study investigating patients with PSA at any level32. The current guidelines do not clearly delineate the management strategy for these uncertain lesions33. Previous report34 has investigated the usefulness of radiomics in detecting PCa in transition zone lesions with PI-RADS score of 3 (n = 41) and 4 (n = 32). However, the datasets were small and unbalanced, which limited the generalizability of the results. To the best of our knowledge, this is the first study that verified the predictive ability of the radiomics model for lesions associated with serum PSA levels of 4–10 ng/mL and PI-RADS v2.1 score of 3. Our study found that the radiomics model may potentially help predict PCa in lesions with PI-RADS v2.1 score of 3, thus facilitating the decision-making process for biopsy. Moreover, the radiomics model performed better for lesions with PI-RADS score of 3 assigned by junior radiologist (AUC: 0.854). This suggests that the radiomics model may be more useful for inexperienced radiologists in the PI-RADS v2.1 evaluation.Management decision-making for patients with lesions assigned PI-RADS v2.1 score of 3 is a clinical challenge for urologists: whether to follow up or conduct immediate biopsySome limitations of our study should be acknowledged. First, this was a retrospective single-center study without external validation. In future studies, our model should be assessed and verified in more centers with larger samples. Second, manual segmentations of VOIs are inevitably subjective bias. The VOIs were manually delineated by two radiologists in our study, and robust features were retained for further analysis to minimize the bias. Further studies should adopt automatic or semi-automatic segmentation. Third, we did not explore the performance of radiomics model in different zones because of the limited samples. Therefore, further investigations are needed to expand the samples and verify the predictive performance for PCa in different zones. Fourth, the pathological results were confirmed by biopsy. Nevertheless, we conducted multidisciplinary discussions before and after the biopsy to improve the accuracy and credibility. Additionally, we performed systematic TRUS-guided prostate biopsies plus targeted biopsies and identified lesions by comparing specimens with MRI results to reduce the deviation.In this study, the radiomics model quantitatively outperformed PI-RADS v2.1 for noninvasive prediction of PCa in men with PSA levels of 4–10 ng/mL. The radiomics model can help improve the diagnostic performance of junior radiologists and facilitate better management decision-making by urologists for lesions with different PI-RADS v2.1 score.This retrospective study was approved by our institutional ethics committee. The need for informed consent was waived by the Zhejiang Provincial People’s Hospital ethics committee.Between January 2019 and June 2021, data pertaining to 225 patients were reviewed from the institutional picture archiving and communication system. The inclusion criteria were: (a) PSA levels 4–10 ng/mL; (b) prostate MRI scan performed at our institution; (c) ≤ 1 month interval between determination of PSA level and MRI; and (d) a suspicious abnormality in MRI eligible for targeted biopsy (PI-RADS v2.1 score ≥ 3). Out of 225 patients, 54 were excluded for the following reasons: (a) lack of clinical or pathological data (n = 26); (b) history of prostate therapies before MRI scan (n = 21); or (c) poor imaging quality, such as motion or metal artifacts (n = 7). Finally, 171 patients were enrolled and divided into training group (n = 119) and testing group (n = 52) at a ratio of 7:3 according to the time of MRI scan. The study flowchart is shown in Fig. The clinical data, including patient’s age, PSA level, lesion location, and PI-RADS v2.1 score were assessed.1-weighted imaging, axial and sagittal T2-weighted imaging (T2WI), axial diffusion weighted imaging (DWI), and dynamic contrast enhanced imaging (DCE). The temporal resolution for DCE imaging was 11 s, and the total scan time was 2 min. Apparent diffusion coefficient (ADC) sequence was automatically generated from the DWI sequence. The detailed parameters for each sequence are shown in Supplementary Materials Prostate MRI with body phased-array coil was performed using a 3.0 T MRI scanner , including axial TThe PI-RADS v2.1 rates the likelihood of clinically significant cancer using a five-point scale: 1, very low; 2, low; 3, intermediate; 4, high; 5, very high. The assessment of PI-RADS v2.1 was performed by two radiologists: one junior radiologist with 5 years of experience (Radiologist A) and one senior radiologist with > 20 years of experience (Radiologist B) in urogenital imaging. Both radiologists were blinded to the initial MRI reports, clinical, and pathological data and respectively reviewed anonymized MRI images to assess the PI-RADS v2.1 score. For patients with multiple lesions, the largest or the most aggressive lesion in the PI-RADS v2.1 was assigned to the patient. Chi-squared test was used to analyze the PI-RADS v2.1 score assigned by the two radiologists, and weighted Cohen's kappa was used to assess inter-reader agreement.35. Any differences in evaluation were resolved by consensus. The histopathological information, such as pathological type  and Gleason score (GS) were recorded. The pathological results were divided into two categories: PCa set  and Non-PCa set (benign prostatic hyperplasia and prostatitis).A multidisciplinary team comprising of radiologist, urologist, sonographer, and pathologist evaluated each case, before and after the biopsy, to guarantee that each patient's segmentation, PI-RADS v2.1 score, and biopsy results were matched to ensure accurate results. All patients underwent at least 12-core systematic transrectal ultrasound (TRUS)-guided prostate biopsy plus targeted biopsy of the suspicious lesion on MRI or prostatectomy within 3 months of MRI scan in order to obtain pathological results. At least two cores were obtained from targeted biopsy of the suspicious lesion. All biopsy or prostatectomy specimens were further processed and evaluated by two pathologists using the International Society of Urological Pathology-modified Gleason score classification38. Image preprocessing was performed by resampling the images with a resolution of 1 × 1 × 1 mm3 through the linear interpolation method. And we used a z-score normalization to make the image intensities fit a standard normal distribution with μ = 0 and σ = 1, where μ is the mean value, and σ is the variance. The desired minimum value of the initial setting of gray-level discretization is 0 and the desired maximum value is 255. Firstly, A.K. software was used to rigorously register the images of T2WI, DWI and ADC sequences in order to reduce the potential influence of the parameters of a scanning scheme. After that, the standardized T2WI images were imported into the ITK-SNAP software to manually segment the prostate index lesion of each patient layer by layer and to determine the three-dimensional (3D) volume of interest (VOI). Since the three sequences have been rigorously registered, tumor VOI obtained from T2WI can be applied directly to other sequences. Segmentations were performed by two radiologists (Radiologist A and Radiologist B). For patients with multiple tumor foci, only the index lesion with the highest PI-RADS v2.1 score or the largest volume was selected for analysis. All segmentations were performed completely blinded to the clinical and pathological data. Subsequently, Spearman rank correlation test was used to calculate the correlation coefficient (CC) for each feature between the feature sets from Radiologist A and Radiologist B, according to a previous study39. Features with CC values > 0.80 were considered as robust features40. The quantified values of robust features were averaged for further analysis.Some preprocessing steps were carried out prior to feature extraction, namely resampling, normalization, and gray-level discretization using AK software  to match the resolutionA total of 1188 radiomics features were extracted for each patient using AK software. The details of all radiomics features are described in Supplementary Materials The diagnostic performance of PI-RADS v2.1 and radiomics model was evaluated using the receiver operative characteristic (ROC) curve analysis. The area under the curve (AUC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated in the training group, testing group, and the entire cohort, respectively. Respective AUC values were compared using the DeLong method to determine any significant difference. The agreement between radiomics model-predicted and expected probabilities was assessed using calibration curves and Hosmer–Lemeshow test. The clinical usefulness and the benefits of the radiomics model were evaluated using decision curve analysis (DCA). Five-fold cross-validation was also utilized for radiomics model evaluation. Finally, we used ROC curves to verify the predictive ability of radiomics model for lesions with different PI-RADS v2.1 score assigned by Radiologist A and Radiologist B, respectively. The radiomics workflow is shown in Fig. t test. Non-normally distributed continuous variables are presented as median (interquartile range) and between-group differences assessed using Mann–Whitney U test. Categorical variables are presented as frequencies and between-group differences assessed using Pearson’s Chi-squared test or Fisher’s exact test. Two-sided P values < 0.05 were considered indicative of statistical significance.Statistical analyses were performed using the SPSS software (version 24.0), MedCalc software (version 17.9.4), and R software (version 3.6.2). Normally distributed continuous variables with homogeneous variance are expressed as mean ± standard deviation and between-group differences assessed using independent sample The study was approved by the institutional review board and the Zhejiang Provincial People’s Hospital ethics committee. This study was conducted in accordance with the relevant guidelines, national legislation and the institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Supplementary Information.


Microscopic cracks in the cuticle (microcracks) are the first symptom of the strawberry fruit disorder ‘water soaking’ in which the fruit surface appears watery, translucent, and pale. Water soaking severely impacts fruit quality. The objective was to investigate the factors and mechanisms of cuticular microcracking in strawberry. Fluorescence microscopy revealed numerous microcracks in the achene depressions, on the rims between depressions and at the bases of trichomes. Microcracks in the achene depressions and on the rims were either parallel or transversely oriented relative to a radius drawn from the rim to the point of attachment of the achene. In the achene depression, the frequency of microcracks with parallel orientation decreased from the calyx end of the fruit, towards the fruit tip, while the frequency of those with transverse orientation remained constant. Most microcracks occurred above the periclinal cell walls of the epidermal cells. The long axes of the epidermal cells were primarily parallel-oriented. Microcracking increased during fruit development. Cuticle mass per fruit remained constant as fruit surface area increased but cuticle thickness decreased. When fruit developed under high relative humidity (RH) conditions, the cuticle had more microcracks than under low RH conditions. Exposing the fruit surface to increasing RHs, increased microcracking, especially above 75% RH. Liquid-phase water on the fruit surface was markedly more effective in inducing microcracking than high vapor-phase water (high RH). The results demonstrate that a combination of surface area growth strain and water exposure is causal in inducing microcracking of the strawberry cuticle. The CM fulfills important barrier functions. It helps restrict uncontrolled water and gas exchanges, inhibits pathogen entry and preserves fruit quality4. Maintaining all these functions requires an intact CM throughout fruit development.The cuticular membrane (CM) is an extracellular lipophilic membrane deposited on all primary above ground plant surfaces. The receptacle of the strawberry, a false fruit, is no exception. The CM comprises epicuticular and embedded cuticular waxes, polymeric cutin and phenolics. It is encrusted with cell-wall polysaccharides5. Microcracking is economically important in commercial fruit production and marketing. Microcracks impair the cuticle’s barrier function, thereby compromising fruit quality and, hence, fruit quantity (by necessary rejection). Moreover, microcracking is the first visible symptom in a number of fruit-surface disorders such as russeting in apple and pear6 and mango7, cracking in sweet cherry8 and grapes9, neck shrivel in plum10 and desiccation of lychee11. In strawberries, microcracking is the first symptom of impending water soaking12 and of cracking13. Microcracking allows rapid water uptake by viscous flow, that bypasses the cuticle barrier. The incidence of water soaking and cracking is high in open-field cultivation of strawberry and is substantially eliminated in protected cultivation14. Microcracking also increases fruit transpiration and, hence, accelerates fruit shrivel12. Lastly, microcracks serve as entry points for fruit rot pathogens causing diseases such as grey mold and anthracnose15. Despite the importance of cuticle integrity to the highly perishable strawberry fruit, remarkably little is known about cuticle deposition and microcrack formation in strawberries.The integrity of fruit cuticles is sometimes compromised by growth strains, resulting from rapid increases in fruit surface area and/or hostile environmental factors. Cuticular microcracks result from the failure of an overly-strained CM. Microcracks are minute fractures in the cuticle, not visible to the naked eyeThe objective of this study was: (1) to characterize microcracking of the cuticle of strawberry skin and (2) to identify the key factors affecting cuticular microcracking.Fluorescence microscopy of the strawberry fruit (receptacle) surface revealed numerous microcracks Fig. a. MicrocIrrespective of whether an achene was situated in the distal or proximal region of the fruit, microcrack orientation in all achene depressions showed a frequency distribution with three dominant peaks: at + 90°, 0° and −90°  was less effective in increasing microcracking than liquid-phase water. Exposure of the fruit surface to liquid water, increased cuticular microcracking rapidly, compared to the opposing fruit surface (control) that remained dry Fig. a. SurfacThe main findings of this study were: (1) Growth strain is the primary driver of microcracking of strawberry’s very-thin cuticle and (2) exposure of the fruit surface to water—either vapor or liquid—greatly exacerbates cuticular microcracking.16. The fixation of strain (elastic) essentially prevented/reduced microcracking. Beyond the breakpoint, the microcracking increased markedly, probably as a result of the continuing increases in strain in the face of the absences of any deposition of new cutin and wax and, hence, the absence of any further strain ‘fixation’. Unfortunately, the thin, and extremely fragile strawberry cuticle, prevented a strain-relaxation analysis18. However, it is expected that the increase in strain and the concurrent decrease in structural support by the underlying tissue due to fruit softening, induced failure and microcracking of the CM8. This cause and effect relationship is not unique to strawberry but is also seen in grapes19, sweet cherries20 and plums21.Growth strain causes cuticular microcracking. First, cuticle synthesis and deposition cease at about 28 DAFB, when the fruit has achieved about half its later maximum surface area (at ripeness). The subsequent doubling of fruit surface area to the fully-ripe stage, has the effect of distributing an approximately constant amount of cuticle over about twice the surface area. As a result, the cuticle is significantly strained and this strain is associated with significant thinning and failure. Interestingly, the cessation of cuticle deposition (28 DAFB) coincides with a breakpoint in microcracking incidence and severity. Before this stage, the deposition of cutin and the impregnation of the cutin network with wax partially ‘fixed’ the accumulating strain14. This makes it very susceptible to failure. Second, microcracks tend to be more frequent in the fruit’s proximal region  and equatorial region, than in its distal region (tip) (P = 0.06). The proximal and equatorial regions have larger diameters than the narrower tip region, due to the higher rates of radial expansion of the fruit. For similar reasons one would predict larger fruit will show more microcracking than smaller fruit. However, this relationship was not evident in our results—perhaps because of high fruit:fruit variability in microcracking. Third, the unique surface topography of the strawberry causes a characteristic pattern of microcracking. To better understand the pattern of microcracking, it is important to understand the origins of the unusual ‘corrugated’ topography of the strawberry surface. The corrugation of the surface results from restricted flesh expansion growth in the immediate vicinity of an achene and the lack of such a restriction in the spaces between achenes. The restriction in flesh expansion is probably a result of tensile forces in the radially orientated vascular bundles serving the achenes, with the result that the achenes are ‘pulled’ into the surface as flesh expansion growth proceeds. This results in the ‘funnel type’ structure of the achene cavity where the vascular bundles form the center of the funnel. This interpretation is consistent with a pronounced parallel radial orientation of the epidermal cells (in depressions and on the rim) irrespective of the position of the achene on the fruit surface. As a consequence, microcracks form transverse to this extension growth as the cuticle is dragged along by the underlying epidermal cells. This explains the peaks in the orientation frequency at ± 90° relative to any radius drawn from the achene axis.The above interpretation is also consistent with the following arguments. First, the strawberry cuticle is the thinnest fruit cuticle ever reported across large number of fruit species22.However, the above explanation does not account for the about 20% incidence of microcracks in the achene depression orientated parallel to the radii (0°) and the about 30% incidence on the rims between the depressions. Interestingly, in the achene depression these parallel-orientated microcracks occurred in both the proximal  and the equator regions but not in the distal (tip) region. A parallel orientation of the microcracks would result from a ‘flattening’ of the funnel-shape of the achene depression and thus an increase in the depression diameter. This would be consistent with a tangential strain as fruit volume and, hence, fruit circumference increased8. This is different from apple, where the pattern of microcracking aligns with the underlying anticlinal epidermal cell walls, indicating that a failure of cell–cell adhesion is a factor in microcracking23.Finally, there was preferential location of microcracks above the periclinal epidermal cell walls  in the achene depressions and on the rims between depressions. This distribution indicates strain is greater above the periclinal walls. This result is similar to that in sweet cherry, where microcracking is not related to an underlying anticlinal cell wall24, mango7, grape9 and sweet cherry25.Surface moisture plays a critical role in cuticular microcracking in strawberry fruit. Microcracking was especially increased when the fruit surface was exposed to liquid-phase water and less so, but still significantly, when it was exposed to high concentrations of vapor-phase water. These findings are not unique to strawberry but have also been reported in apple12. Hence, water uptake by the epidermal cells is judged unlikely to play a direct role. What other factors might be involved in water-induced microcracking?The mechanism of water-induced microcracking is not known. Earlier experiments demonstrated that strawberry fruit incubated in an isotonic polyethylene glycol solution did not take up water but they did develop microcracks26. It is already known that hydration of a strained cuticle alters its rheological properties, leading to a general weakening. Both the cuticle’s stiffness and its fracture force decrease, while its fracture strain increases. This phenomenon has been demonstrated for the cuticles of sweet cherry25, tomato27 and for apple and pear28.First, exposure to liquid water, or even to high concentrations of vapor-phase water, hydrates the cuticle29. Here, the swelling is accompanied by an increase in intercellular spaces, presumably due to a reduction in cell–cell adhesion and to partial cell separation in the strained fruit skin29. In sweet cherry, cell wall swelling results in decreased cell–cell adhesion30. Additional evidence for a role for cell wall swelling in microcracking in strawberry comes from an effect of Ca ions on microcracking. Microcracking is reduced if fruit are incubated in a CaCl2 solution31. In sweet cherry, Ca ions decrease cell wall swelling by increasing crosslinking of the cell wall constituents. This particularly applies to the homogalacturonans of the middle lamella32. As a consequence, cell–cell adhesion is increased and along with the skin’s fracture force33. These interpretations are consistent with the absence of a role for the cuticle in fruit-skin mechanics. It is the epidermal and hypodermal cell layers that represent main structural member in most fruit skins—not the cuticle36.Second, it is possible that surface wetness (liquid-phase water) may induce swelling of the epidermal cell walls immediately underlying the cuticle. Cell wall swelling would strain the immediately overlying area of cuticle and so could result in microcracking. Epidermal cell wall swelling has been reported in strawberry during ripening37. It is not known if such downregulation also occurs in strawberries.Lastly, surface wetness (liquid-phase water) decreases the rate of cuticle deposition (both wax and cutin) and the decrease favors cuticle failure during fruit expansion growth. In apple, the genes involved in cuticle synthesis and deposition are downregulated when the fruit surface is exposed to surface moistureThe strawberry cuticle is subject to marked growth strain during fruit development. First, this is because the rate of cuticle deposition in strawberry is low, compared with other fruit crop species, and it is also limited to the early stages of fruit development. Second, strawberries develop to considerable size during quite a short period of time. As a result, the amount of cuticle present at the stage deposition ceases must be stretched over a much larger area during quite a short period of time. In the light of this information, the high incidence of cuticular microcracking in strawberry is hardly surprising. Taking both a whole-fruit perspective, and also a more local achene-depression perspective, the incidences and orientations of microcracking are fully consistent with the idea that growth strain is the dominant driver of cuticular microcracking. Along with this, as in many other fruit crop species, the exposure of the strained cuticle of strawberry to either liquid-phase or vapor-phase water exacerbates microcracking. Projecting these mechanistic understandings to the production issues facing strawberry growers, the cultivation of strawberries in protected environments or in regions with low rainfall and low RH and the maintenance of open canopy structures should all reduce cuticular microcracking and hence also reduce the incidences of water soaking, flesh cracking and fruit rots.Fragaria × ananassa Duch., ‘Clery’, ‘Elsanta’ and ‘Florentina’) were cultivated in a growth chamber or in a greenhouse on the Herrenhausen Campus of the Leibniz University, Hannover, Germany . Plants in the growth chamber were grown in pots (14 cm diam.) filled with a peat/moss-based growing medium . The growing conditions in the growth chamber were set at 20/16 °C day/night temperature, 60/80% day/night relative humidity (RH), and a 16 h photoperiod. In the greenhouse, plants were grown in boxes (size 25 × 120 × 20 cm) with 8–10 plants per box. Fruits were selected for uniformity of size and color and absence of macroscopically visible defects.Strawberry  aqueous acridine orange  for 5 min. This incubation time is sufficiently long to allow dye uptake, but not long enough to induce microcracks. After incubation, fruit was rinsed with deionized water, blotted dry with soft tissue paper, and the fruit surface viewed under a fluorescence binocular microscope . Calibrated images were taken randomly  in the equatorial region of the fruit, where diameter is at its maximum. The number of individual fruit replicates was 20. Microcracking was indexed by recording the percentage of the surface area in the microscope window infiltrated by acridine orange. The areas infiltrated by acridine orange were quantified using image analysis . Fluorescing areas associated with achenes and trichomes were subtracted.Microcracking of the cuticle of strawberry fruit was studied following infiltration with the fluorescent tracer acridine orange using fluorescence microscopy. When incubating fruit in an aqueous solution of acridine orange, penetration of the tracer is restricted to openings in the cuticle including those associated with microcracks, lenticels, and (sometimes) stomata and trichomesOrientations of microcracks and epidermal cells in the achene depressions and on the rims between depressions. The achene depression, and the rim between adjacent depressions, were selected because they are representative locations on the ‘corrugated’ strawberry fruit surface. The orientations of microcracks and epidermal cells were quantified as the angle made between the microcrack, or of the long axis of an epidermal cell, and a radius drawn from that point to the point of attachment of the achene—like the spokes of a bicycle wheel, with the point of attachment of the achene being the hub . Depending on the orientation of a microcrack or cell, the angle value might range from − 90° (anticlockwise) to + 90° (clockwise). The exact point of attachment of an achene was determined in a preliminary experiment in which achenes were excised and viewed under a microscope with the lower side up so that the point of attachment was visible . Calibrated images were taken and the maximum length (ymax) and maximum width (xmax) of the achene determined using image analysis. The point of attachment was then expressed relative to the ymax and the xmax. This point had the x and y coordinates of 0.5·xmax and 0.7·ymax ; the equatorial region (maximum fruit diameter and about the center of the fruit), and the tip region .The dimensions of the achene depressions  were measured on 3D-images using a digital microscope . The areas of the fruit surface investigated were the proximal, equatorial, and distal regions of the fruit. The fruit used for these measurements were selected to be of almost the same mass (10.9 ± 0.2 g). The number of replicates was 16 per region.This was studied by harvesting 20 fruits of the same ripeness stage (color) but of a broad range of different masses (5.1–26.4 g per fruit). The soluble solids of the expressed juices were quantified  using a refractometer . To record microcracks, the procedure described above was used.Microcracking was also quantified in three different regions of the fruit surface along its length: within the region carrying achenes near the calyx , near the equator (the point of maximum diameter) and near the tip . A total of 14 fruits were investigated.To identify the sites of initiation of microcracking, it was assumed the shortest microcracks had only recently been initiated. We selected only those shorter than the dimensions of the underlying epidermal cells. The percentages of short microcracks lying just above the anticlinal cell walls and those just above the periclinal cell walls were recorded in the achene depressions and also on the rim between adjacent depressions.38. Juice of the fruit was extracted using a ‘garlic press’ and its osmotic potential quantified by water vapor pressure osmometry .The time course of cuticular microcracking was investigated in fruit sampled at 20, 28, 33 and 37 days after full bloom of the respective flower (DAFB). Younger fruit (< 20 DAFB) were too small to measure. Microcracks were quantified following infiltration with acridine orange as described above. Fruit developmental stage was characterized by measuring fruit mass, color and the osmotic potential of the expressed juice. Color was quantified in the equatorial region using a spectrometer . The hue angle was calculated−1; Panzym Super E flüssig, Novozymes A/S, Krogshoejvej, Bagsvaerd, Denmark), and cellulase  buffered in 50 mM citric acid buffer39. The pH was adjusted to pH 4.0 using NaOH. Sodium azide (NaN3) was added at a final concentration of 30 mM to prevent microbial growth. The isolation medium was refreshed once. Six CM discs per fruit were collected from a total of 25 fruits. There was no significant difference in CM area after isolation when only one ES (7.2 ± 0.2 mm2) was excised per fruit or when several ES (7.4 ± 0.2 mm2) were excised per fruit. Following isolation, the CMs were rinsed three times with deionized water. Adhering cellular debris was removed by ultrasonication at 35 kHz for 10 min . Achenes were carefully removed from the CM discs by hand. The CM samples (n = 5 discs per rep) were dried above silica gel for 48 h and weighed on a microbalance . Wax was extracted from the CMs by incubating in CHCl3/MeOH  for 24 h at room temperature. The DCM were dried above silica gel and re-weighed. The wax mass per unit area was obtained by subtracting the mass of the DCM from that of the CM. The wax content was calculated. The number of replicates was 10.The change in mass of the CM, the mass of the dewaxed CM (the DCM) and the wax fraction were determined on the same batches of fruit. Epidermal skin segments (ES) comprising cuticle, epidermis, and adhering flesh were excised in the equatorial region of the fruit using a biopsy punch . Care was taken to include not more than one achene per ES. The CMs were enzymatically isolated by incubating the ESs in a solution of pectinase . Pottle humidities were adjusted using dry silica gel, or saturated solutions of CaCl2, NaCl or deionized water41. Using our setup, the RH in the pottle equilibrated at about 23% RH when the bottom of the pottle was covered with dry silica gel, at 48% RH when using CaCl2, at 83% RH when using NaCl and at 100% RH when using deionized water. There was no contact between the salts / solutions and the fruit . This was done as follows: at 10 DAFB fruitlets were introduced through a 12-mm-diameter hole in the lid of the pottle. The space between the hole and the peduncle was then sealed with soft plastic-foam plug. This prevented damage of the peduncle and also reduced water–vapor transfer into/out of the pottle25. Briefly, stainless steel washers (6.4 mm inner diameter) were glued onto the fruit surface in the equatorial region of a mature strawberry using a fast-curing epoxy glue . The washer prevented any strain relaxation of the fruit skin before recording microcracking. The extent of microcracking was quantified before and after exposure to the different humidities. Before exposure, acridine orange was applied to the ES surface. After 5 min, the dye solution was removed, the surface rinsed with deionized water and the ES viewed under a fluorescence microscope. The area infiltrated by dye was quantified. Thereafter, the ES were incubated in polypropylene boxes above either dry silica gel, or saturated salt slurries of CaCl2 or NaCl, or deionized water41 . After 0, 0.25, 0.50, 1, 2, 4, 8, and 10 h, 10 fruit were examined by staining with acridine orange, blotting dry, and inspecting for microcracks using fluorescence microscopy. The fluorescing areas were quantified as described above.Moisture effect on microcracking was studied at four stages of fruit development. Fruit was harvested at 20, 28, 33 or 37 DAFB. Individual fruits were incubated for 4 h in deionized water as described above with half being submerged in deionized water and half remaining dry (control). Microcracks were quantified as described above.P < 0.05. Data in the Tables and Figures are presented as means ± standard errors.All experiments comply with relevant institutional, national, and international guidelines and legislation. All experiments had completely randomized designs. Data were subjected to analysis of variance and regression analysis using R . Means were compared using Tukey’s studentized range test at Supplementary Information 1.Supplementary Figures.Supplementary Table S1.


Tumor necrosis factor-α (TNF-α) is an important mediator of the immune response. At present, the improvement of TNF-α after continuous positive airway pressure (CPAP) treatment of obstructive sleep apnea-hypopnea syndrome (OSAHS) is still controversial.We conducted a systematic review of the present evidence based on a meta-analysis to elucidate the effects of TNF-α on OSAHS after CPAP treatment.P < 0.001) based on the REM since there is highly significant heterogeneity (I2 = 90%) among the studies. Therefore, we used the subgroup and sensitivity analyses to investigate the source of heterogeneity. The findings of the sensitivity analysis revealed that the pooled WMD ranged from 0.91  to 1.18 . The findings were not influenced by any single study. Notably, there was homogeneity in the Asia subgroup and publication year: 2019, implying that these subgroups could be the source of heterogeneity.To measure TNF-α, ten studies used enzyme-linked immunosorbent assay (ELISA), and one used radioimmunoassay. The forest plot outcome indicated that CPAP therapy would lower the TNF-α levels in OSAHS patients, with a weighted mean difference (WMD) of 1.08 (95% CI: 0.62–1.55; Our meta-analysis recommends that CPAP therapy will decrease the TNF-α level in OSAHS patients, but more related research should be conducted. Obstructive sleep apnea-hypopnea syndrome (OSAHS) is characterized by repeated episodes of partial or complete upper airway obstruction during sleep, resulting in chronic intermittent hypoxemia, excessive daytime sleepiness, irregular snoring at night, and increased nocturia. It is recognized as a new controllable risk factor for cerebrovascular and cardiovascular disease , 2. Numeet al. [et al. [Tumor necrosis factor-α (TNF-α) is a multidirectional pro-inflammatory cytokine secreted by various cells, including adipocytes, activated monocytes, macrophages, B cells, T cells, and fibroblasts. TNF-α is an important mediator of the immune response. Chronic inflammatory injury involving numerous inflammatory factors such as TNF-α, C-reactive protein, interleukin-6 (IL-6), and interleukin-8 (IL-8) is the leading cause of cardiovascular and cerebrovascular complications , 11. In et al.  revealed [et al.  demonstrTo determine potential applicable original articles, a thorough search in the PubMed, EMBASE, and Web of Science databases up to January 2, 2022, using the following words: "CPAP", "continuous positive airway pressure", "tumor necrosis factor-α", "TNF-α", "OSA", "obstructive sleep apnea-hypopnea syndrome", "obstructive sleep apnea" and "OSAHS". We first screened the article’s title and abstract and reviewed the included research reference lists to find additional relevant articles.The present study had no national limitations. All OSAHS patients received CPAP treatment; plasma TNF-α was measured before and after the CPAP; studies were limited to humans, published in English, and included detailed raw data. Cancers and other illnesses that could be associated with TNF-α were not included. The present study excluded repeated studies, letters, case reports, abstracts, and comments. We extracted the first author’s name and publication year, sample size, country, CPAP time, AHI, BMI, and age from the final included studies. After removing duplicates, two independent reviewers (ZFR and LY) assessed the studies against the inclusion and exclusion criteria. The third author (JJX) resolves conflicts.I2 index determines heterogeneity. I2 values of 75–100%, 50–75%, 25–50%, and < 25% indicated high, medium, and low heterogeneity and homogeneity. If the I2 value is > 50%, the random effect model (REM) is used; otherwise, the fixed effect model (FEM) is used. The weighted mean difference (WMD) of TNF-α levels was calculated for each study. The sensitivity analysis was repeated several times to evaluate the impact of each study in the analysis, each time excluding a different individual study. By excluding different individual studies each time, the sensitivity analysis was repeated to evaluate the impact of each study in the analysis.The value of the Sixty-three articles were identified as relevant. The removal of repeated studies reduced the number of studies to thirty-seven. The remaining thirty-seven studies were screened, resulting in the exclusion of seventeen studies. After reviewing the full text of the twelve studies, nine articles were excluded. Finally, eleven articles were included in the meta-analysis , 17–26. P < 0.001) based on the REM since there is highly significant heterogeneity (I2 = 90%) among the studies P < 0.001) to 1.18 . The findings were not influenced by any single study.The forest plot outcome indicated that CPAP therapy would lower the TNF-α levels in OSAHS patients, with a WMD of 1.08  and Egger’s test (P = 0.154) indicated a significant negative publication bias.Change in TNF-α levels in OSAHS patients was also investigated using subgroup analysis. Patient number and CPAP duration time were also significant in the region subgroup, and the complete information is presented in Our meta-analysis suggests that CPAP therapy reduces TNF-α levels in OSAHS patients. Simultaneously, the present study illustrates high heterogeneity. In addition, subgroup analysis was performed to investigate the differences between TNF-α level and OSAHS. Furthermore, sensitivity analysis revealed that the overall results remained unchanged when any single study was excluded or REM was converted to FEM. Therefore, we believe the data obtained from the present study are reliable.et al. [Long-term CPAP therapy decreases circulating levels of TNF-αin OSA patients , which iet al.  describeet al. . Studieset al. . Moreoveet al. . BMI waset al. . Due to et al. . HoweverOSA is a risk factor for obesity and cardiovascular disease. The cytokine TNF-α gene is also linked to OSAHS susceptibility; the frequency of the ’-308A’ allele in the TNF-α gene was significantly higher in obese patients with OSA compared to obese subjects without OSA . In addiSo far, this is the first meta-analysis to determine whether OSAHS patients are related to TNF-α and CPAP treatment. Simultaneously, our study also has some limitations. First, we lack information on BMI, AHI, age, gender, and CPAP time due to a lack of data. Second, the results may be biased due to the differences in TNF-α susceptibility and measurement methods. Finally, primary prevention necessitates a large number of subjects and a more extended follow-up period, which can cause information deviation and affect the accuracy of the results.Our meta-analysis recommends that CPAP therapy decrease the TNF-α level in OSAHS patients, but more related research should be conducted.S1 Checklist(DOCX)Click here for additional data file.


Diabetic wound healing poses a significant clinical dilemma. Bacterial infection and immune dysregulation are the predominant reasons. However, conventional wound dressings with a single treatment approach often limit therapeutic efficacy and continue working with difficulty. These limitations cause high treatment failure for diabetic wounds. In this study, we developed a multiple drug-loaded carbomer hydrogel containing Que/Van/Rif (QVR-CBMG) for the simultaneous treatment of infection and immune dysregulation. Honeycomb-like QVR-CBMG hydrogel exhibits excellent abilities to eliminate bacterial infection and biofilms in vitro. Moreover, QVR-CBMG hydrogel possesses an immunomodulatory capacity via affecting the Sirt3/SOD2 signaling pathway to promote M2 macrophages. Furthermore, QVR-CBMG hydrogel effectively promotes wound healing in diabetic rats through several mechanisms. The multidrug-loaded wound dressing not only eliminates bacterial infection and facilitated angiogenesis but also promotes collagen deposition and remodulates the local immune microenvironment in the areas of wounds. In summary, this synthetic strategy to eliminate infection and regulate immune disorders has potential translational value for the prevention and management of diabetic wounds. The management of diabetic wounds presents a significant clinical challenge due to their difficulty in healing and the potential for severe complications, leading to substantial economic and societal burdens . The micAppropriate antibiotic therapy remains the most common choice to effectively control infection . RifampiIn addition to addressing bacterial infections, the dysfunctional immune microenvironment in diabetic wounds is of equal concern . The secHydrogels have been widely used in wound healing ,18. AnotTherefore, we developed a carbomer hydrogel (CBMG) loaded with Rif/Van/Que (QVR-CBMG) to treat diabetic-infected wounds. We first evaluated its antibacterial effects against both planktonic bacteria and biofilms in vitro. Then, we investigated the influence of this hydrogel on macrophage polarization and its potential mechanisms. A diabetic-infected wound model was performed to evaluate the antibacterial and immunomodulatory properties of QVR-CBMG to promote wound healing in vivo . Our stuThe microstructures of the hydrogels were examined using SEM. The biocompatibility of hydrogels was assessed using the CCK-8 assay. CCK-8 analysis showed that the hydrogels had no effect on BMDMs at any time point . The excWe then conducted an in vitro evaluation of the MRSA biofilm-clearing capabilities of QVR-CBMG hydrogel. Initially, we utilized 1% crystal violet staining to demonstrate the potential of the hydrogel for clearing biofilms. The hydrogel loaded with Rif showed superior efficacy in biofilm clearing compared to the hydrogel loaded with Van. However, the QVR-CBMG and VR-CBMG hydrogels exhibited the most effective biofilm clearance, as confirmed by IF stains A,C. AddiThe dysfunctional immune microenvironment is a significant obstacle to the repair of diabetic wounds . NumerouThe underling mechanism of Que in regulating macrophage polarization is still unclear. Previous reports indicated that Que plays a corresponding role by activating Sirt3 ,33. SirtSOD2, which is downstream from Sirt3, plays a significant role as an antioxidant enzyme in mitigating cellular damage and the progression of diseases triggered by inflammation ,38. HoweThe healing process of diabetic-infected wounds is complex and is impeded by various factors, such as drug-resistant bacterial infections, vascular impairment, oxidative damage, and immune microenvironment alterations ,43. To eThe MRSA in infected skin tissues is represented in In the main structure of the skin, collagen fibers are mainly composed of thick collagen type I, which is a major component of skin tissue, and thin collagen type III, which is a major component of reticular fibers . The norThe immune microenvironment plays a crucial role in the process of diabetic wound healing ,50. CD86In conclusion, the QVR-CBMG hydrogel, which incorporates Rif, Van, and Que, has exhibited significant potential in enhancing the healing of diabetic wounds. The ease of preparation and sustained release of the loaded drugs make this multifunctional hydrogel highly advantageous. Moreover, the QVR-CBMG hydrogel effectively eliminates biofilms and provides comprehensive and long-lasting antibacterial protection for diabetic wounds. Additionally, this hydrogel promotes the polarization of macrophages towards the anti-inflammatory M2 phenotype by activating the Sirt3/SOD2 signaling pathway in macrophages. This mechanism creates a favorable microenvironment for tissue repair. Simultaneously, the hydrogel induces vascularization, enhances proliferation, promotes the formation of granulation tissue, and facilitates collagen accumulation, showcasing its immense potential in promoting diabetic skin regeneration. Overall, the QVR-CBMG hydrogel represents a multifunctional therapeutic approach capable of eliminating biofilm infection, fostering an anti-inflammatory microenvironment, and facilitating tissue regeneration.Staphylococcus aureus  was a gift from the microbiology lab at the author’s institute.Que, Van, and Rif were procured from Solarbio . Carbomer 940  and triethanolamine  were obtained from Macklin Reagent Co. Ltd. . Methicillin-resistant 2O to prepare 1 wt% carbomer gel (CBMG) [2O and stirred at 60 °C for 30 min. Then, 0.25% triethanolamine  was added into the carbomer solution to adjust the final pH to 6.5–7.5. After stirring for 10 min, CBMG was prepared. Afterwards, drugs  in powders of different weights were mixed with CBMG under stirring for 30 min and left overnight at room temperature to form combined hydrogels. The crude hydrogels were washed three times with ddH2O to remove the uncombined material and stored in a sealed bottle at 4 °C.Carbomer 940 was dissolved in ddHl (CBMG) . SpecifiThe microstructure of the hydrogel was analyzed via a SEM . As for the freeze-drying process, the hydrogels were frozen at −80 °C for 24 h and then subjected to freeze-drying at a pressure of 0.05 mbar for 24 h. Regarding the SEM imaging parameters, the voltage used was 5 kV, the current was 10 μA, and the detector used was SEM (SU-3500). 2O to remove the uncombined materials. The washed hydrogels were then stored in a sealed bottle at 4 °C.The hydrogel samples were prepared by mixing the drugs  in a powdered form with the CBMG solution. The mixture was stirred for 30 min and left overnight at room temperature to form combined hydrogels. The resulting crude hydrogels were washed three times with ddHTo analyze the rheological properties of the hydrogels, rotary rheometer measurements were conducted. The hydrogel samples were placed between the rotor and the base of the rheometer to prevent slipping. A frequency of 1 Hz and a constant strain rate of 1% were maintained during the experiments. The measurements were performed within the linear viscoelastic range of the hydrogel. Three repetitions of the experiment were conducted, and a representative plot was selected to illustrate the changes in the elastic modulus, storage modulus, and the storage/loss modulus ratio of the hydrogel as a function of the temperature.CCK-8 kits  were used to investigate the cell viability of BMDMs with samples at different concentrations. In brief, BMDMs were cultured with the control, CBMG, VR-CBMG, and QVR-CBMG for 1 day and 3 days, respectively. The cells were then washed and stained with 10% CCK-8 solution for 4 h. The relative cell density was measured using a microplate reader  at an absorbance value of 450 nm.g for 5 min. Subsequently, the cells were suspended in a red blood cell (RBC) lysis buffer for 15 min at 4 °C to purify the bone marrow cells. The suspended cells were then centrifuged at 500× g for 10 min to collect the BMDMs and eliminate the RBCs. Then, the cells were resuspended in 10% FBS/DMEM (high-glucose formulation) and cultured at 37 °C with 5% CO2. Nonadherent cells (monocytes) were collected and resuspended in an antibiotic-free complete medium containing 10% FBS and 15 ng/mL M-CSF after 48 h of culture. Macrophages were generated for 4 days in the presence of M-CSF.Primary rat bone marrow-derived macrophages (BMDMs) were obtained from Sprague–Dawley (SD) rats aged 28 days. Initially, bone marrow cells were extracted from the femur, followed by centrifugation at 450× IF staining was used to detect the protein levels of Sirt3 (1:400), CD68 (1:400), CD206 (1:400), CD31 (1:400), α-SMA (1:400), and F-actin (1:1000). Briefly, cells or tissues were immobilized with 4% paraformaldehyde for 15 min and then treated with 4% bovine serum albumin for 1 h. Afterward, the samples were incubated with antibodies overnight at 4 °C and labeled with secondary fluorophore antibodies  for 1.5 h. Nuclear staining was done using DAPI  for 15 min. Finally, all samples were observed via a confocal microscope .RIPA lysis buffer  containing 1 mM phenylmethanesulfonylfluoride  and phosphatase inhibitors  was used to extract cells. Following the determination of the protein concentrations using the BCA protein assay kit , 50 µg of protein extract was separated through gel electrophoresis and then transferred onto polyvinylidene fluoride membranes . After being blocked with StartingBlock  for 15 min, the membranes were incubated overnight at 4 °C with primary antibodies such as Sirt3 (1:1000), SOD2 (1:1000), and GAPDH (1:5000). Subsequently, the membranes underwent rinsing and were incubated for 2 h with horseradish peroxidase-labeled secondary antibodies . Following rinsing, the protein signals were visualized using Image Lab 3.0 software.−∆∆Ct method.The total RNAs were isolated from BMDMs following the provided guidelines . The purity of the RNAs was assessed by measuring the OD at 260 and 280 nm, and a ratio of A260/A280 over 1.8 was considered satisfactory. The integrity of the RNAs was confirmed by agarose gel electrophoresis. cDNA synthesis was performed using the First-Strand Synthesis for qPCR kit . RT-PCR was carried out using SYBR Premix Ex Taq kits  and the Bio-Rad CFX96 Real-Time System . Relative expression levels of the target genes were determined using the 26 CFU/mL) was added to 900 µL fresh TSB medium. After incubation for 48 h, the culture medium was removed, and the 48-h-old biofilm was treated with different hydrogels (10% in 1 mL fresh TSB) overnight. Then, the biofilm in each group was washed with PBS twice. The antibiofilm effect of QVR-CBMG was evaluated using crystal violet staining , the spread plate method, and Live/Dead bacterial staining . In brief, 100 µL of bacterial suspension (MRSA at 1 × 10The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) for Van-CBMG. Firstly, 100 µL of diluted MRSA suspension (100×) was added to 10 wells in a 96-well plate, and then, 100 µL of Van-CBMG at a content of 512 μg/g was added to the first well. After two-fold serial dilution, the bacteria were grown overnight with different contents of Van-CBMG. Finally, the MIC was defined as the lowest Van-CBMG concentration at which no turbidity was observed in the corresponding well.v/v) overnight. The treated biofilm was rinsed, fixed, and then stained with 1% crystal violet.To determine the reciprocal reinforcement of Van combined with Rif to eradicate a mature biofilm, VR-CBMG with Van (4 μg/g) and various contents of Rif from 4 to 256 μg/g were used to treat a 48-h-old biofilm. Briefly, a 48-h-old biofilm was cultured in 1 mL fresh TSB medium with 50% VR-CBMG . The wound sites were then treated with approximately 200 µL of the hydrogel at 48 h post-infection. The optical images of the wound were captured using a digital camera on days 0, 3, 7, and 14, respectively. Five rats from each group were randomly selected for bacteriological assessments on day 3.Female Sprague–Dawley (SD) rats weighing 200 g were obtained from the Laboratory of Animal Center . These rats were housed in a specific pathogen-free (SPF) environment at a temperature of 22 ± 1 °C with a 12-h light–dark cycle. Diabetic animal models were induced by administering streptozotocin in a citric acid buffer (50 mg/kg). Fasting blood glucose levels above 16.67 mmol/L after one week were considered a successful model. Then, the rats were anesthetized with an intraperitoneal administration of pentobarbital (40 mg/kg). After shaving and disinfection, circular full-thickness skin defects with a diameter of 10.0 mm were created on the dorsal side of each rat. Next, each defect area was infected with 10.0 µL of MRSA suspension  staining and Sirius red staining (polarized light). Immunohistochemical staining was performed using antibodies against CD31 (1:400) and α-SMA (1:400) to investigate angiogenesis during the wound-healing process. Additionally, CD68 (1:400)/CD206 (1:400) antibodies were utilized to identify polarized macrophages.p-value less than 0.05 was considered statistically significant.Each experiment was performed at least three times. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test were conducted using SPSS 26 . All data are shown as the mean ± standard deviation (SD), and a


Pityriasis amiantacea (PA) is a rare but distinctive scalp condition. It is characterised by thick, silvery scales that bind together causing matting of multiple hair shafts. This can ultimately result in temporary or permanent hair loss. PA is thought to be a reaction pattern rather than a specific diagnosis and common associated conditions include: scalp psoriasis, seborrhoeic dermatitis, tinea capitis and atopic dermatitis. We report a case of PA presenting in a patient with longstanding rheumatoid arthritis (RA), associated with tumour necrosis factor-α inhibitor (anti-TNF-α) therapy.A 42-year-old was reviewed at her routine rheumatology follow up appointment. She has a 15-year history of seropositive, erosive RA managed with monthly golimumab injections for the last eight years. Prior to this she had been on etanercept for four years, but this lost efficacy. Unfortunately, she had poor disease control on methotrexate, leflunomide and sulphasalazine.She developed a sore area on her scalp nine months previously with accompanying itch. This then spread into an extensive confluent area of redness, associated with soreness, scale, matting onto hair and hair loss. She had been treated unsuccessfully with multiple antibiotic courses, including flucloxacillin, doxycycline and clarithromycin, oral fexofenadine, topical diprosalic and ketoconazole shampoo. The pruritic skin lesions continued to progress and even spread to behind the ears. This had been managed by her general practitioner (GP) as otitis externa. The patient was otherwise well with no joint pains, fever, weight loss or other systemic features.Staphylococcus aureus and Psuedomonas aeruginosa were identified. Dermatology consult was sought, and she was diagnosed with extensive impetiginized pityriasis amiantacea associated with otitis externa. This was thought to be secondary to her anti-TNF-α therapy. Initial management was with scalp oils to soften the scales, Betnovate scalp ointment and a course of antibiotics. However, the mainstay of treatment was stopping the golimumab. Within one month of these interventions she made a dramatic improvement.On examination, thickened, hyperkeratotic scaling plaques were seen with clumped hair shafts and underlying erythema of the entire scalp. There were no skin lesions over the rest of the body and nail changes were absent. Swab culture from the scalp was negative for fungal infection but Staphylococcus isolates found in skin swabs taken from the affected scalp. Both S. aureus and S.epidermidis were identified; this could suggest incidental colonisation or that these bacteria play a part in development of PA. Multiple studies have looked at distinguishing microbial biofilms and their structure and physical properties. Resilient biological scaffolding of microbial biofilms, rich in polysaccharides, proteins, lipids, and nucleic acids, is triggered by the bacteria and develops on the scalp and hair shafts. As this continues it promotes accumulation of adherent scale and the hair shafts are increasingly bound together and matted to the scalp.The pathogenesis of PA is yet to be definitively established; however, it has been thought that PA may be a manifestation of an underlying inflammatory skin disease. Most commonly, PA is associated with psoriasis and seborrheic dermatitis. Drug induced cases have also been described. There are case reports of PA occurring in patients treated with valproic acid, vemurafenib and minoxidil. The skin symptoms seen in PA are likely a reaction of hair-bearing scalp to various inflammatory and microbial insults. Moreover, a study undertaken in Egypt found that over 96% of patients with PA had TNF-a is an inflammatory cytokine produced by macrophages during acute inflammation. It causes a range of signalling events within cells, leading to necrosis or apoptosis, which contributes to the pathogenesis of many chronic inflammatory diseases, like psoriasis. However, in some cases TNF-a inhibition can paradoxically lead to new psoriasis or worsening of existing psoriasis. This has been observed with the three major anti-TNF-a agents . Often it is seen in patients receiving treatment for rheumatoid arthritis or inflammatory bowel disease. On average, cutaneous adverse effects are seen approximately 17 months after starting anti-TNF-a therapy. The most frequently seen presentations are of plaque psoriasis and palmoplantar pustulosis.Inhibition of TNF-a causes increased production of interferon-a by dendritic cells, which has been associated with induction of psoriasis. Interleukin-1 (IL-1) has also been implicated in the development of psoriasis. Furthermore, TNF-a can activate apoptosis of potentially autoreactive T-cells. Blockade of this action could lead to T-cell mediated tissue and psoriatic pathology.Anti-TNF-a induced PA is a rare adverse reaction that usually requires drug discontinuation. The exact pathogenetic mechanism is unclear but an imbalance of the cytokine environment has been theorised. Our patient highlights the need to stay vigilant for cutaneous adverse reactions and the evolving diverse spectrum of psoriasiform conditions that can be seen secondary to anti-TNF-a therapy.


Legionella pneumophila. Legionella causes the severe pneumonia Legionnaires’ disease and predominantly affects individuals with a suppressed immune system, including those receiving therapeutic TNF blockade to treat autoinflammatory disorders. TNF induces pro-inflammatory gene expression, cellular proliferation, and survival signals in certain contexts, but can also trigger programmed cell death in others. It remains unclear, however, which of the pleiotropic functions of TNF mediate control of intracellular bacterial pathogens like Legionella. In this study, we demonstrate that TNF signaling licenses macrophages to die rapidly in response to Legionella infection. We find that TNF-licensed cells undergo rapid gasdermin-dependent, pyroptotic death downstream of inflammasome activation. We also find that TNF signaling upregulates components of the inflammasome response, and that the caspase-11-mediated non-canonical inflammasome is the first inflammasome to be activated, with caspase-1 and caspase-8 mediating delayed pyroptotic death. We find that all three caspases are collectively required for optimal TNF-mediated restriction of bacterial replication in macrophages. Furthermore, caspase-8 is required for control of pulmonary Legionella infection. These findings reveal a TNF-dependent mechanism in macrophages for activating rapid cell death that is collectively mediated by caspases-1, -8, and -11 and subsequent restriction of Legionella infection.The inflammatory cytokine tumor necrosis factor (TNF) is necessary for host defense against many intracellular pathogens, including  Legionella pneumophila, which causes the severe pneumonia Legionnaires’ disease, readily invade and replicate inside innate immune cells known as macrophages. While it is clear that TNF signaling is required for protection of macrophages against pathogens like L. pneumophila, the mechanisms by which this signaling protects cells from infection remain unclear. Here, we show that one of the methods by which TNF protects macrophages from L. pneumophila infection is by promoting the death of infected cells. TNF signaling can lead to the production of anti-microbial factors and inflammation, but it can also prompt cells to engage programmed forms of cell death. We demonstrate that in L. pneumophila infection, TNF specifically licenses cells to more rapidly and robustly activate a kind of cell death called pyroptosis, which causes inflammation and warns nearby uninfected cells. We additionally demonstrate several cooperative pathways downstream of TNF by which this death occurs. Our work shows that TNF protects macrophage populations from L. pneumophila by licensing cells to rapidly self-sacrifice upon infection and limit the replicative niche of the bacteria.The immune signaling molecule tumor necrosis factor (TNF) is critical for defense against many bacterial infections. In the absence of TNF, opportunistic bacterial pathogens such as  The innate immune system is generally capable of preventing and controlling infection. However, pathogens are able to establish infection when key immune factors are evaded or deficient . InflammLegionella pneumophila. The etiologic agent of the severe pneumonia Legionnaire’s disease, L. pneumophila is an opportunistic pathogen that causes disease predominantly in immunocompromised hosts  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Mary X. O'RiordanAssociate EditorPLOS PathogensRaphael ValdiviaSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens  orcid.org/0000-0002-7699-2064  ***********************The reviewers appreciated the impact and rigor of your study, and felt that it has strong potential to advance the field. Many of the comments relate to providing additional context or more carefully examining interpretation of the data. In addition to any text modifications, please be sure to address the major points below:(1) clarification as to the contribution of ROS to host cell death(2) additional assays to better identify the cell death pathway(s) involved(3) Activation assays specific to caspase-11 as noted by reviewer #3.Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: In this study by Pollock et al., the authors address the role of TNF in mediating control of intracellular Legionella by pathways that lead to death of infected macrophages. The manuscript is very well-written and the data are convincing that at least one major mechanism by which TNF contributes to host defense against Legionella is licensing macrophages to undergo rapid caspase-1, -11, and -8-mediated macrophage death. In addition, they show that TNFR1 signaling is required for restriction of Legionella infection of macrophages and primes cells to rapidly undergo cell death. They go on to demonstrate that the T4SS is somehow triggering caspase-1 and -11 activation in TNF-primed macrophages and that TNF upregulated the expression of IL1a, IL1b, and IL18 mRNA as well as the expression of Caspase-11 protein. They also show that in macrophages from GbpChr3-/- mice that cell death and bacterial control were not abrogated. These findings suggest that TNF-dependent upregulation of inflammasome components leads to rapid engagement of non-canonical inflammasome activation independent of GBPs on chromosome 3. Finally, in a series of elegant experiments utilizing various BMDMs from KO and transgenic mice that caspase-8 contributes to cell death independently of its autocleavage downstream of TNF signaling. Finally, they demonstrate that TNR1 and caspse-8 are required for control of pulmonary Legionella infection in a mouse model. The manuscript is excellent and the findings are a significant contribution to the field and I have only minor suggestions to address before publication in Plos Pathogens.Reviewer #2: In this manuscript, Pollock et al demonstrate a novel role for TNF in caspase-mediated cell death in response to Legionella pneumophila infection. It is clear that TNF is important for pulmonary control of L. pneumophila, and the data presented reveal that caspase-1, -11, and 8 are essential for NAIP5/NLRC4-independent TNF-mediated restriction. The manuscript is well-written and the data are rigorous. I have some comments on interpretation of the data and further discussion of these data in the context of the published literature, which will further support/refine the model. Overall, this work is an important contribution to the field of antimicrobial innate immunity and host response to intracellular bacterial pathogens.Reviewer #3: This is a comprehensive study focused or the role of TNF licensing in mediating macrophage death in response to L. pneumophila. The authors demonstrated that TNF priming triggers cell death, however, the downstream mechanisms remain unclear. They show that TNF signaling licenses macrophages to die rapidly in response to Legionella infection mainly by rapid gasdermin-dependent, pyroptotic death downstream of inflammasome activation. They also show that TNF signaling upregulates components of the inflammasome response. They suggest that caspase-11 is rapidly activated, while caspase-1 and caspase-8 mediate delayed pyroptotic death. Furthermore, caspase-8 is required for control of pulmonary Legionella infection. Together, they propose that all three caspases are collectively required for optimal TNF-mediated restriction of L. pneumophila replication in macrophages. These findings reveal a TNF-dependent mechanism in bone marrow derived macrophages for the activation of rapid cell death that is collectively mediated by caspases-1, -8, and -11 and subsequent restriction of Legionella infection. While the findings are mechanistically important, the authors need to address some points.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: NONEReviewer #2: Lines 144-154: These data do not rule out a role for ROS since mtROS are not evaluated . To support this conclusion, additional data are required (antioxidant treatment) or the text should be modified to specify NOX2-mediated ROS . Is the quantity of TNF secreted consistent between wt and Nos2-/- and cybb-/- BMDMs? Do iNOS or NOX2 contribute to bacterial restriction in TNF-primed BMDMs? The authors should also consider discussing their work in the context of the study by Roca et al (PMID: 31474371) showing a role for mtROS in TNF-mediated mediated cell death and restriction of Mycobacterium spp.Lines 306-310: It's difficult to compare cell death between Ripk3-/-Casp8DA, Ripk3-/-, and Ripk3-/-Casp8-/- since they are plotted on separate histograms. Plotting all of the PI-uptake data on the same histogram would be a mess, so showing LDH data here would be more intuitive for comparison between these BMDMs. The authors could consider including the PI uptake data as supplemental information.Lines 312-315/Fig 4D: Could decreased Casp3/7 activity in primed cells be a consequence of less viable cells? I'm not sure how well this assay detects enzymatic activity in cell supernatants. Can the authors comment on this? Perhaps it would be worth normalizing these data to viable cells?Lines 344-345: It seems that gasdermin-dependent cell death is not responsible for L. pneumophila restriction. How do the authors reconcile this with their overall model whereby TNF-mediated cell death is responsible for bacterial restriction? It would seem that the 'additional TNF-mediated protective mechanisms' are quite important here. In Fig 5B, it seems that there is some gasdermin-independent cell death at 8h post-infection. I suggest modifying lines 357-359 to address this. Based on the work of Oxenius and colleagues, it may be phagolysosomal fusion (PMID: 27105352). It would be interesting to see whether this is the case, but I recognize it is likely out of the scope of the study. Please provide additional discussion on potential alternative mechanisms of bacterial restriction.Reviewer #3: - The authors did not examine other specific forms of cell death including PANoptosis and the downstream repercussions. LDH release and PI uptake can occur in response to several forms of cell death.- The authors mention in the legends of several figures using non-replicating fla mutants. They did not explain the characteristic of this unusual fla mutant. Typically, Legionella fla mutants replicate very well even in restrictive cells.- Please add the name of the different mutants above every panel. It is confusing to follow which ones included WT legionella, DotA mutant or Fla mutant.- In several figures, they focus on IL-1a and not IL-1B. Although IL-1B is the well characterized inflammasome-dependent cytokine whereas IL-1a is not.- The authors conclude that TNF licenses macrophages to undergo caspase-1 and 11-mediated cell death to restrict Legionella, yet in figure 2 E, primed Casp-1-/-/casp11-/- macrophages still restrict Legionella replication.- The authors consider the increase of caspase-11 expression as a sign of activation, which is incorrect. They also use the cleaved caspase-11 band on western blots as a sign of activation, which is also incorrect, since it is known in the field that cleavage of caspase-11 does not signify activation. Instead, they need to use activation assays specific to caspase-11 as published by others.- It is not clear why caspase-11 cleavage products are detected in culture supernatants and not in cell lysates .- There is a difference between the secretion of cytokines and the passive release of cytokines from dying cells. The authors need to accurately state these differences and use them appropriately through the text while explaining their results.- SFig 4B is confusing. Why is there no actin in the cell lysates? What is the band that they show as casp11? Why does it disappear when actin disappears?- The authors need to explain how can “uncleavable” caspase-8 modulate Legionella replication?**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: 1. The authors show that triggering cell death pathways in macrophages is dependent on the T4SS. Can they speculate what the host might be sensing that then leads to activation of caspases-1 and -11 and finally caspase-8? What is upstream that is dependent on TNF, but independent of GBPs on chrom3?2. In Figure 6, is it possible to look in infected lungs and quantitate macrophage death to see differences in WT compared to KO mice? And does this correlate with differences in influx of cells that can kill Legionella e.g., perhaps higher levels of neutrophils in WT compared to KO’s?Reviewer #2: Lines 180-182: It seems that only PI uptake assays are shown. Please also include the LDH-based cytotoxicity data.Lines 202-205: The statement '… other caspase-1/11-independent factors contribute to TNF-mediated cell death…' is confusing in light of the data in Fig 2B-C and the statement in lines 188-190. Can the authors please clarify what the statement is based on?Lines 251-263: Is there evidence that these GBPs are actually present in infected BMDMs in the absence of IFN-g priming? Fig S3B: Why are fold CFU at 48 h shown as opposed to 72 h like the other figures?Figure 4D: It would be helpful to make the Tnf-/- primed bars a different color since it looks a lot like the red used for Tnf-/- mock.Figure 5B. Please comment on gasdermin-independent cell death observed at 8h post-infection in TNF-primed cellsLines 343-345: The data in Fig 5D suggest to me that bacterial restriction is independent of cell deathLines 460-462: What do the authors think the pro-inflammatory genes could be?Line 509: What was the MOI used for Fig 1H?Reviewer #3: - The sentence: “Using this priming and infection model alongside genetic tools, our data demonstrate that TNF signaling through TNFR1 licenses L. pneumophila-infected cells to rapidly and robustly undergo flagellin and NAIP/NLRC4-independent cell death in a T4SS-dependent manner” Is somewhat misleading, since it is known that T4SS is indispensable for Legionella replication under all circumstances.- “TNF is able to license this pyroptotic death in part by upregulating components of the caspase-11 non-canonical inflammasome ahead of infection” is inaccurate since the authors did not exclude other types of cell death such as necrosis and apoptosis (PANoptosis).- Please make sure to add the appropriate references for the role of flagellin in Legionella replication and its recognition by Nlrc4/Ipaf.- Please discuss the findings in relation to other similar studies in the field such as Mascarenhas et al 2017, Santic ela al 2007, kawamoto et al 2017,**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. 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Read more information on sharing protocols at  22 Feb 2023AttachmentTP_SS_ResponseLetter.docxSubmitted filename: Click here for additional data file. 25 May 2023Dear Dr. Shin,Legionella pneumophila infection' has been provisionally accepted for publication in PLOS Pathogens.We are pleased to inform you that your manuscript 'TNF licenses macrophages to undergo rapid caspase-1, -11, and -8-mediated cell death that restricts Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Raphael H. ValdiviaSection EditorPLOS PathogensRaphael ValdiviaSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens  orcid.org/0000-0002-7699-2064  ***********************************************************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The authors have done an excellent job of addressing the reviewer's concerns.Reviewer #2: The authors have addressed all of my concerns and I congratulate them on a rigorous and impactful study.Reviewer #3: the authors responded to all my comments.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: (No Response)Reviewer #2: (No Response)Reviewer #3: none**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: (No Response)Reviewer #2: (No Response)Reviewer #3: none**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064